Co-reporter:Lihua Du, Huan Qin, Teng Ma, Tao Zhang, and Da Xing
ACS Nano September 26, 2017 Volume 11(Issue 9) pp:8930-8930
Publication Date(Web):September 11, 2017
DOI:10.1021/acsnano.7b03226
Developing multifunctional phototheranostics with nanoplatforms offers promising potential for effective eradication of malignant solid tumors. In this study, we develop a multifunctional phototheranostic by combining photothermal therapy (PTT) and photoacoustic therapy (PAT) based on a tumor-targeting nanoagent (DBCO-ZnPc-LP). The nanoagent DBCO-ZnPc-LP was facilely prepared by self-assembling of a single lipophilic near-infrared (NIR) dye zinc(II)-phthalocyanine (ZnPc) with a lipid-poly(ethylene glycol) (LP) and following modified further with dibenzyl cyclootyne (DBCO) for introducing the two-step chemical tumor-targeting strategy based on metabolic glycoengineering and click chemistry. The as-prepared DBCO-ZnPc-LP could not only convert NIR light into heat for effective thermal ablation but also induce a thermal-enhanced ultrasound shockwave boost to trigger substantially localized mechanical damage, achieving synergistic antitumor effect both in vitro and in vivo. Moreover, DBCO-ZnPc-LP can be efficiently delivered into tumor cells and solid tumors after being injected intravenously via the two-step tumor-targeting strategy. By integrating the targeting strategy, photoacoustic imaging, and the synergistic interaction between PTT and PAT, a solid tumor could be accurately positioned and thoroughly eradicated in vivo. Therefore, this multifunctional phototheranostic is believed to play an important role in future oncotherapy by the enhanced synergistic effect of PTT and PAT under the guidance of photoacoustic imaging.Keywords: click chemistry; nanoparticle; photoacoustic imaging; photoacoustic therapy; photothermal therapy; tumor-targeting;
Co-reporter:Yujiao Shi, Sihua Yang, and Da Xing
The Journal of Physical Chemistry C March 16, 2017 Volume 121(Issue 10) pp:5805-5805
Publication Date(Web):February 27, 2017
DOI:10.1021/acs.jpcc.6b12498
Efficient contrast agents such as plasmonic nanoparticles (PNPs) are highly desirable for good-performance photoacoustic (PA) imaging. Owing to the small size effect, PNPs have a unique microscopic mechanism of PA conversion from photons to ultrasound. Here, by quantitatively modeling the optical absorption, the time-resolved temperature field, and thermal expansion based on the analytical and finite element analysis (FEA) method, we obtained the quantitative size-dependent PA conversion efficiency of nanospheres/nanorods spanning a comprehensive range of particle sizes, which also provides a deep understanding of the microscopic PA conversion mechanism for nanoprobes. Results show that both the plasmon-mediated absorption and energy conversion from absorbed laser energy to ultrasound are strongly dependent on the PNPs’ size, which comes from their sharply increased surface-to-volume ratios. The gold nanospheres and nanorods possess peak size values for maximizing the PA conversion efficiency. Our work gives theoretical guidelines for constructing highly sensitive PA nanoprobes through rational optimization of the PNPs’ size.
Co-reporter:Aiguo Zhou, Yanchun Wei, Baoyan Wu, Qun Chen, and Da Xing
Molecular Pharmaceutics June 4, 2012 Volume 9(Issue 6) pp:
Publication Date(Web):April 25, 2012
DOI:10.1021/mp200590y
Near-infrared (NIR)-to-visible upconversion nanoparticle (UCNP) has shown promising prospects in photodynamic therapy (PDT) as a drug carrier or energy donor. In this work, a photosensitizer pyropheophorbide a (Ppa) and RGD peptide c(RGDyK) comodified chitosan-wrapped NaYF4:Yb/Er upconversion nanoparticle UCNP-Ppa-RGD was developed for targeted near-infrared photodynamic therapy. The properties of UCNP-Ppa-RGD, such as morphology, stability, optical spectroscopy and singlet oxygen generation efficiency, were investigated. The results show that covalently linked pyropheophorbide a molecule not only is stable but also retains its spectroscopic and functional properties. In vitro studies confirm a stronger targeting specificity of UCNP-Ppa-RGD to integrin αvβ3-positive U87-MG cells compared with that in the corresponding negative group. The photosensitizer-attached nanostructure exhibited low dark toxicity and high phototoxicity against cancer cells upon 980 nm laser irradiation at an appropriate dosage. These results represent the first demonstration of a highly stable and efficient photosensitizer modified upconversion nanostructure for targeted near-infrared photodynamic therapy of cancer cells. The novel UCNP-Ppa-RGD nanoparticle may provide a powerful alternative for near-infrared photodynamic therapy with an improved tumor targeting specificity.Keywords: 980 nm laser; c(RGDyK); chitosan; PDT; pyropheophorbide a; upconversion nanoparticle;
Co-reporter:Yuhui Liao, Xiaoming Zhou, Yu Fu, and Da Xing
Analytical Chemistry December 5, 2017 Volume 89(Issue 23) pp:13016-13016
Publication Date(Web):November 2, 2017
DOI:10.1021/acs.analchem.7b04142
Electrochemiluminescence (ECL) has been engineered to perform various tasks in the area of immunoassays and molecular diagnosis. However, there is still substantial potential for developments of ECL assay with high efficiency to achieve trace analysis. Herein, we demonstrate a polymer-amplified ECL assay via construction of linear Ru(bpy)32+–polymer. This new polymer material compensates for the relatively low ECL intensity from single ECL luminophore and realizes a stable and controllable labeling process. The polymer-amplified ECL assay achieved a remarkable sensitivity of 100 amol. The wide-ranging applications of the polymer-amplified ECL assay for Hepatitis B virus, carcinoembryonic antigen, 16sRNA, and thrombin also demonstrate its superiority. Hence, the polymer-amplified ECL assay possesses the potential to create a new paradigm in amplified ECL assays that could provide outstanding performance for biomedical analysis.
Co-reporter:Xiaotong Gu, Lei Liu, Qi Shen, Da Xing
Cellular Signalling 2017 Volume 37(Volume 37) pp:
Publication Date(Web):1 September 2017
DOI:10.1016/j.cellsig.2017.06.007
•LPLI exerts neuroprotection against MPP+-induced neurotoxicity.•LPLI induces VMAT2 expression to protect against MPP+ toxicity.•LPLI-induced activation of CREB enhances VMAT2 transcription.•Photoactivated ERK is responsible for CREB-dependent upregulation of VMAT2.The vesicular monoamine transporter 2 (VMAT2) pumps dopamine from cytoplasm into synaptic vesicles for subsequent release, and the deficits of VMAT2 has been implicated in the dopaminergic neuronal cell loss which is considered as a typical pathological feature of Parkinson's disease (PD). Low-power laser irradiation (LPLI), a potent noninvasive physiotherapy approach, is capable of penetrating into nerve tissue to exert beneficial effects such as promoting nerve regeneration and ATP production. In the present study, we demonstrated that LPLI protects against MPP+-induced neurotoxicity via upregulation of VMAT2 in SH-SY5Y human dopaminergic neuroblastoma cells. The photoactivation of ERK phosphorylated cAMP-response element binding protein (CREB) at Ser133, and thus increased the ability of CREB binding to the promoter region of VMAT2, leading to elevated VMAT2 expression, which contributes to dopamine release and cell survival. Taken together, for the first time to our knowledge, the results showed that LPLI attenuates MPP+-induced neurotoxicity through activation of ERK/CREB/VMAT2 pathway, suggesting that the manipulation of VMAT2 by LPLI may provide a potential therapeutic strategy for PD.
Co-reporter:Bowen Shu, Chunsun Zhang, Da Xing
Biosensors and Bioelectronics 2017 Volume 97(Volume 97) pp:
Publication Date(Web):15 November 2017
DOI:10.1016/j.bios.2017.06.014
•A pocket-size, real-time convective PCR system capable of sample-to-answer genetic analysis was firstly proposed.•The VMBs-based bacteria enrichment and photothermal conversion was firstly applied to the microfluidic PCR field.•A wireless video camera-enabled fluorescence imaging was integrated for monitoring PCR products in real time.•The dynamic linear range of 101-106 copies/µL and detection sensitivity of 1 copies/µL has been obtained.•The proposed system may become a potential candidate for fast, accurate and affordable POC molecular diagnostics.Timely and accurate molecular diagnostics at the point-of-care (POC) level is critical to global health. To this end, we propose a handheld convective-flow real-time polymerase chain reaction (PCR) system capable of direct sample-to-answer genetic analysis for the first time. Such a system mainly consists of a magnetic bead-assisted photothermolysis sample preparation, a closed-loop convective PCR reactor, and a wireless video camera-based real-time fluorescence detection. The sample preparation exploits the dual functionality of vancomycin-modified magnetic beads (VMBs) for bacteria enrichment and photothermal conversion, enabling cell pre-concentration and lysis to be finished in less than 3 min. On the presented system, convective thermocycling is driven by a single-heater thermal gradient, and its amplification is monitored in real-time, with an analysis speed of less than 25 min, a dynamic linear range from 106 to 101 copies/µL and a detection sensitivity of as little as 1 copies/µL. Additionally, the proposed PCR system is self-contained with a control electronics, pocket-size and battery-powered, providing a low-cost genetic analysis in a portable format. Therefore, we believe that this integrated system may become a potential candidate for fast, accurate and affordable POC molecular diagnostics.
Co-reporter:Guohai Liang, Xudong Jin, Shuxu Zhang, Da Xing
Biomaterials 2017 Volume 144(Volume 144) pp:
Publication Date(Web):1 November 2017
DOI:10.1016/j.biomaterials.2017.08.017
Radiotherapy is a leading treatment approach of cancer therapy. While it is effective in killing tumor cells, it can also cause serious damage to surrounding normal tissue. Targeted radiotherapy with gold nanoparticle-based radiosensitizers is actively being investigated, and considered as a promising means to enhance the efficacy of radiotherapy against tumors under a relatively low and safe radiation dose. In this work, we report a green and one-step strategy to synthesize fluorescent gold nanoclusters by using a commercialized cyclic arginine-glycine-aspartic acid (c(RGDyC)) peptide as the template. The nanoclusters inherit special properties of both the Au core (red/NIR fluorescence emission and strong radiosensitizing effect) and c(RGDyC) shell (active cancer cell-targeting ability and good biocompatibility), and can be applied as fluorescent probes to stain αvβ3 integrin-positive cancer cells, as well as radiosensitizing agents to boost the killing efficacy of radiotherapy. Our data suggest that the as-designed gold nanoclusters have excellent biocompatibility, bright red/NIR fluorescence, active tumor targeting property, and strong radiosensitizing effect, making them highly promising towards potential clinical translation.
Co-reporter:Na Zhao, Baoyan Wu, Xianglong Hu, Da Xing
Biomaterials 2017 Volume 141(Volume 141) pp:
Publication Date(Web):1 October 2017
DOI:10.1016/j.biomaterials.2017.06.031
Stimuli-responsive nanoparticles with multiple therapeutic/diagnostic functions are highly desirable for effective tumor treatment. Herein novel caspase-3 responsive functionalized upconversion nanoparticles (CFUNs) were fabricated with three-in-one functional integration: near-infrared (NIR) triggered photodynamic damage along with caspase-3 activation, subsequent caspase-3 responsive drug release, and cascade chemotherapeutic activation. CFUNs were formulated from the self-assembly of caspase-3 responsive doxorubicin (DOX) prodrug tethered with DEVD peptide (DEVD-DOX), upconversion nanoparticles (UCNP), a photosensitizer (pyropheophorbide-a methyl ester, MPPa), and tumor-targeting cRGD-PEG-DSPE to afford multifunctional CFUNs, MPPa/UCNP-DEVD-DOX/cRGD. Upon cellular uptake and NIR irradiation, the visible light emission of UCNP could excite MPPa to produce reactive oxygen species for photodynamic therapy (PDT) along with the activation of caspase-3, which further cleaved DEVD peptide to release DOX within tumor cells, thus accomplishing NIR-triggered PDT and cascade chemotherapy. CFUNs presented silent therapeutic potency and negligible cytotoxicity in the dark, whereas in vitro and in vivo experiments demonstrated the NIR-triggered cascade therapeutic activation and tumor inhibition due to consecutive PDT and chemotherapy. Current NIR-activated cascade tumor therapy with two distinct mechanisms is significantly favorable to overcome multidrug resistance and tumor heterogeneity for persistent tumor treatment.
Co-reporter:Yu Fu;Xiaoming Zhou
Lab on a Chip (2001-Present) 2017 vol. 17(Issue 24) pp:4334-4341
Publication Date(Web):2017/12/05
DOI:10.1039/C7LC01107E
In this work, we describe for the first time a genetic diagnosis platform employing a polydiallyldimethylammonium chloride (PDDA)-modified capillary and a liquid-based thermalization system for rapid, simple and quantitative DNA analysis with minimal user interaction. Positively charged PDDA is modified on the inner surface of the silicon dioxide capillary by using an electrostatic self-assembly approach that allows the negatively charged DNA to be separated from the lysate in less than 20 seconds. The capillary loaded with the PCR mix is incorporated in the thermalization system, which can achieve on-site real-time PCR. This system is based on the circulation of pre-heated liquids in the chamber, allowing for high-speed thermalization of the capillary and fast amplification. Multiple targets can be simultaneously analysed with multiplex spatial melting. Starting with live Escherichia coli (E. coli) cells in milk, as a realistic sample, the current method can achieve DNA extraction, amplification, and detection within 40 min.
Co-reporter:Guohai Liang;Xudong Jin;Huan Qin
Journal of Materials Chemistry B 2017 vol. 5(Issue 31) pp:6366-6375
Publication Date(Web):2017/08/09
DOI:10.1039/C7TB01517H
The development of functional nanomaterials that undergo renal clearance is of fundamental importance to their in vivo biomedical applications. In this work, we report a one-pot method for the preparation of ultrasmall copper sulfide nanodots capped with a small natural tripeptide glutathione (GSH-CuS NDs). The GSH-CuS NDs had a hydrodynamic diameter of 5.8 nm, smaller than the reported polymer-coated CuS NDs with similar core sizes, and exhibited strong optical absorption and conversion at the near-infrared (NIR) region, leading to a sufficient photohyperthermic effect under the irradiation of a 980 nm laser. In vivo studies showed that the GSH-CuS NDs could induce significant photoacoustic imaging signal enhancement and remarkable photothermal therapy efficacy. Importantly, biodistribution studies and MRI imaging showed that the GSH-CuS NDs could freely circulate in the blood pool without undesirable accumulation in the liver and spleen, and could be naturally removed from the body through renal clearance, making them attractive for practical theranostic applications.
Co-reporter:Ru Huang, Yuhui Liao, Xiaoming Zhou, Yu Fu, Da Xing
Sensors and Actuators B: Chemical 2017 Volume 247(Volume 247) pp:
Publication Date(Web):1 August 2017
DOI:10.1016/j.snb.2017.03.055
•The paper describes an optical biosensor to simultaneously detect multiple miRNAs.•QD and GO were used as a homogeneous nano-photon switch.•Detecting two miRNAs from tumor cells realized a high sensitivity of 102 cells.•The performance for tumor tissue samples confirmed the reliability of this platform.MicroRNAs (miRNAs) have been served as the promising biomarkers for tumor diagnosis. Generally, miRNAs undergo synergistic adjustment in tumor cells, for instance, both miRNA21 and miRNA155 are highly expressed in breast cancer cells. Thus, the combinational detection of multiple miRNAs has practical significance. In this study, we constructed a homogeneous nano-photon switch based on quantum dots (QDs) and graphene oxide (GO) for the combinational detection of multiple miRNAs in tumor cells and tissues. Moreover, multiple enzyme-free DNA circuits were constructed to perform the function of signal amplification, and the homogeneous nano-photon switch could emit characteristic signals corresponding to specific miRNAs. The results of miRNA detection from tumor cells indicated that this platform realized the high sensitivity of 102 cells. Its excellent performance in tumor tissue also confirmed this platform as a potentially reliable, multivariate assay that could reduce the false-negative and false-dismissal rates of routine tests.Download high-res image (193KB)Download full-size image
Co-reporter:Yujiao Shi;Sihua Yang
Nano Research 2017 Volume 10( Issue 8) pp:2800-2809
Publication Date(Web):06 May 2017
DOI:10.1007/s12274-017-1483-9
The probe-assisted integration of imaging and therapy into a single modality offers significant advantages in bio-applications. As a newly developed photoacoustic (PA) mechanism, plasmon-mediated nanocavitation, whereby photons are effectively converted into PA shockwaves, has excellent advantages for image-guided therapy. In this study, by simulating the laser absorption, temperature field, and nanobubble dynamics using both finite-element analysis and computational fluid dynamics, we quantified the cavitation-induced PA conversion efficiency of a water-immersed gold nanosphere, revealing new insights. Interestingly, sequential multi-bubble emission accompanied by high PA signal production occur under a single high-dose pulse of laser irradiation, enabling a cavitation-induced PA conversion efficiency up to 2%, which is ~50 times higher than that due to thermal expansion. The cavitation-induced PA signal has unique frequency characteristics, which may be useful for a new approach for in vivo nanoparticle tracking. Our work offers theoretical guidance for accurate diagnosis and controllable therapy based on plasmon-mediated nanocavitation.
Co-reporter:Yanchun Wei, Feifan Zhou, Da Zhang, Qun Chen and Da Xing
Nanoscale 2016 vol. 8(Issue 6) pp:3530-3538
Publication Date(Web):11 Jan 2016
DOI:10.1039/C5NR07785K
Subcellular organelles play critical roles in cell survival. In this work, a novel photodynamic therapy (PDT) drug delivery and phototoxicity on/off nano-system based on graphene oxide (NGO) as the carrier is developed to implement subcellular targeting and attacking. To construct the nanodrug (PPa-NGO-mAb), NGO is modified with the integrin αvβ3 monoclonal antibody (mAb) for tumor targeting. Pyropheophorbide-a (PPa) conjugated with polyethylene-glycol is used to cover the surface of the NGO to induce phototoxicity. Polyethylene-glycol phospholipid is loaded to enhance water solubility. The results show that the phototoxicity of PPa on NGO can be switched on and off in organic and aqueous environments, respectively. The PPa-NGO-mAb assembly is able to effectively target the αvβ3-positive tumor cells with surface ligand and receptor recognition; once endocytosized by the cells, they are observed escaping from lysosomes and subsequently transferring to the mitochondria. In the mitochondria, the ‘on’ state PPa-NGO-mAb performs its effective phototoxicity to kill cells. The biological and physical dual selections and on/off control of PPa-NGO-mAb significantly enhance mitochondria-mediated apoptosis of PDT. This smart system offers a potential alternative to drug delivery systems for cancer therapy.
Co-reporter:Junping Zhong, Sihua Yang, Liewei Wen, Da Xing
Journal of Controlled Release 2016 Volume 226() pp:77-87
Publication Date(Web):28 March 2016
DOI:10.1016/j.jconrel.2016.02.010
Here, a novel triggered drug release modality was developed for oncotherapy. Paclitaxel (PTX), perfluorohexane (PFH) and gold nanorods (AuNRs) loaded nanoparticles (PTX-PAnP) were synthesized. Folic acid (FA) conjugated PTX-PAnP (PTX-PAnP-FA) could be selectively taken into folate receptor-overexpressed tumor cells. Upon pulsed laser irradiation, the PTX-PAnP-FA could be rapidly destructed because of the PFH vaporization, resulting in fast drug release, which induced apoptosis of cancer cells efficiently. Stimulated fragmentation of the PTX-PAnP-FA nanoparticles can facilitate multiple mechanisms such as bubble implosion, shockwave generation, and sonoporation that further enhance the therapeutic efficiency. The in vivo therapy study further confirmed this new approach resulted in efficient tumor suppression. The results demonstrate a unique drug release mechanism based on photoacoustic effect. It provides an all-in-one platform for photoacoustic image-guided drug release and synergistic chemo-photoacoustic therapy.Paclitaxel, perfluorohexane and gold nanorods loaded nanoparticle is synthesized as an all-in-one platform for photoacoustic imaging-guided drug release and synergistic chemo-photoacoustic therapy.
Co-reporter:Hongxing Liu, Xiaoming Zhou, Weipeng Liu, Xiaoke Yang, and Da Xing
Analytical Chemistry 2016 Volume 88(Issue 20) pp:10191
Publication Date(Web):September 16, 2016
DOI:10.1021/acs.analchem.6b02772
Genetic analysis is of great importance for the detection of pathogenic bacteria. Bacterial identification must become simpler, less expensive, and more rapid than the traditional methods. In this study, a low-cost, label-free, and wireless paper-based bipolar electrode electrochemiluminescence (pBPE-ECL) analysis system was constructed for the rapid and sensitive genetic detection of pathogenic bacteria. Wax-screen printing was used to form hydrophilic channels on filter paper, and a carbon ink-based bipolar electrode and driving electrodes were screen-printed into the channels. The “light-switch” molecule [Ru(phen)2dppz]2+ (phen = 1,10-phenanthroline; dppz = dipyridophenazine) was used to intercalate into the base pairs of the double-stranded DNA PCR amplification products, and the complexs were then applied to the paper-based bipolar electrode to perform the ECL assays; the ECL of [Ru(phen)2dppz]2+ is quenched in aqueous solution, but this molecule displays intense ECL when intercalated into double-stranded DNA. Under optimized experimental conditions, as little as 10 copies/μL of the genomic DNA of Listeria monocytogenes could be detected. Additionally, the system could also specifically distinguish Listeria monocytogenes from Salmonella, Escherichia coli O157:H7, and Staphylococcus aureus. This label-free, simple, and rapid method has potential in point-of-care applications for pathogen detection.
Co-reporter:Weipeng Liu, Huiheng Yu, Xiaoming Zhou, and Da Xing
Analytical Chemistry 2016 Volume 88(Issue 17) pp:8369
Publication Date(Web):August 9, 2016
DOI:10.1021/acs.analchem.6b02338
The CRISPR/Cas9 system is a revolutionary genome-editing tool that enables targeted and efficient gene knockouts. However, the off-target effects and loci-dependent enzyme activity limit its uses on the field of research and treatment. In this study, we designed a convenient and sensitive in vitro test method, which was based on electrochemiluminescence (ECL) technology for evaluating cleavage activity of the CRISPR/Cas9 system. It was find that Cas9 can tolerate some common genetic modifications to its target DNA. It was also find that target DNA/sgRNA with single-base mismatch and UV damages of target DNA resulted in significantly reduction of Cas9 cleavage efficiency. Comparing with traditional method, the proposed method reduced the evaluation time from weeks to 2 h. Therefore, our study provides a versatile in vitro method for a priori analysis of CRISPR/Cas9 system and highlights the potential to guide in vivo genome editing.
Co-reporter:Weipeng Liu, Minjun Zhu, Hongxing Liu, Jitao Wei, Xiaoming Zhou, Da Xing
Biosensors and Bioelectronics 2016 81() pp: 309-316
Publication Date(Web):15 July 2016
DOI:10.1016/j.bios.2016.02.073
•We develop a universal primer design strategy for enhancing amplification efficiency.•This strategy can exquisite discrimination of microRNA at the single base level.•The strategy is based on stacking hybridization and toehold-mediated strand exchange.•It is the first time to study the effect of an invading stacking primer.Searching for a strategy to enhance the efficiency of nucleic acid amplification and achieve exquisite discrimination of nucleic acids at the single-base level for biological detection has become an exciting research direction in recent years. Here, we have developed a simple and universal primer design strategy which produces a fascinating effect on isothermal strand displacement amplification (iSDA). We refer to the resultant primer as “invading stacking primer (IS-Primer)” which is based on contiguous stacking hybridization and toehold-mediated exchange reaction and function by merely changing the hybridization location of the primer. Using the IS-Primer, the sensitivity in detecting the target miR-21 is improved approximately five fold compared with the traditional iSDA reaction. It was further demonstrated that the IS-Primer acts as an invading strand to initiate branch migration which can increase the efficiency of the untwisting of the hairpin probe. This effect is equivalent to reducing the free energy of the stem, and the technique shows superior selectivity for single-base mismatches. By demonstrating the enhanced effect of the IS-Primer in the iSDA reaction, this work may provide a potentially new avenue for developing more sensitive and selective nucleic acids assays.
Co-reporter:Huan Qin, Yue Zhao, Jian Zhang, Xiao Pan, Sihua Yang, Da Xing
Nanomedicine: Nanotechnology, Biology and Medicine 2016 Volume 12(Issue 7) pp:1765-1774
Publication Date(Web):October 2016
DOI:10.1016/j.nano.2016.02.016
Elevated expression of matrix metalloproteinase-2 (MMP2) is one of the clinical features of high-risk atherosclerotic plaques. Many methods such as fluorescence imaging methods have been used to detect MMP2 for evaluating plaque vulnerability. However, so far no imaging method has been able to resolve the expression of MMP2 with high fidelity and resolution beyond microscopic depths. In this study, gold nanorods conjugated with MMP2 antibody (AuNRs-Abs) were developed as a highly efficient photoacoustic imaging (PAI) probe for mapping MMP2 in atherosclerotic plaques. AuNRs-Abs could specifically target MMP2 as demonstrated by scanning electron microscope and immunofluorescence imaging. After labeling with AuNRs-Abs, area of distribution of MMP2 from the surface to the depths of the atherosclerotic plaques was revealed using intravascular PAI. AuNRs-Abs has the ability to enable quantitative detection of MMP2 in atherosclerotic plaques.Elevated expression of matrix metalloproteinase-2 (MMP2) is one of the features of high-risk atherosclerotic plaques. In this paper, gold nanorods conjugated with MMP2 antibody (termed here AuNRs-Abs) are developed as a high-efficient photoacoustic imaging probe for mapping MMP2 in atherosclerotic plaques.
Co-reporter:Hongjiang Chen, Sihua Yang, Ting Zhou, Jiankun Xu, Jun Hu, Da Xing
Nanomedicine: Nanotechnology, Biology and Medicine 2016 Volume 12(Issue 6) pp:1453-1462
Publication Date(Web):August 2016
DOI:10.1016/j.nano.2016.02.023
Co-reporter:Yujiao Shi;Huan Qin;Sihua Yang
Nano Research 2016 Volume 9( Issue 12) pp:3644-3655
Publication Date(Web):2016 December
DOI:10.1007/s12274-016-1234-3
Efficient probes/contrast agents are highly desirable for good-performance photoacoustic (PA) imaging, where the PA signal amplitude of a probe is dominated by both its optical absorption and the conversion efficiency from absorbed laser energy to acoustic waves. Nanoprobes have a unique micromechanism of PA energy conversion due to the size effect, which, however, has not been quantitatively demonstrated and effectively utilized. Here, we present quantitative simulations of the PA signal production process for plasmonmediated nanoprobes based on the finite element analysis method, which were performed to provide a deep understanding of their PA conversion micromechanism. Moreover, we propose a method to amplify the PA conversion efficiency of nanoprobes through the use of thermally confined shell coating, which allows the active control of the conversion efficiency beyond that of conventional probes. Additionally, we deduced the dependence of the conversion efficiency on the shell properties. Gold-nanoparticles/polydimethylsiloxane nanocomposites were experimentally synthesized in the form of gel and microfilms to verify our idea and the simulation results agreed with the experiments. Our work paves the way for the rational design and optimization of nanoprobes with improved conversion efficiency.
Co-reporter:Lisha Zhang, Ru Huang, Weipeng Liu, Hongxing Liu, Xiaoming Zhou, Da Xing
Biosensors and Bioelectronics 2016 Volume 86() pp:1-7
Publication Date(Web):15 December 2016
DOI:10.1016/j.bios.2016.05.100
•Nanoparticle cluster catalyzed signal amplification biosensor.•Fe3O4 nanoparticle cluster possesses high efficient peroxidase-like activity.•A detection limit of 5.4×103 cfu/mL L. Monocytogenes was obtained.•The developed biosensor can be used for visual detection of foodborne pathogens.Foodborne pathogens pose a significant threat to human health worldwide. The identification of foodborne pathogens needs to be rapid, accurate and convenient. Here, we constructed a nanoparticle cluster (NPC) catalyzed signal amplification biosensor for foodborne pathogens visual detection. In this work, vancomycin (Van), a glycopeptide antibiotic for Gram-positive bacteria, was used as the first molecular recognition agent to capture Listeria monocytogenes (L. monocytogenes). Fe3O4 NPC modified aptamer, was used as the signal amplification nanoprobe, specifically recognize to the cell wall of L. monocytogenes. As vancomycin and aptamer recognize L. monocytogenes at different sites, the sandwich recognition showed satisfied specificity. Compared to individual Fe3O4 nanoparticle (NP), NPC exhibit collective effect-enhanced catalytic activity for the color reaction of chromogenic substrate. The change in absorbance or color could represent the concentration of target. Using the Fe3O4 NPC-based signal amplification method, L. monocytogenes whole cells could be directly assayed within a linear range of 5.4×103–108 cfu/mL and a visual limit of detection of 5.4×103 cfu/mL. Fe3O4 NPC-based method was more sensitive than the Fe3O4 NP-based method. All these attractive characteristics of highly sensitivity, visual and labor-saving, make the biosensor possess a potential application for foodborne pathogenic bacteria detection.
Co-reporter:Liewei Wen, Wenzheng Ding, Sihua Yang, Da Xing
Biomaterials 2016 Volume 75() pp:163-173
Publication Date(Web):January 2016
DOI:10.1016/j.biomaterials.2015.10.028
Co-reporter:Minjun Zhu, Weipeng Liu, Hongxing Liu, Yuhui Liao, Jitao Wei, Xiaoming Zhou, and Da Xing
ACS Applied Materials & Interfaces 2015 Volume 7(Issue 23) pp:12873
Publication Date(Web):May 25, 2015
DOI:10.1021/acsami.5b02374
Infectious diseases, especially pathogenic bacterial infections, pose a growing threat to public health worldwide. As pathogenic bacteria usually exist in complex experimental matrixes at very low concentrations, developing a technology for rapid and biocompatible sample enrichment is essential for sensitive diagnosis. In this study, an Fe3O4/Vancomycin/PEG magnetic nanocarrier was constructed for efficient sample enrichment and in situ nucleic acid preparation of pathogenic bacteria for subsequent gene sensing. We attached Vancomycin, a well-known broad-spectrum antibiotic, to the surface of Fe3O4 nanoparticles as a universal molecular probe to target bacterial cells. Polyethylene glycol (PEG) was introduced to enhance the nanocarrier’s water solubility and biocompatibility. Results show that the proposed nanocarrier achieved a 90% capture efficiency even if at a Listeria monocytogenes concentration of 1 × 102 cfu/mL. Contributing to the good water solubility achieved by the employment of modified PEG, highly efficient enrichment (enrichment factor 10 times higher than PEG-free nanocarrier) can be completed in 30 min. Moreover, PEG would also develop the nanoparticles’ biocompatibility by passivating the positively charged unreacted amines on the magnetic nanoparticles, thus helping to release the negatively charged bacterial genome from the nanocarrier/bacteria complexes when an in situ nucleic acids extraction step was executed. The outstanding bacterial capture capability and biocompatibility of this nanocarrier enabled the implementation of a highly sensitive gene-sensing strategy of pathogens. By employing an electrochemiluminescence-based gene-sensing assay, L. monocytogenes can be rapidly detected with a limit of detection of 10 cfu/mL, which shows great potential for clinical applications.Keywords: electrochemiluminescence; Fe3O4/Vancomycin/PEG magnetic nanocarrier; gene sensing; pathogenic bacterial infections; sample enrichment;
Co-reporter:Bowen Shu, Chunsun Zhang and Da Xing
Lab on a Chip 2015 vol. 15(Issue 12) pp:2597-2605
Publication Date(Web):21 Apr 2015
DOI:10.1039/C5LC00139K
A handheld flow genetic analysis system (FGAS) is proposed for rapid, sensitive, multiplex and real-time quantification of nucleic acids at the point-of-care (POC) level. The FGAS includes a helical thermal-gradient microreactor and a microflow actuator, as well as control circuitry for temperature, fluid and power management, and smartphone fluorescence imaging. All of these features are integrated into a field-portable and easy-to-use molecular diagnostic platform powered by lithium batteries. Due to the unique design of the microreactor, not only steady temperatures for denaturation and annealing/extension but also a linear thermal gradient for spatial high-resolution melting can be achieved through simply maintaining a single heater at constant temperature. The smartphone fluorescence imaging system has a wide field of view that captures all PCR channels of the microreactor in a single snapshot without the need for any mechanical scanning. By these designs, the FGAS enables real-time monitoring of the temporal and spatial fluorescence signatures of amplicons during continuous-flow amplification. On the current FGAS, visual detection of as little as 10 copies per μL of genomic DNA of Salmonella enterica was achieved in 15 min, with real-time quantitative detection of the DNA over 6 orders of magnitude concentration from 106 to 101 copies per μL also completed in 7.5–15 min. In addition, multiple pathogenic DNA targets could be simultaneously discriminated with direct bar-chart readout or multiplex spatial melting in serial flow. We anticipate that the FGAS has great potential to become a next-generation gene analyzer for POC molecular diagnostics.
Co-reporter:Wei-Yan Hu;Zhi-Yong He;Lu-Jun Yang;Ming Zhang;Zhi-Cheng Xiao
British Journal of Pharmacology 2015 Volume 172( Issue 9) pp:
Publication Date(Web):
DOI:10.1111/bph.13048
Background and Purpose
At the early stage of Alzheimer's disease (AD), the accumulation of β-amyloid (Aβ) oligomers disturbs intracellular Ca2+ homeostasis and disrupts synaptic plasticity of brain neurons. Prevention of Aβ-induced synaptic failure remains an unsolved problem for the treatment of AD. Here, the effects of 2-aminoethoxydiphenyl borate (2-APB), a non-specific, but moderately potent Ca2+ channel inhibitor, on Aβ-induced deficit of synaptic long-term potentiation (LTP) and the underlying molecular mechanisms were explored.
Experimental Approach
We used hippocampal slices and primary cultures of hippocampal neurons from C57BL/6 mice. Methods applied in our study included electrophysiological recording, membrane protein extraction, Western blot assay and Ca2+ imaging.
Key Results
2-APB at 10 μM effectively reversed suppression by oligomeric Aβ1–42 (500 nM) of LTP in hippocampal slices. 2-APB also restored phosphorylation and trafficking of the glutamate receptor subunit GluA1 in Aβ-treated hippocampal slices, supporting its protective action on synaptic function. Aβ-mediated abnormal neuronal [Ca2+]i elevation and hyperactivation of the mitochondrial apoptotic proteins BAX, caspase-3, and glycogen synthase kinase-3β, were blocked by 2-APB pretreatment. Moreover, the defict in long term potentiation deficit in hippocampal slices from APPswe/PS1ΔE9 gene mutant mice was rescued by 2-APB at 10 μM.
Conclusions and Implication
These data demonstrate that 2-APB is a potentially useful chemical to protect synaptic plasticity against neurotoxic effects of Aβ in AD.
Co-reporter:Ru Huang, Yuhui Liao, Xiaoming Zhou, Da Xing
Analytica Chimica Acta 2015 Volume 888() pp:162-172
Publication Date(Web):12 August 2015
DOI:10.1016/j.aca.2015.07.041
•This paper explored the interaction mechanism of TMNA products with GO surface.•This homogeneous and isothermal system permits a detection limit of 10 pM for microRNA.•This nonenzymatic strategy can avoid nonspecific desorption caused by enzyme protein.•The interaction model can be used to explore the application ability of nonenzymatic circuit.A novel graphene oxide (GO) fluorescence switch-based homogenous system has been developed to solve two problems that are commonly encountered in conventional GO-based biosensors. First, with the assistance of toehold-mediated nonenzymatic amplification (TMNA), the sensitivity of this system greatly surpasses that of previously described GO-based biosensors, which are always limited to the nM range due to the lack of efficient signal amplification. Second, without enzymatic participation in amplification, the unreliability of detection resulting from nonspecific desorption of DNA probes on the GO surface by enzymatic protein can be avoided. Moreover, the interaction mechanism of the double-stranded TMNA products contains several single-stranded toeholds at two ends and GO has also been explored with combinations of atomic force microscopy imaging, zeta potential detection, and fluorescence assays. It has been shown that the hybrids can be anchored to the surface of GO through the end with more unpaired bases, and that the other end, which has weaker interaction with GO, can escape GO adsorption due to the robustness of the central dsDNA structures. We verified this GO fluorescence switch-based detection system by detecting microRNA 21, an overexpressed non-encoding gene in a variety of malignant cells. Rational design of the probes allowed the isothermal nonenzymatic reaction to achieve more than 100-fold amplification efficiency. The detection results showed that our strategy has a detection limit of 10 pM and a detection range of four orders of magnitude.
Co-reporter:Lei Huang;Xiaoxiao Jiang;Longlong Gong
Journal of Cellular Biochemistry 2015 Volume 116( Issue 8) pp:1741-1754
Publication Date(Web):
DOI:10.1002/jcb.25133
ABSTRACT
Promotion of insulin-secreting β-cell regeneration in patients with diabetes is a promising approach for diabetes therapy, which can contribute to rescue the uncontrolled hyperglycemia. Low-power laser irradiation (LPLI) has been demonstrated to regulate multiple physiological processes both in vitro and in vivo through activation of various signaling pathways. In the present study, we showed that LPLI promoted β-cell replication and cell cycle progression through activation of Akt1/GSK3β isoform-specific signaling axis. Inhibition of PI3-K/Akt or GSK3 with specific inhibitors dramatically reduced or increased LPLI-induced β-cell replication, revealing Akt/GSK3 signaling axis was involved in β-cell replication and survival upon LPLI treatment. Furthermore, the results of shRNA-mediated knock down of Akt/GSK3 isoforms revealed that Akt1/GSK3β isoform-specific signaling axis regulated β-cell replication and survival in response to LPLI, but not Akt2/GSK3α. The mechanism by which LPLI promoted β-cell replication through Akt1/GSK3β signaling axis involved activation of β-catenin and down-regulation of p21. Taken together, these observations suggest that Akt1/GSK3β isoform signaling axis play a key role in β-cell replication and survival induced by LPLI. Moreover, our findings suggest that activation of Akt1/GSK3β isoform signaling axis by LPLI may provide guidance in practical applications for β-cell regenerative therapies. J. Cell. Biochem. 116: 1741–1754, 2015. © 2015 Wiley Periodicals, Inc.
Co-reporter:Yuhui Liao, Yu Fu, Yunxia Wu, Ru Huang, Xiaoming Zhou, and Da Xing
Biomacromolecules 2015 Volume 16(Issue 11) pp:
Publication Date(Web):October 13, 2015
DOI:10.1021/acs.biomac.5b00959
Nucleic acids have been engineered to participate in a wide variety of tasks. Among them, the enzyme-free amplification modes, enzyme-free DNA circuits (EFDCs), and hybridization chain reactions (HCRs) have been widely applied in a series of studies of bioanalysis. We demonstrated here an ultrasensitive hairpin probe-based circulation for continuous assemble of DNA probe. This strategy improved the analyte stability-dependent amplification efficiency of EFDC and signal enhancement without being limited by the analyte’s initial concentration, and it was used to produce a novel microRNA (miRNA) trace analysis assay with ultrasensitive amplification properties. Through the detection of standard miRNA substances, 1 amol-level sensitivity and satisfactory specificity were achieved. Compared with EFDCs and HCRs, the sensitivity of ultrasensitive hairpin probe-based circulation was higher by 3 or 4 orders of magnitude. Furthermore, the excellent performance of this platform was also demonstrated in the detection of miRNAs in tumor cells. The sensitivities for the detection of miRNAs in HepG2, A549 and MCF-7 tumor cells were 10, 10, and 100 cells, respectively. In addition, a high detection rate of 83% was achieved for tumor tissues. Thus, this ultrasensitive hairpin probe-based circulation possesses the potential to be a technological innovation in the field of tumor diagnosis.
Co-reporter:Lijuan Liu, Yanchun Wei, Shaodong Zhai, Qun Chen, Da Xing
Biomaterials 2015 62() pp: 35-46
Publication Date(Web):
DOI:10.1016/j.biomaterials.2015.05.036
Co-reporter:Yuhui Liao, Xiaoming Zhou, and Da Xing
ACS Applied Materials & Interfaces 2014 Volume 6(Issue 13) pp:9988
Publication Date(Web):June 5, 2014
DOI:10.1021/am503230h
Graphene oxide (GO) and quantum dots (QDs), as burgeoning types of nanomaterials, have gained tremendous interest in the biosensor field. In this work, we designed a novel multivariate testing strategy that depends on the fluorescence resonance energy transfer (FRET) effect between quantum dots (QDs) and graphene oxide (GO). It integrates the QD–GO FRET principle and QD probes with different emission peaks into a platform, aims at multiplex gene detection of a human infectious and highly pathogenic pathogen, Listeria monocytogenes (L. monocytogenes). With the development of a multiplex linear-after-the-exponential (LATE) polymerase chain reaction (PCR) system, the single-stranded DNA (ssDNA) products of hlyA genes and iap genes are obtained by simultaneous amplification of the target genes. Then with the hybridization of ssDNA products and QD probes, simultaneous homogeneous detection of two gene amplification products can be achieved by using GO as a fluorescence switch and monitoring the relevant emissions excited by a single light source. Distinguishable signals corresponding to target genes are obtained. With this developed approach, genomic DNA from L. monocytogenes can be detected as low as 100 fg/μL. Moreover, this platform has a good dynamic range from 102 to 106 fg/μL. It is indicated that this platform has potential to be a reliable complement for rapid gene detection technologies and is capable of reducing the false-negative and false-dismissal probabilities in routine tests.Keywords: foodborne pathogenic bacteria; graphene oxide; linear-after-the-exponential PCR; multivariate testing strategy; quantum dots
Co-reporter:Jitao Wei, Hongxing Liu, Fang Liu, Minjun Zhu, Xiaoming Zhou, and Da Xing
ACS Applied Materials & Interfaces 2014 Volume 6(Issue 24) pp:22577
Publication Date(Web):November 20, 2014
DOI:10.1021/am506695g
Plant viruses cause significant production and economic losses in the agricultural industry worldwide. Rapid and early identification of contagious plant viruses is an essential prerequisite for the effective control of further spreading of infection. In this work, we describe a miniaturized paper-based gene sensor for the rapid and sensitive identification of a contagious plant virus. Our approach makes use of hybridization-mediated target capture based on a miniaturized lateral flow platform and gold nanoparticle colorimetric probes. The captured colorimetric probes on the test line and control line of the gene sensor produce characteristic red bands, enabling visual detection of the amplified products within minutes without the need for sophisticated instruments or the multiple incubation and washing steps performed in most other assays. Quantitative analysis is realized by recording the optical intensity of the test line. The sensor was used successfully for the identification of banana bunchy top virus (BBTV). The detection limit was 0.13 aM of gene segment, which is 10 times higher than that of electrophoresis and provides confirmation of the amplified products. We believe that this simple, rapid, and sensitive bioactive platform has great promise for warning against plant diseases in agricultural production.Keywords: contagious plant virus; gold nanoparticle colorimetric probes; low-cost diagnostics; paper-based gene sensor; visual detection
Co-reporter:Yuhui Liao, Ru Huang, Zhaokui Ma, Yunxia Wu, Xiaoming Zhou, and Da Xing
Analytical Chemistry 2014 Volume 86(Issue 9) pp:4596
Publication Date(Web):April 2, 2014
DOI:10.1021/ac5007427
MicroRNAs (miRNAs) participate in important processes of life course. Because of their characters of small sizes, vulnerable degradabilities, and sequences similarities, the existing detection technologies mostly contain enzymatic amplification reactions for acquisition of high sensitivities and specificities. However, specific reaction conditions and time-dependent enzyme activities are caused by the accession of enzymes. Herein, we designed a target-triggered enzyme-free amplification platform that is realized by circulatory interactions of two hairpin probes and the integrated electrochemiluminescence (ECL) signal giving-out component. Benefiting from outstanding performances of the enzyme-free amplification system and ECL, this strategy is provided with a simplified reaction process, high sensitivity, and operation under isothermal conditions. Through detection of the miRNA standard substance, the sensitivity of this platform reached 10 fmol, and a splendid specificity was achieved. We also analyzed three tumor cell lines (human lung adenocarcinoma, breast adenocarcinoma, and hepatocellular liver carcinoma cell lines) through this platform. The sensitivities of 103 cells, 104 cells, and 104 cells were, respectively, achieved. Furthermore, clinical tumor samples were tested, and 21 of 30 experimental samples gave out positive signals. Thus, this platform possesses potentials to be an innovation in miRNA detection methodology.
Co-reporter:Bowen Shu, Chunsun Zhang, Da Xing
Analytica Chimica Acta 2014 Volume 826() pp:51-60
Publication Date(Web):15 May 2014
DOI:10.1016/j.aca.2014.04.017
•A high-throughput and rapid microfluidic method for pathogen detection is proposed.•The method offers a simple and convenient way toward high-throughput DNA analysis.•Important parameters of segmented continuous-flow multiplex PCR were investigated.•The proposed method is suitable for high-throughput biomedical monitoring.High-throughput and rapid identification of multiple foodborne bacterial pathogens is vital in global public health and food industry. To fulfill this need, we propose a segmented continuous-flow multiplex polymerase chain reaction (SCF-MPCR) on a spiral-channel microfluidic device. The device consists of a disposable polytetrafluoroethylene (PTFE) capillary microchannel coiled on three isothermal blocks. Within the channel, n segmented flow regimes are sequentially generated, and m-plex PCR is individually performed in each regime when each mixture is driven to pass three temperature zones, thus providing a rapid analysis throughput of m × n. To characterize the performance of the microfluidic device, continuous-flow multiplex PCR in a single segmented flow has been evaluated by investigating the effect of key reaction parameters, including annealing temperatures, flow rates, polymerase concentration and amount of input DNA. With the optimized parameters, the genomic DNAs from Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7 and Staphylococcus aureus could be amplified simultaneously in 19 min, and the limit of detection was low, down to 102 copies μL−1. As proof of principle, the spiral-channel SCF-MPCR was applied to sequentially amplify four different bacterial pathogens from banana, milk, and sausage, displaying a throughput of 4 × 3 with no detectable cross-contamination.On a spiral-channel microfluidic platform, the high-throughput and rapid amplification for multiple foodborne bacterial pathogens was developed via the segmented continuous-flow multiplex PCR. Fig. 1 showed the design principle of spiral-channel segmented continuous-flow multiplex PCR amplification.
Co-reporter:Weipeng Liu, Xiaoming Zhou, Da Xing
Biosensors and Bioelectronics 2014 Volume 58() pp:388-394
Publication Date(Web):15 August 2014
DOI:10.1016/j.bios.2014.02.082
•ECL detection of miRNA by stacking hybridization is developed for the first time.•The miRNA detection is convenient by combining microfluidic system with ECL.•The proposed miRNA assay has demonstrated satisfactory sensitivity and reliability.MicroRNAs play pivotal roles in many fundamental aspects of life. Because microRNAs have the characteristics of small size, similar sequence, and low abundance, it is challenging to identify microRNAs rapidly and specifically with high sensitivity. Herein, we developed an electrochemiluminescent (ECL) chip system for microRNA detection based on base-stacking hybridization and magnetic microparticle enrichment technology. In the designed system, the integration of the microfluidic system with ECL detection made it easy to assemble the multiple assay steps and allowed the construction of a device that is convenient to carry. A limit of detection of 1 fmol was achieved with this assay. The proposed direct optical microRNA detection technique demonstrated an acceptable sensitivity combined with the advantages of reliability and rapidity.Rapid and reliable microRNA detection is achieved by stacking hybridization on electrochemiluminescent chip system, which demonstrated an acceptable sensitivity and accompany the advantages of reliability and rapidity.
Co-reporter:Jiaxing Song, Yanchun Wei, Qun Chen, Da Xing
Journal of Photochemistry and Photobiology B: Biology 2014 134() pp: 27-36
Publication Date(Web):
DOI:10.1016/j.jphotobiol.2014.03.015
Co-reporter:Ting Zhou, Xiaoming Zhou, Da Xing
Biomaterials 2014 35(13) pp: 4185-4194
Publication Date(Web):
DOI:10.1016/j.biomaterials.2014.01.044
Co-reporter:Hongxing Liu, Fangfang Zhan, Fang Liu, Minjun Zhu, Xiaoming Zhou, Da Xing
Biosensors and Bioelectronics 2014 Volume 62() pp:38-46
Publication Date(Web):15 December 2014
DOI:10.1016/j.bios.2014.06.020
•A paper platform for visual detection of viable pathogenic bacteria is constructed.•This method can overcome the false-positive results.•This platform is low-cost, simple and can be used for point-of-care diagnosis.•The results can be turned into a two-dimensional bar code for further analysis.Food-borne pathogens have been recognized as a major cause of human infections worldwide. Their identification needs to be simpler, cheaper and more reliable than the traditional methods. Here, we constructed a low-cost paper platform for viable pathogenic bacteria detection with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper-based platform to perform a visual test using sandwich hybridization assays. When the RNA products migrated along the platform by capillary action, the gold nanoparticles accumulated at the designated area. Under optimized experimental conditions, as little as 0.5 pg/μL genomic RNA from L. monocytogenes could be detected. It could also be used to specifically detect 20 CFU/mL L. monocytogenes from actual samples. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. This method is suitable for point-of-care applications to detect food-borne pathogens, as it can overcome the false-positive results caused by amplifying nonviable L. monocytogenes. Furthermore, the results can be imaged and transformed into a two-dimensional bar code through an Android-based smart phone for further analysis or in-field food safety tracking.
Co-reporter:Fangfang Zhan, Xiaoming Zhou, Da Xing
Analytica Chimica Acta 2013 Volume 761() pp:71-77
Publication Date(Web):25 January 2013
DOI:10.1016/j.aca.2012.11.014
A novel method for detection of rotavirus has been developed by integrating magnetic primer based reverse transcription-polymerase chain reaction (RT-PCR) with electrochemiluminescence (ECL) detection. This is realized by accomplishing RT of rotavirus RNA in traditional way and performing PCR of the resulting cDNA fragment on the surface of magnetic particles (MPs). In order to implement PCR on MPs and achieve rapid ECL detection, forward and reverse primers are bounded to MPs and tris-(2,2′-bipyridyl) ruthenium (TBR), respectively. After RT-PCR amplification, the TBR labels are directly enriched onto the surface of MPs. Then the MPs–TBR complexes can be loaded on the electrode surface and analyzed by magnetic ECL platform without any post-modification or post-incubation process. So some laborious manual operations can be avoided to achieve rapid yet sensitive detection. In this study, rotavirus in fecal specimens was successfully detected within 1.5 h. Experimental results showed that the detection limit of the assay was 0.2 pg μL−1 of rotavirus. The ECL intensity was linearly with the concentration from 0.2 pg μL−1 to 400 pg μL−1. What's more, the specificity of this method was confirmed by detecting other fecal specimens of patients with nonrotavirus-associated gastroenteritis. We anticipate that the proposed magnetic primer based RT-PCR with ECL detection strategy will find numerous applications in food safety field and clinical diagnosis.Graphical abstractIn this work, we have developed and demonstrated a magnetic primer based RT-PCR assay for ECL detection of rotavirus. In the presence of two functional primers (magnetic primer and TBR-primer) and PCR reagents, cDNA from RT was amplified directly onto MPs during PCR cycles of denaturation, annealing and extension. The resulting MPs–TBR complexes were easily loaded on the electrode surface and produced a concentrated ECL signal. The figure shows the schematic illustration of magnetic primer RT-PCR based ECL assay for rotavirus detection.Highlights► A novel method for detection of rotavirus has been developed. ► In the presence of magnetic primer, TBR-primer and PCR reagents, cDNA form RT was amplified directly onto MPs. ► To obtain the best sensing and efficient performance, important parameters associated with the efficiency were investigated carefully. ► The proposed method will find numerous applications in food safety field and clinical diagnosis.
Co-reporter:Bowen Shu;Chunsun Zhang
Microfluidics and Nanofluidics 2013 Volume 15( Issue 2) pp:161-172
Publication Date(Web):2013 August
DOI:10.1007/s10404-013-1138-4
Highly sensitive detection of foodborne pathogens such as Listeria monocytogenes (L. monocytogenes) is crucial to the prevention and recognition of problems related to public health and legal repercussions, due to “zero tolerance” standard adopted for food safety in many countries. Here we first propose a single-phase continuous-flow nested polymerase chain reaction (SP-CF-NPCR) strategy for identification of the low level of L. monocytogenes on an integrated microfluidic platform. The PCR reactor is constructed by a disposable capillary embedded in the grooved heating column, coupled with a fluorescence microscopy for on-line semi-quantitative end-point fluorescence detection. As a proof-of-concept microfluidic system, the nested PCR is performed in a continuous-flow format without the need of any non-aqueous oil or solvent. On this device, the performance of nested PCR amplification has been evaluated by investigating the effect of reaction parameters, including polymerase concentration, flow rates, and template DNA concentration. In addition, the types of samples the presented system can accept, such as the unpurified DNA samples and artificially contaminated clinical stool samples were also evaluated. With the optimized reaction parameters, 0.2 copies/μL of genomic DNA from L. monocytogenes can be detected on the presented device. To our knowledge, this is the highest detection sensitivity in single-phase continuous-flow PCR microsystems reported so far. The high sensitivity of the analysis method, combined with the flexibility of reaction volumes and convenience of continuous operation, renders it to be further developed for potential analytical and diagnostic applications.
Co-reporter:XiaoMing Zhou;YuHui Liao
Science Bulletin 2013 Volume 58( Issue 21) pp:2634-2639
Publication Date(Web):2013 July
DOI:10.1007/s11434-012-5584-2
Catalytic transfer of genetic information from DNA to RNA is very important in life activities. The unconventional RNA transcription level may be related to the source of genetic diseases. At present, conventional methods for detection of RNA transcripts usually involve cumbersome preparative steps, or require sophisticated laboratory equipments. In this study, we presented a rapid, sensitive nano-detection platform for monitoring of RNA transcript levels. T7 RNA polymerase transcription reaction is employed as the example to test the feasibility of this method. In this design, in vitro synthesized RNA products can be hybridized to the FAM labeled single strand DNA (ssDNA) probes which can be adsorbed onto the graphene oxide (GO) surface. Using GO as the fluorescence switch, excellent capacity of the signal-on fluorescence platform for detection of RNA transcripts level is demonstrated. Transcription levels sensing with this nano-platform achieved a sensitivity of 5 pmol/L transcription template. It is anticipated that current developed RNA transcript nano-detection mode has the potential to be an alternative to the conventional RNA transcript detection methods.
Co-reporter:Yi Long, Xiaoming Zhou, Da Xing
Biosensors and Bioelectronics 2013 Volume 46() pp:102-107
Publication Date(Web):15 August 2013
DOI:10.1016/j.bios.2013.02.003
Sequence-specific nucleic acid detection is playing a more and more important role in modern life sciences. Traditional rolling circle amplification (RCA) involves multiple distinct reaction steps and the experiment result is influenced by multiple factors. What's more, a main limitation of traditional RCA is that each target strand hybridizes with only one padlock probe, and this 1:1 hybridization ratio limits the sensitivity. Here we have proposed target sequence recycled rolling circle amplification (TR-RCA) to increase sensitivity by one step. We demonstrated that our method can not only make RCA occur, but also one target DNA can be reused and thus achieving self-recycle. In TR-RCA, the dumbbell probe recognizes the target DNA and hybridizes with it, and then the stem of the dumbbell probe is opened, after that the opened area anneals with the primer and triggers RCA. At the same time, after a target is displaced, it recognizes and hybridizes with another dumbbell probe, triggering the next cycle of RCA. This amplification method is achievable at a constant temperature simply by mixing dumbbell probes, target DNA, primers, and other chemical complexes together in one tube. Our method has significant advantages in ease of operation. And the results indicate that the target DNA can be detected at fM level with high specificity.Highlights► We proposed TR-RCA for isothermal, high sensitive and specific nucleic acids sequence detection. ► We confirm the self-ligation of dumbbell probe and verify the TR-RCA is high specific. ► We prove the target can be reused and meanwhile the rolling circle amplification can occur by TR-RCA. ► We optimize the TR-RCA experiment condition. ► We examine the detection limit of the TR-RCA and found that it can detect as low as 100 fM DNA.
Co-reporter:Zhongyu Fu, Xiaoming Zhou, Da Xing
Sensors and Actuators B: Chemical 2013 Volume 182() pp:633-641
Publication Date(Web):June 2013
DOI:10.1016/j.snb.2013.03.033
Foodborne pathogens have been recognised as the major cause for infection of people worldwide with vital health damage. Thus the development of rapid food pathogen detection method has become an urgent task. It is described here, a simple, rapid gold nanoparticle (GNP) colorimetric assay for Listeria monocytogenes and Salmonella enterica detection. The method is based on polymerase chain reaction (PCR) using thiol-labelled primers. PCR is applied to amplify hly gene of L. monocytogenes and hut gene of S. enterica, and the products with thiol-label at one end were acquired. After mixing PCR products and GNPs, the sulphur-gold linkage resulted in the formation of GNP-PCR products. Due to the GNP-PCR products are more salt-tolerant than primers linked GNPs, detection of the bacteria can be achieved by a facilitated GNP based colorimetric testing using naked eyes. Further, more accurate analysis could be executed by spectrum measurement. The limit of detection of analyzing genome DNA of L. monocytogenes and S. enterica are 0.015 ng mL−1 and 0.013 ng mL−1, respectively. The specificity is evaluated by distinguishing target pathogenic bacteria from other bacterias. The artificial contaminated food samples were also detected for its potential applications in real food samples.
Co-reporter:Xiao Zhu;Dr. Xiaoming Zhou ;Dr. Da Xing
Chemistry - A European Journal 2013 Volume 19( Issue 17) pp:5487-5494
Publication Date(Web):
DOI:10.1002/chem.201204605
Abstract
A label-free approach with multiple enhancement of the signal for microRNA detection has been introduced. The key idea of this strategy is achieved by taking advantage of a novel graphene oxide (GO)/intercalating dye based fluorescent hairpin probe (HP) and an isothermal polymerization reaction. In this paper, we used microRNA-21 (mir-21) as the target to examine the desirable properties of this assay. When the target, as a “trigger”, was hybridized with the HP and caused a conformation change, an efficient isothermal polymerization reaction was activated to achieve the first step of the “signal” amplification. After incubation with the platform of GO/intercalating dye, the formed complex of DNA interacted with the high-affinity dye and then detached from the surface of the GO, a process that was accompanied by distinguishable fluorescence recovery. Further signal enhancement has been accomplished by a mass of intercalating dye inserting into the minor groove of the long duplex replication product. Due to the efficient and multiple amplification steps, this approach exerted a substantial enhancement in sensitivity and could be used for rapid and selective detection of Mir-21 at attomole levels. Proof-of-concept evidence has been provided for the proposed cost-effective strategy; thus, this strategy could expand the application of GO-material-based bioanalysis for nucleic acid studies.
Co-reporter:Xiaoming Zhou and Da Xing
Chemical Society Reviews 2012 vol. 41(Issue 13) pp:4643-4656
Publication Date(Web):01 May 2012
DOI:10.1039/C2CS35045A
Human telomerase is a ribonucleoprotein complex that functions as a telomere terminal transferase by adding multiple TTAGGG hexamer repeats using its integral RNA as the template. There is a very strong association between telomerase activity and malignancy in nearly all types of cancer, suggesting that telomerase could be used not only as a diagnostic and prognostic marker but also as a therapeutic target for managing cancer. The significant progress in biomedical telomerase research has necessitated the development of new bioanalytical methods for the rapid, sensitive, and reliable detection of telomerase activity in a particular cell or clinical tissue and body fluids. In this review, we highlight some of the latest methods for identifying telomerase activity and inhibition and discuss some of the challenges for designing innovative telomerase assays. We also summarise the current technologies and speculate on future directions for telomerase testing.
Co-reporter:Yanchun Wei, Qun Chen, Baoyan Wu, Aiguo Zhou and Da Xing
Nanoscale 2012 vol. 4(Issue 13) pp:3901-3909
Publication Date(Web):02 May 2012
DOI:10.1039/C2NR30804E
Fluorescein has been used for in vivo imaging to identify tumors. However, this technique presents several limitations, mainly due to its limited targeting efficiency, tissue autofluorescence and poor light penetration in tissue. In the present study, an alternative fluorescence imaging technique to localize tumors has been developed by using up-conversion nanoparticles (UCNs) and enhanced targeting approaches. A folic acid molecule is conjoined with UCNs (NaYF4: Yb3+, Er3+) to improve the tumor-specificity; the UCN is also loaded with the microtubule inhibitor CA4P, to further improve the local delivery of particles in the tumor. The proposed imaging technique combines several well-established individual concepts into one novel integrated procedure and significantly improves its tumor-imaging capability: the near-infrared excitation for UCNs minimizes tissue autofluorescence and allows imaging into deeper tissue; the improvement in the signal to noise ratio (SNR) is at least a magnitude better than that of a conventional fluorescence imaging technique, and the modification of UCNs with folic acid significantly improves the tumor targeting efficiency by utilizing its affinity for the folic acid receptor that is often over expressed in tumors. The loading of CA4P further helps UCNs to cross blood vessel walls to reach tumor cells by depolymerizing the microtubules of endothelial cells. The integrated nanoparticle possesses the near-infrared-identical optical properties of UCNs alone, thus achieving a highly effective fluorescence imaging probe. The results demonstrated that the proposed method provides an excellent alternative for tumor localization and a potential traceable vehicle for highly efficient drug delivery.
Co-reporter:Ting Zhou, Baoyan Wu and Da Xing
Journal of Materials Chemistry A 2012 vol. 22(Issue 2) pp:470-477
Publication Date(Web):09 Nov 2011
DOI:10.1039/C1JM13692E
Multimodal imaging based on complementary detection principles has great potential for improving the accuracy of tumor diagnosis. Fe3O4 core/Au shell nanoparticles (Fe3O4@Au NPs) can be used as an effective multimodal contrast agent due to combining the advantageous and highly complementary features of the Fe3O4 core and gold shell. In the present work, we have developed a novel Fe3O4@Au NP based probe for targeting and multimodal imaging of cancer cells. The prepared Fe3O4@Au NPs have been characterized by transmission electron microscopy (TEM), visible spectra and SERS. The potential use of the prepared Fe3O4@Au NPs as a multimodal contrast probe for magnetic resonance imaging (MRI), microwave-induced thermoacoustic imaging and photoacoustic imaging was then evaluated. Importantly, when conjugated with a cancer cell targeted molecular and fluorescent dye, the Fe3O4@Au NPs could be internalized by the corresponding cancer cells selectively and sensitively, and fluorescence imaging was realized at the same time. The bio-modified Fe3O4@Au NPs incorporating multiple functionalities into one single nano-structured system can be effectively used for targeting and multimodal imaging of cancer cells simultaneously.
Co-reporter:Guojia Huang, Zhan Si, Sihua Yang, Cong Li and Da Xing
Journal of Materials Chemistry A 2012 vol. 22(Issue 42) pp:22575-22581
Publication Date(Web):10 Sep 2012
DOI:10.1039/C2JM33990K
A dextran based pH-sensitive near-infrared nanoprobe was prepared and applied as a contrast agent for photoacoustic imaging to identify a tumor in vivo. The nanoprobe has two resonance absorption peaks in the near-infrared region and the cleavage of hydrazone bonds in the low pH value environment can cause their coefficients to change, making a different ratio of photoacoustic signal output. By comparing the photoacoustic signal ratio under the two wavelengths, in vitro photoacoustic images show that the nanoprobe has the capacity to distinguish cancer cells and normal cells. Moreover, an in vivo mouse breast tumor model (EMT-6 cells) can be identified from peripheral normal tissues by dual-wavelength photoacoustic imaging. These results suggest the potential application of this pH-sensitive near-infrared nanoprobe as a contrast agent to noninvasively detect tumors in their early stage with high optical contrast by dual-wavelength photoacoustic imaging based on the tunable absorbance ratio in normal physiological and tumor acidic microenvironments.
Co-reporter:Liyun Xue, Xiaoming Zhou, and Da Xing
Analytical Chemistry 2012 Volume 84(Issue 8) pp:3507
Publication Date(Web):March 28, 2012
DOI:10.1021/ac2026783
Specific and sensitive detection of proteins in biotechnological applications and medical diagnostics is one of the most important goals for the scientific community. In this study, a new protein assay is developed on the basis of hairpin probe and nicking enzyme assisted signal amplification strategy. The metastable state hairpin probe with short loop and long stem is designed to contain a protein aptamer for target recognition. A short Black Hole Quencher (BHQ)-quenching fluorescence DNA probe (BQF probe) carrying the recognition sequence and cleavage site for the nicking enzyme is employed for fluorescence detection. Introduction of target protein into the assay leads to the formation change of hairpin probe from hairpin shape to open form, thus faciliating the hybridization between the hairpin probe and BQF probe. The fluorescence signal is amplified through continuous enzyme cleavage. Thrombin is used as model analyte in the current proof-of-concept experiments. This method can detect thrombin specifically with a detection limit as low as 100 pM. Additionally, the proposed protein detection strategy can achieve separation-free measurement, thus eliminating the washing steps. Moreover, it is potentially universal because hairpin probe can be easily designed for other proteins by changing the corresponding aptamer sequence.
Co-reporter:Debin Zhu, Xiaoming Zhou, Da Xing
Analytica Chimica Acta 2012 Volume 725() pp:39-43
Publication Date(Web):6 May 2012
DOI:10.1016/j.aca.2012.03.006
An ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence (IM-ECL) method for the detection of protein is developed. The target protein is captured by biotin-labeled aptamer (biotin probe) and [Ru(bpy)3]2+ (TBR)-Au bio bar code-labeled aptamer (ECL nanoprobe), to form a double aptamer–protein sandwich complex. The complex is then immobilized on the streptavidin microbeads through biotin–streptavidin linkage and detected by ECL assay. The ECL signal of the target protein is amplified by the TBR-bio bar code DNAs. As an example, platelet-derived growth factor B-chain homodimer (PDGF-BB) was detected by the method. Experimental results show that the detection limit of the assay is 1 pM of PDGF-BB. A calibration curve with a linearity range from 1 pM to 10 nM is established, thus, make quantitative analysis possible. The method has been used to detect PDGF-BB in fetal calf serum with minimum background interference. Due to the wide availability of aptamer for numerous proteins, this aptamer-based bio bar code IM-ECL method holds great promise in protein detection.Graphical abstractHighlights► The ECL nanoprobe was synthesized by bifunctioning AuNPs with the capture aptamer and the TBR-bio bar code DNAs with a ratio of 1:100. ► The ECL nanoprobe and the biotin-labeled aptamer were incubated with the target protein to form a double aptamer–protein sandwich complex. ► The sandwich complex was captured by the streptavidin microbeads through biotin–streptavidin linkage and detected by ECL assay.
Co-reporter:Xiaoming Zhou, Qiang Su, Da Xing
Analytica Chimica Acta 2012 Volume 713() pp:45-49
Publication Date(Web):3 February 2012
DOI:10.1016/j.aca.2011.11.003
In this study, we firstly demonstrated that Bst DNA polymerase shows specific recognition and function on the T–Hg2+–T biomimetic structure. Based on this, a novel available electrochemiluminescence (ECL) sensor for Hg2+ has been developed. In this strategy, magnet beads tagged primer was designed to complementary to the region of the circular padlock probe but with two T–T mismatches at the 3′ end. The mismatched primers cannot be extended by Bst DNA polymerase in the absence of Hg2+. Stable T–Hg2+–T can be formed in the presence of Hg2+, thus induces the elongation and amplification reaction by DNA polymerase with a rolling circular amplification (RCA) mechanism. Subsequently, the resulted RCA products are hybridized with the tris (bipyridine) ruthenium (TBR)-tagged probes and detected by ECL platform. Current method shows a sub-nanomolar sensitivity and excellent selectivity over a spectrum of interference metal ions.Graphical abstractHighlights► Bst DNA polymerase shows specific function on the T–Hg2+–T biomimetic structure. ► T–Hg2+–T can be formed in the presence of Hg2+, thus induces the RCA reaction. ► Sub-nanomolar sensitivity and excellent selectivity were achieved for Hg2+ detection.
Co-reporter:Xiaohui Zheng, Feifan Zhou, Baoyan Wu, Wei R. Chen, and Da Xing
Molecular Pharmaceutics 2012 Volume 9(Issue 3) pp:514-522
Publication Date(Web):February 14, 2012
DOI:10.1021/mp200526m
Indocyanine green (ICG) is a conventional dye that can be used in clinical near-infrared (NIR) imaging, and it is also an effective light absorber for laser-mediated photothermal therapy. However, applications of ICG were limited due to its fast degradation in aqueous media and quick clearance from the body. Herein, an ICG-containing nanostructure, ICG-PL-PEG, was developed for photothermal therapy, which was self-assembled by ICG and phospholipid-polyethylene glycol (PL-PEG). Our in vitro and in vivo experiments demonstrated that ICG-PL-PEG suspension was more efficient in producing a NIR-dependent temperature increase than ICG alone, due to the increase of ICG monomers from the addition of PL-PEG to match the central wavelength of the 808 nm laser. When conjugated with integrin αvβ3 monoclonal antibody (mAb), ICG-PL-PEG could be selectively internalized and retained in target tumor cells. Irradiation of an 808 nm laser after intravenous administration of ICG-PL-PEG-mAb resulted in tumor suppression in mice, while ICG alone had only limited effect. This is the first time an ICG-containing nanostructure has been used through systemic administration to achieve an efficient in vivo photothermal effect for cancer treatment. Therefore, ICG-PL-PEG could be used as a fluorescent marker as well as a light-absorber for imaging-guided photothermal therapy. All the components of ICG-PL-PEG have been approved for human use. Therefore, this unique ICG-containing nanostructure has great potential in clinical applications.Keywords: ICG-containing nanostructure; laser-mediated photothermal therapy; NIR-dependent temperature increase; photothermal tumor suppression; self-assembly;
Co-reporter:Xiaoming Zhou and Da Xing
Analyst 2012 vol. 137(Issue 18) pp:4188-4192
Publication Date(Web):26 Jun 2012
DOI:10.1039/C2AN35367A
Here, we present a straightforward method for isothermal amplified detection of nucleic acids. In this proof-of-concept study, a specific DNA sequence is amplified through hairpin probe-based isothermal strand-displacement polymerization reaction and then detected via a sensitive and commercially available ECL detection platform. Results show that the DNA sequence derived from the Listeria monocytogenes hly gene can be detected down to 10 pM in solution, together with correlation of the detected signal with the initial concentration of target DNA. Moreover, the designed stem–loop structured hairpin probe shows single-base variation differentiating ability. Considering the superior sensitivity and specificity, as well as the simple-to-implement features, the developed assay demonstrates a great potential of becoming a first-line tool for quantitative analysis of nucleic acids for biomedical research.
Co-reporter:Xiaoming Zhou, Ruixue Duan and Da Xing
Analyst 2012 vol. 137(Issue 8) pp:1963-1969
Publication Date(Web):15 Feb 2012
DOI:10.1039/C2AN00020B
Amplified optical detection of biomolecules using nanoparticle as the carrier has attracted considerable interest in the scientific community. In this study, a promising aptasensor was developed for highly sensitive detection of protein and small molecules based on the construction of aptamer-modified electrochemiluminescence (ECL) nanoprobe. Specifically, thrombin and ATP serve as the examples for detection. By taking advantage of sandwich binding of two affinity aptamers for high specificity, tris-(2,2′-bipyridyl)ruthenium (TBR)-cysteamine loaded in gold nanoparticle (GNP) as barcodes for signal amplification, and micromagnetic particles (MMPs) based ECL technology for rapid detection, a novel assay for biomolecules quantification was developed. The sandwich complex containing targets could be selectively captured by MMPs and then quantified by ECL intensity. We have demonstrated that the detection limits of human thrombin and ATP are 1 pM and 10 pM, respectively, with high specificity. The proposed technology is expected to become a powerful tool for biomolecule analysis.
Co-reporter:Feifan Zhou, Shengnan Wu, Sheng Song, Wei R. Chen, Daniel E. Resasco, Da Xing
Biomaterials 2012 33(11) pp: 3235-3242
Publication Date(Web):
DOI:10.1016/j.biomaterials.2011.12.029
Co-reporter:Zhen Zhao, Xiaoming Zhou, Da Xing
Biosensors and Bioelectronics 2012 Volume 31(Issue 1) pp:299-304
Publication Date(Web):15 January 2012
DOI:10.1016/j.bios.2011.10.035
Herein, we describe a novel electrochemiluminescence (ECL) biosensor for protein kinase activities and inhibition monitoring based on the magnetic beads (MB) technology and signal enhancement of gold nanoparticles (GNP). In this design, ECL nanoprobes were prepared by conjugating GNP with phosphorylated DNA capture probes and tris-(2,2′-bipyridyl) ruthenium (TBR)–cysteamine. Zirconium cations, a specific bridging agent, mediate the linkage between biotin modified phosphorylated peptides and ECL nanoprobes. The complexes were then captured and enriched on the electrode surface by streptavidin-coated MB for ECL reaction. To confirm the feasibility of this biosensor, we employed protein kinase A (PKA) as the model kinase to validate the assay and a satisfactory detection limit of 0.005 U/mL was achieved. The combination of ECL and GNP lays a solid foundation for highly sensitive assay, meanwhile, the coupling of MB surfaces used for separation and capture with unmodified ECL electrode detection results in a greatly simplified and reusable protocol. Thus, our biosensor offers great promise for a highly sensitive and simple assay for protein kinase activity. Furthermore, the inhibition of PKA activity was monitored on the basis of the ECL signals change in response to the concentration of PKA inhibitor.Highlights► ECL nanoprobe based on cysteamine is used for the first time to enzyme activity. ► The sensor is simple and rapid based on Zr4+ linkage without chemical activation. ► Coupling ECL with magnetic beads results in simplicity and low background signal. ► Based on the ingenious design, we achieved high sensitivity of 0.005 U/mL.
Co-reporter:Xiao Zhu, Xiaoming Zhou, Da Xing
Biosensors and Bioelectronics 2012 Volume 31(Issue 1) pp:463-468
Publication Date(Web):15 January 2012
DOI:10.1016/j.bios.2011.11.016
Here we have developed a novel nano-magnetic primer based electrochemiluminescence-polymerase chain reaction (NMPE-PCR) strategy for detection of genome. The key idea of this method is integrating the two in situ processes: PCR on the surface of magnetic nanoparticles (MNPs) and magnetic beads based ECL readout platform, to avoid some laborious manual operations and achieve rapid yet sensitive detection. At first, the approach employs a pair of functional primers for amplification: one is tris-(2,2′-bipyridyl) ruthenium (TBR) labeled primer; the other one is nano-magnetic primer which is prepared by attaching the primer to the surfaces of MNPs. With the presence of DNA analyte and PCR mixture, the TBR labeled products are directly loaded and enriched on the surface of MNPs during PCR cycling. Then the MNPs–TBR complexes can be analyzed by a magnetic ECL platform without any post-modification or post-incubation. Finally, we used Listeria monocytogenes as the target to examine these desirable properties of this assay, reaching a detection limit of 500 fg/μL for genome in 1 h. The proposed study has provided the evidence as a proof-of-concept, thus having potential for development of automatic mode for detection of specific gene.Highlights► We are the first to combine PCR and ECL directly on magnetic nanoparticles. ► The assay achieves rapid, specific, yet sensitive detection of genome. ► The method is simple, and most of the reagents and equipments are commercial. ► Because of avoiding some manual operation, the probable error could be reduced. ► The method has potential for development of automatic equipment for gene analysis.
Co-reporter:Xiaoming Zhou, Yonghong Tang, and Da Xing
Analytical Chemistry 2011 Volume 83(Issue 8) pp:2906
Publication Date(Web):March 17, 2011
DOI:10.1021/ac1028648
A new protein assay based on fluorescence cross-correlation spectroscopy (FCCS) and aptamer probe is developed. In this assay, two spectrally distinct fluorophores labeled aptamer probes are used to recognize and detect thrombin through a sandwich reaction. The sandwich complexes are diffused through a confocal detection volume. The cross-correlation signals can be observed only at the presence of the aptamer probes-protein sandwich complexes. Thrombin is selected as a target to validate the assay. The whole detection process can be completed within an hour with low-nanomolar sensitivity and high specificity. The novel aptamer-based FCCS detection offers a simple, rapid and sensitive method for protein analysis in a homogeneous format.
Co-reporter:Xiaohui Zheng, Da Xing, Feifan Zhou, Baoyan Wu, and Wei R. Chen
Molecular Pharmaceutics 2011 Volume 8(Issue 2) pp:447-456
Publication Date(Web):January 3, 2011
DOI:10.1021/mp100301t
Indocyanine green (ICG) is a near-infrared (NIR) imaging agent and is also an ideal light absorber for laser-mediated photothermal therapy. This NIR dye could serve as a basis of a dual-functional probe with integrated optical imaging and photothermal therapy capabilities. However, applications of ICG remain limited by its concentration-dependent aggregation, poor aqueous stability, nonspecific binding to proteins and lack of target specificity. To overcome these limitations, a novel ICG-containing nanostructure is designed utilizing the noncovalent self-assembly chemistry between phospholipid-polyethylene glycol (PL-PEG) and ICG. The interactions between both amphiphilic ICG and PL-PEG were studied using absorption and fluorescence spectroscopy. The properties of ICG-PL-PEG nanoprobe, such as absorption and fluorescence spectra, stability, morphology and size distribution, were also investigated. Two representative targeting molecules, namely, a small molecule, folic acid (FA), and a large protein, integrin αvβ3 monoclonal antibody (mAb), were conjugated to the surface of ICG-PL-PEG nanoprobe, displaying the diversity of ligand conjugation. The target specificity was confirmed using three cell lines with different levels of available folate receptors (FRs) or integrin αvβ3 expression via laser scanning confocal microscope and flow cytometry. This targeting ICG-PL-PEG nanoprobe could be internalized into targeted cells via ligand−receptor mediated endocytosis pathway. Our in vitro experiments showed that internalized ICG-PL-PEG could be used for cell imaging and selective photothermal cell destruction. These results represent the first demonstration of the dual functionality of ICG-containing nanostructure for targeted optical imaging and photothermal therapy of cancerous cells. This novel ICG-PL-PEG nanostructure, when conjugated with other therapeutic and imaging agents, could become a multifunctional probe for cancer diagnosis and treatment.Keywords (keywords): amphiphilic; indocyanine green; phospholipid-polyethylene glycol; self-assembly;
Co-reporter:Yuyuan Li, Chunsun Zhang, Da Xing
Analytical Biochemistry 2011 Volume 415(Issue 2) pp:87-96
Publication Date(Web):15 August 2011
DOI:10.1016/j.ab.2011.04.026
The development of microfluidic tools for nucleic acid analysis has become a burgeoning area of research during the postgenome era. Here we have developed a microfluidic device that integrates reverse transcription (RT) and polymerase chain reaction (PCR) with online fluorescence detection to realize a rapid detection system for performing both genetic amplification and product analysis. The microfluidic device mainly comprises a grooved copper heating block for RT and a heated cylinder for amplification. To expedite the analysis process, we combined the continuous-flow PCR with an online fluorescence detection system that allows analysis of amplification products within 1 min. Rotaviruses are worldwide enteric pathogens in humans and animals responsible for a significant burden of disease through person-to-person transmission and exposure to contaminated foods and water. In this study, rotavirus from stool specimens was successfully amplified and detected using the RT-PCR microfluidic system within 1 h, and the limit of detection of the RNA concentration was estimated to be 3.6 × 104 copies μl−1. Compared with a large-scale apparatus, the integrated microfluidic system presented here can perform rapid nucleic acid amplification and analysis, possibly making it a crucial platform for future diagnosis application.
Co-reporter:Yuyuan Li;Chunsun Zhang
Microfluidics and Nanofluidics 2011 Volume 10( Issue 2) pp:367-380
Publication Date(Web):2011 February
DOI:10.1007/s10404-010-0675-3
Being beneficial from dramatic process in bio-chemical and micro-electro-mechanical technologies, this study presents an integrated microfluidic system capable of performing a continuous-flow reverse transcription-polymerase chain reaction (RT-PCR) combined with fluorescence microscopy for rapid diagnosis of RNA-based viruses. The device consists of two heated cylinders for heating the different reaction zones of the reverse transcription and the amplification. In the amplification cylinder, bath refrigerating is used for thermal protection of the annealing region, which is heated possibly due to the thermal effects of radiation, conduction, and/or convection from denaturation and extension regions, thus resulting in a space-saving design of the whole device. Detection of the amplified products is performed on-line by a fluorescence microscopy with SybrGreen I, a widely used intercalating dye. In this article, the proposed miniature RT-PCR system is used to amplify and detect two RNA-based viruses (Noroviruses (NVs) and Rotaviruses (RVs)), which are now recognized as the most common etiological agents of acute viral gastroenteritis causing numerous outbreaks worldwide. The experimental data have demonstrated the ability of the presented system to perform a two-step or one-step RT-PCR process. On this device, the NVs and RVs RNA samples were successfully reverse transcribed and amplified within 1 h, and the limit of detection of the RNA concentration was 6.4 × 104 copies μl−1 using one-step RT-PCR process. Consequently, the developed microfluidic system can provide a promising platform for fast diagnosis of RNA-based viruses.
Co-reporter:Chunsun Zhang;Haiying Wang
Biomedical Microdevices 2011 Volume 13( Issue 5) pp:885-897
Publication Date(Web):2011 October
DOI:10.1007/s10544-011-9558-y
In the field of continuous-flow PCR, the amplification throughput in a single reaction solution is low and the single-plex PCR is often used. In this work, we reported a flow-based multiplex PCR microfluidic system capable of performing high-throughput and fast DNA amplification for detection of foodborne bacterial pathogens. As a demonstration, the mixture of DNA targets associated with three different foodborne pathogens was included in a single PCR solution. Then, the solution flowed through microchannels incorporated onto three temperature zones in an oscillatory manner. The effect factors of this oscillatory-flow multiplex PCR thermocycling have been demonstrated, including effects of polymerase concentration, cycling times, number of cycles, and DNA template concentration. The experimental results have shown that the oscillatory-flow multiplex PCR, with a volume of only 5 μl, could be completed in about 13 min after 35 cycles (25 cycles) at 100 μl/min (70 μl/min), which is about one-sixth of the time required on the conventional machine (70 min). By using the presently designed DNA sample model, the minimum target concentration that could be detected at 30 μl/min was 9.8 × 10−2 ng/μl (278-bp, S. enterica), 11.2 × 10−2 ng/μl (168-bp, E. coli O157: H7), and 2.88 × 10−2 ng/μl (106-bp, L. monocytogenes), which corresponds to approximately 3.72 × 104 copies/μl, 3.58 × 104 copies/μl, and 1.79 × 104 copies/μl, respectively. This level of speed and sensitivity is comparable to that achievable in most other continuous-flow PCR systems. In addition, the four individual channels were used to achieve multi-target PCR analysis of three different DNA samples from different food sources in parallel, thereby achieving another level of multiplexing.
Co-reporter:Heng Zhang;Shengnan Wu
Apoptosis 2011 Volume 16( Issue 8) pp:808-821
Publication Date(Web):2011 August
DOI:10.1007/s10495-011-0608-y
Accumulation of amyloid-β-peptide (Aβ) in the brain is considered as a pathological hallmark of Alzheimer’s disease (AD). Previous studies show that p73 is vital for mediating the pathogenic process of AD. Yes-associated protein (YAP) has been shown to positively regulate p73 in promoting apoptosis induced by anti-cancer agents. However, the functional role of YAP and potential relationship between YAP and p73 in AD are unknown. In the present study, we found that YAP accelerated apoptosis in response to Aβ25–35 and the nuclear translocation of YAP was involved in cellular signals that regulated the apoptosis. Aβ25–35 induced YAP translocation from cytoplasm to nucleus accompanied with the increased phosphorylation on Y357, resulting in the enhancement of interaction between YAP and p73. Moreover, inhibition of YAP expression by small hairpin RNA (shRNA) suppressed apoptosis induced by Aβ25–35. More importantly, p73-mediated induction of Bax expression and activation were in a YAP-dependent manner. Overexpression of YAP accelerated Bax translocation, upregulated Bax expression and promoted caspase-3 activation. Taken together, our findings first demonstrated that YAP accelerated Aβ-induced apoptosis through nucleus translocation, leading to the induction of Bax expression and activation. Our results provided a potential therapeutic strategy for the treatment of AD through inhibiting YAP/p73/Bax pathway.
Co-reporter:Chunsun Zhang and Da Xing
Chemical Reviews 2010 Volume 110(Issue 8) pp:4910
Publication Date(Web):April 15, 2010
DOI:10.1021/cr900081z
Co-reporter:Feifan Zhou, Da Xing, Baoyan Wu, Shengnan Wu, Zhongmin Ou and Wei R. Chen
Nano Letters 2010 Volume 10(Issue 5) pp:1677-1681
Publication Date(Web):April 6, 2010
DOI:10.1021/nl100004m
Translocation and localization of single-walled carbon nanotubes (SWNTs) in normal and cancerous cells have significant biomedical implications. In this study, SWNTs functionalized with different biomolecules in cells were observed with confocal laser scanning microscopy. Functionalized with PL-PEG, SWNTs were found to localize exclusively in mitochondria of both tumor and normal cells due to mitochondrial transmembrane potential, but they were found mainly in lysosomes of macrophages due to phagocytosis. However, when conjugated with different molecules, the subcellular localization of the surface-modified SWNT-PL-PEG depended on how SWNTs enter the cells: inside mitochondria if crossing cell membrane or inside lysosomes if being endocytosized. We also show that mitochondrial SWNT-PL-PEG, when irradiated with a near-infrared light, can induce cell apoptosis due to mitochondrial damages. These findings provide a better mechanistic understanding of cellular localization of SWNTs, which could lead to advanced biomedical applications such as the design of molecular transporters and development of SWNT-assisted cancer therapies.
Co-reporter:Liyun Xue, Xiaoming Zhou and Da Xing
Chemical Communications 2010 vol. 46(Issue 39) pp:7373-7375
Publication Date(Web):06 Sep 2010
DOI:10.1039/C0CC02038A
An isothermal and sensitive fluorescence assay for protein detection using aptamer–protein–aptamer conjugates based on nicking enzyme amplification has been developed, which was combined with magnetic microparticles separation, making this assay suitable for protein detection in biological samples.
Co-reporter:Ruixue Duan, Xiaoming Zhou and Da Xing
Analytical Chemistry 2010 Volume 82(Issue 8) pp:3099
Publication Date(Web):March 18, 2010
DOI:10.1021/ac100018z
The recently developed DNA−gold nanoparticle (DNA−GNP) biobarcode assay provides polymerase chain reaction (PCR)-like sensitivity for nucleic acid and protein targets without a need for enzymatic amplification. However, application of the conventional assay is challenged by its complex, expensive, time-consuming, and labor-intense procedure. Herein, we present a new electrochemiluminescence (ECL) biobarcode method based on cysteamine−GNP conjugates. In this strategy, an ECL nanoprobe is fabricated that relies on GNP that is modified with tris-(2,2′-bipyridyl) ruthenium (TBR) labeled cysteamine to boost ECL signals and single strand DNA for target recognition. Specifically, a sandwich complex that consists of a biotin labeled capture probe, target DNA, and cysteamine−GNP conjugate is captured by magnetic microparticles (MMPs) and subsequently identified by the ECL signals from loaded TBR. With the use of the developed probe, a limit of detection as low as 100 fM can be achieved and the assay exhibits excellent selectivity for single-mismatched DNA detection even in human serum. The proposed ECL based method should have wide applications in diagnosis of genetic diseases due to its high sensitivity, simplicity, and low cost.
Co-reporter:Jie Wei, Xiaoming Zhou, Da Xing, Baoyan Wu
Food Chemistry 2010 Volume 123(Issue 3) pp:852-858
Publication Date(Web):1 December 2010
DOI:10.1016/j.foodchem.2010.05.022
Vibrio parahaemolyticus has been considered as one of the most important food-borne bacterial pathogens. Because of the safety concerns, detection and characterization of V. parahaemolyticus have attracted much attention. In this study, electrochemiluminescence polymerase chain reaction (ECL-PCR) method combined with universal probes hybridization technique was applied to rapid detection of V. parahaemolyticus, infected and uninfected sea foods for the first time. Whether the sea food samples were infected was discriminated by detecting the gyrase B (gyrB) gene. We detect V. parahaemolyticus both in artificially contaminated sea foods and natural samples. The experiment results show that the infected and uninfected sea food samples can be clearly identified and the detection limit for V. parahaemolyticus is 1.6 pg purified genomic DNA in the presence of 1 μg non-specific background DNA. The technique may provide a new means in V. parahaemolyticus detection due to its simplicity and high efficiency.
Co-reporter:Qiang Su, Da Xing, Xiaoming Zhou
Biosensors and Bioelectronics 2010 Volume 25(Issue 7) pp:1615-1621
Publication Date(Web):15 March 2010
DOI:10.1016/j.bios.2009.11.025
The identification of point mutations is particularly essential in the fields of medical diagnosis and prognosis of many pathogenic and genetic diseases. In this study, an rolling circle amplification (RCA) based electrochemiluminescence (ECL) assay for highly sensitive point mutation detection was developed. In the assay, an allele-discriminating padlock probe was designed for targeting the sequence in the p53 oncogene locus. A circular template generated by enzymatic ligation upon the recognition of a point mutation (CGT to CAT) on the oncogene could be amplified isothermally by Phi29 DNA polymerase. The elongated products, containing hundreds of copies of the circular DNA template sequence, were hybridized with Ru(bpy)32+ (TBR)-tagged probes and then captured onto streptavidin-coated paramagnetic beads. The resulting products were analyzed by magnetic bead based ECL platform. As low as 2 amol of mutated strands was detected by this assay, which could be attributed to the high amplification efficiency of Phi29 DNA polymerase and current magnetic bead based ECL detection platform. In addition, the positive mutation detection was achieved with a wild-type to mutant ratio of 10 000:1, due to the high fidelity of DNA ligase in differentiating mismatched bases at the ligation site. It is demonstrated that this proposed method provides a highly sensitive and specific approach for point mutation detection.
Co-reporter:Qiang Li, Xiaoming Zhou, Da Xing
Biosensors and Bioelectronics 2010 Volume 26(Issue 2) pp:859-862
Publication Date(Web):15 October 2010
DOI:10.1016/j.bios.2010.07.098
A novel and highly sensitive electrochemiluminescence (ECL) assay based on magnetic beads separation/collection process and thymine–Hg2+–thymine (T–Hg2+–T) coordination chemistry has been designed to detect Hg2+ ions in aqueous solution. In this protocol, two amine-terminated complementary DNA probes with five thymine–thymine (T–T) mismatches were introduced. One was coupled with carboxyl-modified magnetic beads and the other was labeled with tris (2, 2-bipyridine) ruthenium (II) (TBR). The couple of DNA probes, in the presence of Hg2+, can form double-stranded structure via the Hg2+-mediated coordination of T–Hg2+–T base pair. Therefore, they can be collected on the surface of electrode using a magnetic field. On the electrode, TBR labels and tripropylamine (TPA) could react to emit photons, which were detected by a custom-built ECL system. The detection limit for Hg2+ in this assay is 5 nM, which is below the upper limit of Hg2+ for drinkable water mandated by United States Environmental Protection Agency (EPA), 10 nM. To the best of our knowledge, this is the first example of ECL assay applying to detect Hg2+ with highly sensitivity and selectivity.
Co-reporter:Debin Zhu, Xiaoming Zhou, Da Xing
Biosensors and Bioelectronics 2010 Volume 26(Issue 1) pp:285-288
Publication Date(Web):15 September 2010
DOI:10.1016/j.bios.2010.06.028
A new kind of aptamer-based immunomagnetic electrochemiluminescence (IM-ECL) assay for quantitative detection of protein is developed. The assay consists of a double aptamer sandwich format in which a biotin-labeled aptamer is used for rapid and specific capture of target protein, and a [Ru(bpy)3]2+ (TBR)-labeled aptamer is used for ECL detection. As an example, platelet-derived growth factor B-chain homodimer (PDGF-BB) was detected by the method. Experimental results show that the detection limit of the assay is 80 pmol/L of PDGF-BB. A calibration curve with a linearity range from 0.1 to 1000 nmol/L is established, thus, make quantitative analysis possible. The method has been used to detect PDGF-BB in fetal calf serum with minimum background interference. Due to the wide availability of aptamer for numerous proteins, this new method holds great promise in protein detection.
Co-reporter:Debin Zhu, Jinfeng Liu, Yabing Tang, Da Xing
Sensors and Actuators B: Chemical 2010 Volume 149(Issue 1) pp:221-225
Publication Date(Web):6 August 2010
DOI:10.1016/j.snb.2010.05.047
A reusable magnetic bead-based electrochemiluminescence (ECL) DNA biosensor for the detection of genetically modified organism (GMO) is proposed. The sensor method consists of immobilization of biotin-probe using streptavidin-coated magnetic beads, hybridization of target DNA with biotin-probe and ruthenium(II) tris-bipyridal (TBR)-probe, detection of target DNA by direct measuring the ECL emission of TBR. The sensor was applied to detect GMO in engineered tobacco samples. Results indicate that the sensitivity of this sensor method was 5 nmol/L of CaMV35S DNA. A stable calibration curve with a wide dynamic range was established. The calibration curve was linear from 5 nmol/L to 5 μmol/L, thus, made quantitative analysis possible. GM tobacco samples and non-GM tobacco samples were clearly discriminated by the sensor method. Results of the study suggest that the reusable DNA biosensor is a feasible tool for daily GMO detection due to its rapidness, simplicity, safety, sensitivity, reliability and low cost.
Co-reporter:Feifan Zhou;Shengnan Wu;Wei R. Chen;Da Xing
Molecular Imaging and Biology 2010 Volume 12( Issue 1) pp:
Publication Date(Web):2010/02/01
DOI:10.1007/s11307-009-0235-y
The aim of the study is to dynamically and non-invasively monitor the apoptosis events in vivo during photodynamic therapy (PDT) and chemotherapy.A FRET probe, SCAT3, was utilized to determine activation of caspase-3 during tumor cell apoptosis in mice, induced by PDT, and cisplatin treatments. Using this method, dynamics of caspase-3 activation was observed both in vitro and in vivo.Analysis of the fluorescent missions from tumor cells indicated that the caspase-3 activation started immediately after PDT treatment. In contrast, the caspase-3 activation started about 13 and 36 h after cisplatin treatment in vitro and in vivo, respectively.FRET could be used effectively to monitor activation of caspase-3 in living organism. This method could be used to provide rapid assessment of apoptosis induced by anti-tumor therapies for improvement of treatment efficacy.
Co-reporter:Chunsun Zhang
Microfluidics and Nanofluidics 2010 Volume 9( Issue 1) pp:17-30
Publication Date(Web):2010 July
DOI:10.1007/s10404-009-0508-4
Evaporation is of great importance when dealing with microfluidic devices with open air/liquid interfaces due to the large surface-to-volume ratio. For devices utilizing a thermal reaction (TR) reservoir to perform a series of biological and chemical reactions, excessive heat-induced microfluidic evaporation can quickly lead to reaction reservoir dry out and failure of the overall device. In this study, we present a simple, novel method to decrease heat-induced fluid evaporation within microfluidic systems, which is termed as heat-mediated diffusion-limited (HMDL) method. This method does not need complicated thermal isolation to reduce the interfacial temperature, or external pure water to be added continuously to the TR chamber to compensate for evaporation loss. The principle of the HMDL method is to make use of the evaporated reaction content to increase the vapor concentration in the diffusion channel. The experimental results have shown that the relative evaporation loss (Vloss/Vini) based on the HMDL method is not only dependent on the HMDL and TR region’s temperatures (THMDL and TTR), but also on the HMDL and TR’s channel geometries. Using the U-shaped uniform channel with a diameter of 200 μm, the Vloss/Vini within 60 min is low to 5% (THMDL = 105°C, TTR = 95°C). The HMDL method can be used to design open microfluidic systems for nucleic acid amplification and analysis such as isothermal amplification and PCR thermocycling amplification, and a PCR process has been demonstrated by amplifying a 135-bp fragment from Listeria monocytogenes genomic DNA.
Co-reporter:Chunsun Zhang
Biomedical Microdevices 2010 Volume 12( Issue 1) pp:1-12
Publication Date(Web):2010 February
DOI:10.1007/s10544-009-9352-2
This study develops a new microfluidic DNA amplification strategy for executing parallel DNA amplification in the microfluidic gradient polymerase chain reaction (MG-PCR) device. The developed temperature gradient microfluidic system is generated by using an innovative fin design. The device mainly consists of modular thermally conductive copper flake which is attached onto a finned aluminum heat sink with a small fan. In our microfluidic temperature gradient prototype, a non-linear temperature gradient is produced along the gradient direction. On the copper flake of length 45 mm, width 40 mm and thickness 4 mm, the temperature gradient easily spans the range from 97 to 52°C. By making full use of the hot (90–97°C) and cold (60–70°C) regions on the temperature gradient device, the parallel, two-temperature MG-PCR amplification is feasible. As a demonstration, the MG-PCR from three parallel reactions of 112-bp Escherichia coli DNA fragment is performed in a continuous-flow format, in which the flow of the PCR reagent in the closed loop is induced by the buoyancy-driven nature convection. Although the prototype is not optimized, the MG-PCR amplification can be completed in less than 45 min. However, the MG-PCR thermocycler presented herein can be further scaled-down, and thus the amplification times and reagent consumption can be further reduced. In addition, the currently developed temperature gradient technology can be applied onto other continuous-flow MG-PCR systems or used for other analytical purposes such as parallel and combination measurements, and fluorescent melting curve analysis.
Co-reporter:Xiaoming Zhou, Da Xing, Debin Zhu and Li Jia
Analytical Chemistry 2009 Volume 81(Issue 1) pp:255
Publication Date(Web):December 4, 2008
DOI:10.1021/ac801914b
The broad-spectrum expression of telomerase in most malignancies makes it a promising target for a cancer diagnostic and prognostic tool. Conventional polymerase chain reaction (PCR)-based telomerase activity assay is highly sensitive but susceptible to amplification-related errors. Here, we present a novel approach to telomerase activity detection. The detection of telomerase activity is accomplished by the hybridization of electrochemiluminescence (ECL) nanoprobes to telomerase reaction products, subsequent capture by magnetic beads, and in situ measurement of the light signal from ECL nanoprobes. The ECL intensity directly reflects the quantity of telomerase reaction products, thus telomerase activity. The high sensitivity afforded by the current magnetic bead and nanoparticle based ECL detection platform allows measuring of telomerase activity from as little as 500 cultured cancer cells in crude cell extracts without the PCR amplification of telomerase reaction products. In addition, a comparative study of the ECL nanoprobe and linear telomere antisense ECL probe was executed. By the employment of the ECL nanoprobe, a gain of about 100-fold elevation of sensitivity was determined. The method described here is ideal for telomerase activity analysis due to its reliability and high sensitivity.
Co-reporter:Yuyuan Li, Da Xing, Chunsun Zhang
Analytical Biochemistry 2009 Volume 385(Issue 1) pp:42-49
Publication Date(Web):1 February 2009
DOI:10.1016/j.ab.2008.10.028
The ability to perform DNA amplification on a microfluidic device is very appealing. In this study, a compact continuous-flow polymerase chain reaction (PCR) microfluidics was developed for rapid analysis of genetically modified organisms (GMOs) in genetically modified soybeans. The device consists of three pieces of copper and a transparent polytetrafluoroethylene capillary tube embedded in the spiral channel fabricated on the copper. On this device, the P35S and Tnos sequences were successfully amplified within 9 min, and the limit of detection of the DNA sample was estimated to be 0.005 ng μl−1. Furthermore, a duplex continuous-flow PCR was also reported for the detection of the P35S and Tnos sequences in GMOs simultaneously. This method was coupled with the intercalating dye SYBR Green I and the melting curve analysis of the amplified products. Using this method, temperature differences were identified by the specific melting temperature values of two sequences, and the limit of detection of the DNA sample was assessed to be 0.01 ng μl−1. Therefore, our results demonstrated that the continuous-flow PCR assay could discriminate the GMOs in a cost-saving and less time-consuming way.
Co-reporter:Chunsun Zhang, Da Xing
Analytical Biochemistry 2009 Volume 387(Issue 1) pp:102-112
Publication Date(Web):1 April 2009
DOI:10.1016/j.ab.2009.01.017
We present a thermal gradient convective polymerase chain reaction (PCR) for parallel DNA amplification with different annealing temperatures. The thermal gradient for microfluidic gradient PCR is produced by an innovative fin design whose formation principle is given. Without the need for a pump, the buoyancy forces continuously circulate reagents in a closed loop through different thermal zones, which brings self-actuated convective-flow PCR. In our prototype, we measured a temperature difference of about 45 °C along the gradient direction on the copper flake (45 × 40 × 4 mm). When the temperature of the hot zone is 90–97 °C and the temperature of the cold zone is 60–70 °C, the convection triggered two-temperature amplification of 112-bp fragment of Escherichia coli DNA. The time for amplification is less than 45 min. Interestingly, parallel DNA amplification with different annealing temperatures ranging from 60 to 70 °C was performed by this method. The PCR thermocycler demonstrated herein can be further scaled down and the loop length can be further reduced, and therefore the PCR times can be further reduced. These devices are suited as a platform for a new generation of low-power, portable DNA analysis systems.
Co-reporter:Debin Zhu, Da Xing, Yabing Tang, Lan Zhang
Biosensors and Bioelectronics 2009 Volume 24(Issue 11) pp:3306-3310
Publication Date(Web):15 July 2009
DOI:10.1016/j.bios.2009.04.027
A novel mutant allele specific amplification (MASA) and electrochemiluminescence (ECL) method for point mutation detection is proposed. Briefly, the target gene was amplified by a biotinylated mutant specific sense primer and a Ru(bpy)32+ (TBR)-labeled universal antisense primer. Only the mutant allele can be selectively amplified by the mutant specific primer pair. Then, the MASA product was captured onto the streptavidinylated magnetic beads through biotin–streptavidin linkage and detected by measuring the ECL emission of TBR. The method was applied to detect a possible point mutation at codon 12 of K-ras oncogene in 30 colorectal cancer (CAC) clinical samples. The experimental results show that the method can detect K-ras mutant in a 5000-fold excess of wild-type allele. Furthermore, different kinds of mutations can be clearly discriminated. The point mutation was found in 15 (50%) out of 30 CAC samples. This novel MASA–ECL method could potentially become a sensitive, specific, simple, rapid and safe approach for point mutation detection.
Co-reporter:Xuejun Liu, Caiji Gao, Da Xing
Biosensors and Bioelectronics 2009 Volume 24(Issue 6) pp:1537-1542
Publication Date(Web):15 February 2009
DOI:10.1016/j.bios.2008.06.040
Superoxide generated during the early imbibition is an excellent marker for evaluating seed vigor. In this paper, a new principle biosensor for non-invasive detection of seed vigor based on quantitative measurement of superoxide via selective probe 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2α] pyrazin-3-one (MCLA)-mediated chemiluminescence (CL) was developed. The biosensor, which used a compact single-photon counting module (SPCM) to collect the CL signal, could evaluate seed vigor in vivo. Benefiting from the high CL efficiency of MCLA reacting with superoxide and high sensitivity of the SPCM technique, the trace superoxide generated by dry seeds under storage state can be detected to achieve rapid and non-invasive determination of the seed vigor. In comparison with the traditional methods for fast measuring seed vigor based on measurement of physiological and biochemical properties, our proposed technique has significant advantages such as low cost, simplicity, convenient operation and short time consuming. To demonstrate the utility of the system, it was applied to evaluate MCLA-mediated CL of three different plant species wheat (Ze Yu No. 2), maize (Tai Gu No. 1 and 2) and rice (Jing Dao No. 21) seeds with different degrees of aging. The experimental results suggested that there was an excellent positive correlation between the seed vigor assessment from quantitative TTC-test and the detection based on MCLA-mediated CL of superoxide measurement. The new principle of seed vigor measurement is a challenge and breakthrough to conventional method of seed vigor determination and may be a potential technique of the next generation seed vigor detection.
Co-reporter:Huijuan Zhou, Da Xing, Debin Zhu, Xiaoming Zhou
Talanta 2009 Volume 78(4–5) pp:1253-1258
Publication Date(Web):15 June 2009
DOI:10.1016/j.talanta.2009.01.032
The identification of single-base mutations in particular genes plays an increasingly important role in medical diagnosis and prognosis of genetic-based diseases. Here we report a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay, the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target; thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1 cell line, PANC-1 cell line and blood cell in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.
Co-reporter:LingRui Zhang;Feng Wen
Science Bulletin 2009 Volume 54( Issue 21) pp:
Publication Date(Web):2009 November
DOI:10.1007/s11434-009-0157-8
Photosynthetic dysfunction and reactive oxygen species (ROS) production are the common features of plant stress responses. Based on quantitative measurement of ROS production and delayed fluorescence (DF) emission, which is an excellent marker for evaluating photosynthesis, an on-line multi-parameter analyzing optical biosensor for detecting plant stress responses was developed. Performances of the proposed biosensor were tested in the wild type (WT) Arabidopsis and heat shock protein (Hsp) 101 T-DNA knockout mutant (hsp101) plants with different thermotolerance. Results demonstrated that DF intensity correlates with net photosynthesis rate (Pn) in response to elevated temperature in both the WT Arabidopsis and hsp101 mutant plants. The light response characteristics and the recovery dynamics of the DF intensity were also in line with those of Pn in both the WT Arabidopsis and hsp101 mutant plants after heat stress (HS, 40°C for 30 min), respectively. In all experiments discussed above, the hsp101 plant showed the worse photosynthetic performance than the WT plant. Moreover, after HS, more ROS production in the hsp101 mutant than in WT Arabidopsis, which was found to be mainly localized at chloroplasts, could be directly detected by using the proposed biosensor. In addition, the hsp101 mutant showed severer chloroplasts alterations than the WT plant within the first 1 h of recovery following HS. Nevertheless, pre-infiltration with catalase (CAT) reduced ROS production and prevented the declines of the DF intensity. Therefore, HS-caused declines of photosynthetic performance might be due to oxidative damage to photosynthetic organelle. To sum up, we conclude that Hsp101 plays an important role in preventing oxidative stress, and the proposed optical biosensor might be a powerful tool to determine plant stress responses and identify plant resistant difference.
Co-reporter:Yunlong Li;Da Xing;Qun Chen
Molecular Imaging and Biology 2009 Volume 11( Issue 3) pp:
Publication Date(Web):2009/05/01
DOI:10.1007/s11307-008-0195-7
The aim of the study is to dynamically and non-invasively monitor the temporal relationship among caspase-3, BID, and cytochrome c in chemotherapy.ASTC-a-1 cells expressing the corresponding fluorescence reporters were treated with Taxol or cisplatin and imaged using FRET and fluorescence overlapping technique. Western blot was performed to validate the fluorescence analysis.In fluorescence imaging analysis, Taxol-induced apoptosis showed caspase-3 activation (13 h 50 min) was prior to BID cleavage (15 h 10 min) and subsequent significant cytochrome c release (17 ~ 18 h 20 min), whereas the cisplatin-induced apoptosis showed BID cleavage (5 h 40 min) and significant cytochrome c release (7 ~ 8 h 20 min) were prior to caspase-3 activation (14 h 20 min). Western blot further validated the results above.The new approach successfully reveals the difference in temporal signaling apoptosis events between Taxol and cisplatin. It may help us come to a better understanding of the detailed mechanisms in chemotherapeutic-agents-induced apoptosis.
Co-reporter:Debin Zhu, Yabing Tang, Da Xing and Wei R. Chen
Analytical Chemistry 2008 Volume 80(Issue 10) pp:3566
Publication Date(Web):April 3, 2008
DOI:10.1021/ac0713306
A bio bar code assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio bar code assay requires lengthy experimental procedures including the preparation and release of bar code DNA probes from the target−nanoparticle complex and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio bar code assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2,2′-bipyridyl) ruthenium (TBR)-labeled bar code DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products.
Co-reporter:Qingling Wan;Lei Liu;Qun Chen
Photochemistry and Photobiology 2008 Volume 84( Issue 1) pp:250-257
Publication Date(Web):
DOI:10.1111/j.1751-1097.2007.00248.x
Abstract
Photodynamic therapy (PDT) employing photosensiter N-aspartyl chlorin e6 (NPe6) can induce lysosome disruption and initiate the intrinsic apoptotic pathway. Yet the precise signal transduction pathway remains poorly understood. In this study, we have investigated the molecular mechanism in NPe6-PDT-induced apoptosis in human lung adenocarcinoma cells (ASTC-a-1). A recombinant fluorescence resonance energy transfer (FRET) Bid probe was utilized to determine the kinetics of Bid cleavage. The results show that cleavage of the Bid-FRET probe occurred 150 ± 5 min after NPe6-PDT treatment, and this process lasted for 45 ± 5 min. The Bid cleavage coincided with a translocation of tBid from cytoplasm to mitochondria. Remarkably, a significant protection against cell death was observed by using small interfering RNA for Bid. Therefore, our study clearly showed the dynamics of Bid activation and redistribution during NPe6-PDT-induced apoptosis by using real-time analysis in living cells, and the inhibition of cell death by silencing Bid with interference strongly suggested that activation of Bid is required for inducing apoptosis in this experimental model.
Co-reporter:Xiaoming Zhou, Da Xing, Debin Zhu, Yabing Tang, Li Jia
Talanta 2008 Volume 75(Issue 5) pp:1300-1306
Publication Date(Web):15 June 2008
DOI:10.1016/j.talanta.2008.01.040
Enrofloxacin (ENR) is a fluoroquinolone developed exclusively for the use in veterinary practice for the treatment of respiratory and gastrointestinal infections, and ciprofloxacin (CIP) is its main active metabolite. Their contents are regulated by the EU Council Regulation no. 2377/90 in animal edible tissues. We developed a sensitive and rapid method for the determination of ENR and CIP by capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection. The method is based on the detection of aliphatic tertiary or secondary amino moieties in ENR and CIP with end-column tris(2,2-bipyridyl)ruthenium(II) electrochemiluminescence. Parameters that affect separation and detection were optimized. Under the optimized conditions, the calibration functions were linear in the range of 0.03–1 μg ml−1 for ENR and 0.05–1.2 μg ml−1 for CIP. The detection limits of ENR and CIR were 10 ng ml−1 and 15 ng ml−1, respectively, based on the signal-to-noise ratio of 3. The relative standard derivations of the peak height and the migration time for ENR and CIP were less than 4.13%. The developed method was successfully applied to determine ENR and CIP in milk with a solid-phase extraction procedure.
Co-reporter:Lan Zhang;Miaojuan Chen
Apoptosis 2008 Volume 13( Issue 7) pp:950-958
Publication Date(Web):2008 July
DOI:10.1007/s10495-008-0226-5
Bcl-2 family proteins are implicated as essential regulators in tumor necrosis factor-α (TNFα)-induced apoptosis. BimL, a BH3-only member of Bcl-2 family, can directly or indirectly activate the proapoptotic Bax and the subsequent mitochondrial apoptotic pathway. However, the molecular mechanism of BimL activating Bax activation during TNFα-induced apoptosis is not fully understood. In this study, the role of BimL in Bax activation during TNFα-induced apoptosis was investigated in differentiated PC12 and MCF7 cells, with real-time single-cell analysis. The experimental results show that Bax translocated to mitochondria and cytochrome c (Cyt c) released from mitochondria after TNFα treatment. Furthermore, SP600125 (specific inhibitor of JNK) could inhibit the Cyt c release from mitochondria. Co-immunoprecipitation results show that, the interaction between Bcl-xL and Bax decreased after TNFα treatment, while that between Bcl-xL and BimL increased. Bax did not co-immunoprecipitate with BimL before or after the TNFα treatment. In addition, the increased interaction between BimL and Bcl-xL was dynamically monitored by using fluorescence resonance energy transfer (FRET) technique. Most importantly, there was no evidence of BimL redistribution to mitochondria until cell apoptosis. By comprehensively analyzing these data, it is concluded that BimL displaces Bcl-xL in the mitochondria and promotes Bax translocation during TNFα-induced apoptosis.
Co-reporter:Caiji Gao;Lingling Li;Lingrui Zhang
Planta 2008 Volume 227( Issue 4) pp:755-767
Publication Date(Web):2008 March
DOI:10.1007/s00425-007-0654-4
Recent studies have suggested that ultraviolet-C (UV-C) overexposure induces programmed cell death (PCD) in Arabidopsis thaliana (L.) Heynh, and this process includes participation of caspase-like proteases, DNA laddering as well as fragmentation of the nucleus. To investigate possible early signal events, we used microscopic observations to monitor in vivo the behaviour of mitochondria, as well as the production and localization of reactive oxygen species (ROS) during protoplast PCD induced by UV-C. A quick burst of ROS was detected when the protoplasts were kept in continuous light after UV-C exposure, which was restricted in chloroplasts and the adjacent mitochondria. Pre-incubation with ascorbic acid (AsA, antioxidant molecule) or 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU, an inhibitor of photosynthetic electron transport) decreased the ROS production and partially protected protoplasts from PCD. A mitochondrial transmembrane potential (MTP) loss occurred prior to cell death; thereafter, the mitochondria irregularly clumped around chloroplasts or aggregated in other places within the cytoplasm, and the movement of mitochondria was concomitantly blocked. Pre-treatment with an inhibitor of mitochondrial permeability transition pores (MPTP), cyclosporine (CsA), effectively retarded the decrease of MTP and reduced the percentage of protoplasts undergoing PCD after UV-C overexposure. Our results suggest that the MTP loss and the changes in distribution and mobility of mitochondria, as well as the production of ROS play important roles during UV-induced plant PCD, which is in good accordance with what has been reported in many types of apoptotic cell death, both in animals and plants.
Co-reporter:Yinyuan WU;Lei LIU;Tongsheng CHEN;Wei R. CHEN
Acta Biochimica et Biophysica Sinica 2007 Volume 39(Issue 1) pp:37-45
Publication Date(Web):15 JAN 2007
DOI:10.1111/j.1745-7270.2007.00246.x
Ultraviolet (UV) irradiation is a DNA-damaging agent that triggers apoptosis through both the membrane death receptor and mitochondrial apoptotic signaling pathways. Bid, a pro-apoptotic Bcl-2 family member, is important in most cell types to apoptosis in response to DNA damage. In this study, a recombinant plasmid, YFP-Bid-CFP, comprised of yellow and cyan fluorescent protein and a full length Bid, was used as a fluorescence resonance energy transfer analysis (FRET) probe. Using the FRET technique based on YFP-Bid-CFP, we found that Bid activation was initiated at 9±1 h after UV irradiation, and the average duration of the activation was 75±10 min. Bid activation coincided with a collapse of the mitochondrial membrane potential with an average duration of 50±10 min. When cells were pretreated with Z-IETD-fmk (caspase-8 specific inhibitor) the process of Bid activation was completely inhibited, but the apoptosis was only partially affected. Z-DEVD-fmk (caspase-3 inhibitor) and Z-FA-fmk (non asp specific inhibitor) did not block Bid activation. Furthermore, the endogenous Bid activation with or without Z-IETD-fmk in response to UV irradiation was confirmed by Western blotting. In summary, using the FRET technique, we observed the dynamics of Bid activation during UV-induced apoptosis and found that it was a caspase-8 dependent event.
Edite by Xianxi WANG
Co-reporter:Xuejun Liu, Da Xing, Lingling Li and Lingrui Zhang
Photochemical & Photobiological Sciences 2007 vol. 6(Issue 7) pp:767-774
Publication Date(Web):21 May 2007
DOI:10.1039/B704337F
It has been reported that a large amount of reactive oxygen species (ROS) is produced during seed imbibition and this ROS is related to seed vigor. To make this physiological mechanism clear, we have used 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo(1,2-α)pyrazin-3-one (MCLA) as a sensitive and physiologically compatible probe for the determination of superoxide anion (O2˙–) production in vivo. Our results showed that dry rice (Oryzae sativa L.) seed embryo cells possessed the capacity to generate O2˙–. Conversely, the O2˙– production of seed embryo cells was inhibited by quinacrine (QA) and diphenylene iodonium (DPI), two specific inhibitors of NADPH oxidase, and O2˙– induced MCLA-mediated chemiluminescence was also blocked by superoxide dismutase (SOD). Additionally, O2˙–-production ability increased dramatically in a NADPH-dependent way in the plasma membraneprotein abstract from rice seed embryo cells, whereas SOD and the inhibitors mentioned above suppressed O2˙– production. These preliminary results suggested that rice seeds contained intrinsic NADPH oxidase activity. To validate this conclusion, dichlorofluorescein (DCF) fluorescence staining was used (observed under a laser scanning microscope, LSM) to reflect the in situ assessment of O2˙–-generation. The position of O2˙– production located at the plasma membrane. Additionally the ability to synthesize O2˙– was activated directly by calcium ions. These observations are in accord with the character of NADPH oxidase catalyzed O2˙–-generation. All these results indicated that NADPH oxidase contribute to O2˙– production and release to the outside. We concluded that NADPH oxidase plays an intrinsic role as an NADPH sensor, so, measuring the O2˙– one can monitor the NADPH concentration, which is an index of seed vigor. Therefore the O2˙– generation during early imbibition can serve as a rapid measurement of seed vigor.
Co-reporter:Lingrui Zhang, Da Xing, Junsheng Wang and Lingling Li
Photochemical & Photobiological Sciences 2007 vol. 6(Issue 6) pp:635-641
Publication Date(Web):06 Mar 2007
DOI:10.1039/B617893F
Senescence is a phase of leaf ontogeny marked by declining photosynthetic activity that is paralleled by a decline in chloroplast function. The photosystem II in a plant is considered to be the primary site where delayed fluorescence (DF) is produced. We report here a simple, rapid, and non-invasive technique for detecting plants senescence based on quantitative measurements of DF. In the experimental study, various senescence symptoms induced by age or hormones were examined in the Catharanthus roseus L. G. Don plants. Detecting the DF emissions from leaves with a home-made DF biosensor enables DF parameters of C. roseus to be produced in a short time. Meanwhile, evaluations of leaves senescence were made from measurements of chlorophyll content, ion leakage, and net photosynthesis rate (Pn) based on the consumption of CO2 in the tested plants. The results of our investigation demonstrate that the changes in DF intensity of green plants can truly reflect the changes in photosynthetic capacity and chlorophyll content during age-dependent and hormone-modulated senescence. Moreover, the DF intensity negatively correlates with ion leakage in both types of senescence. With proper calibration, DF may provide an important approach for monitoring senescence process in vivo and quantitatively evaluating senescence extent. Therefore, a DF technique could be potentially useful for less time-consuming and automated screening of the interesting mutants with genetic modifications that change the plant senescence progress.
Co-reporter:Bo Li, Da Xing and Lingrui Zhang
Photochemical & Photobiological Sciences 2007 vol. 6(Issue 6) pp:628-634
Publication Date(Web):26 Mar 2007
DOI:10.1039/B618041H
Bisulfite, a major form of SO2 in aqueous phase of apoplast, may reduce photosynthesis rate and thereby crop yield through inducing reactive oxygen species (ROS). In this study, ROS production was directly detected in a living cell of leaf of spinach (Spinacia oleracea L.) using laser scanning confocal microscopes with the assistance of the fluorescence probe dichlorofluorescin diacetate (H2DCF-DA). Results showed that, under bisulfite stress, a large quantity of ROS indicated by DCF fluorescence was produced in epidermic tissue. The role of plasma membrane (PM) NADPH oxidase in bisulfite-induced ROS production was also investigated. Treatment with bisulfite resulted in a significant increase in the content of ROS and the activity of PMNADPH oxidase in spinach leaves. The effects caused by bisulfite were inhibited pronouncedly by pretreatment with two widely used NADPH oxidase inhibitors (diphenyleneiodonium and quinacrine). Moreover, the change patterns of the bisulfite-induced increase and inhibitor-caused decrease in the two parameters were quite similar. Additionally, only a small amount of ROS could be observed on in vitrochloroplasts under bisulfite stress. Based on all the results, we conclude that ROS is involved in bisulfite-induced stress, and the bisulfite-induced enhancements in levels of ROS originate mainly from PMNADPH oxidase.
Co-reporter:Da Xing;Qiang Li;Lingrui Zhang;Chenglong Wang;Lizhang Zeng
Luminescence 2007 Volume 22(Issue 4) pp:387-392
Publication Date(Web):9 MAY 2007
DOI:10.1002/bio.978
The origination of the peak at 730 nm in the delayed fluorescence (DF) spectrum of chloroplasts was studied using various optical analysis methods. The DF spectrum showed that the main emission peak was at about 685 nm, with a small shoulder at 730 nm when the chloroplast concentration was < 7.8 µg/mL. The intensity of the peak at 685 nm decreased, while the intensity of the peak at 730 nm increased, when the chloroplast concentrations were increased from 7.8 to 31.2 µg/mL. With the concentration increasing, the peak at 730 nm became dominant while the peak at 685 nm finally disappeared. The DF decay kinetic curves showed that the intensity of the peak at 730 nm decayed as the same speed as the intensity of the peak at 685 nm during the entire relaxation process (0.5–30.5 s). With the excitation wavelength at 685 nm, the emission intensity was stronger in the excitation spectrum at 730 nm. The absorption spectrum demonstrated that the ratio A685:A730 remained almost constant when the chloroplast concentration increased. The results suggest that the peak at 730 nm appearing in DF is mainly contributed by the fluorescence of photosystem I (PSI), generated by the re-absorption of 685 nm band DF. Copyright © 2007 John Wiley & Sons, Ltd.
Co-reporter:Yihui Pei;Xuejuan Gao;Lei Liu;Tongsheng Chen
Apoptosis 2007 Volume 12( Issue 9) pp:1681-1690
Publication Date(Web):2007 September
DOI:10.1007/s10495-007-0091-7
Bid, a member of the pro-apoptotic Bcl-2 protein family, is activated through caspase-8-mediated cleavage into a truncated form (p15 tBid) during TNF-α(tumor necrosis factor α)-induced apoptosis. Activated tBid can induce Bax oligomerization and translocation to mitochondria, triggering the release of cytochrome c, caspase-3 activation and cell apoptosis. However, it is debatable that whether Bid and tBid can interact directly with Bax in living cells. In this study, we used confocal fluorescence microscope, combined with both FRET (fluorescence resonance energy transfer) and acceptor photobleaching techniques, to study the dynamic interaction between Bid and Bax during TNF-α-induced apoptosis in single living cell. In ASTC-a-1 cells, full length Bid induced Bax translocation to mitochondria by directly interacting with Bax transiently in response to TNF-α treatment before cell shrinkage. Next, we demonstrated that, in both ASTC-a-1 and HeLa cells, Bid was not cleaved before cell shrinkage even under the condition that caspase-8 had been activated, but in MCF-7 cells Bid was cleaved. In addition, in ASTC-a-1 cells, caspase-3 activation was a biphasic process and Bid was cleaved after the second activation of caspase-3. In summary, these findings indicate that, FL-Bid (full length-Bid) directly regulated the activation of Bax during TNF-α-induced apoptosis in ASTC-a-1 cells and that the cleavage of Bid occurred in advanced apoptosis.
Co-reporter:Jing Zhou;Qun Chen
Photochemistry and Photobiology 2006 Volume 82(Issue 4) pp:1058-1064
Publication Date(Web):30 APR 2007
DOI:10.1562/2005-12-02-RA-744
Fluoresceinyl cypridina luciferin analog (FCLA) is a chemiluminescence (CL) probe for detecting reactive oxygen species (ROS). Its efficiency for detecting singlet oxygen (1O2) can be significantly enhanced in the presence of human serum albumin (HSA). This phenomenon may apply to important applications for both research and clinical testing, because of the broad presence of HSA in the human system. In the current study the mechanism of the FCLA-HSA CL system is studied by means of direct CL measurement and spectroscopy techniques. Our results show that FCLA can combine with HSA via a single binding site to form a complex. The CL efficiency of the system is largely governed by an intersystem energy transfer between the two components upon interaction with 1O2. The CL production reaches maximum in a synergetic manner when equal amounts of FCLA and HSA are present simultaneously, but production is less at other ratios. The results also show that the combination of FCLA with HSA does not significantly alter the ROS selectivity of FCLA. In conclusion, our study shows that FCLA and HSA can combine and form a complex with higher CL efficiency. This provides us a new approach in designing CL techniques for studying ROS.
Co-reporter:Jinfeng Liu, Da Xing, Xingyan Shen, Debin Zhu
Analytica Chimica Acta 2005 Volume 537(1–2) pp:119-123
Publication Date(Web):29 April 2005
DOI:10.1016/j.aca.2005.01.011
With the development of biotechnology, more and more genetically modified organisms (GMOs) have entered commercial market. Because of the safety concerns, detection and characterization of GMOs have attracted much attention recently. Electrochemiluminescence (ECL) method is a chemiluminescent (CL) reaction of species generated electrochemically on an electrode surface. It is a highly efficient and accurate detection method. In this paper, ECL polymerase chain reaction (PCR) combined with two types of nucleic acid probes hybridization was applied to detect GMOs for the first time. Whether the organisms contain GM components was discriminated by detecting the cauliflower mosaic virus 35S (CaMV35S) promoter and nopaline synthase (NOS) terminator. The experiment results show that the detection limit is 100 fmol of PCR products. The promoter and the terminator can be clearly detected in GMOs. The method may provide a new means for the detection of GMOs due to its simplicity and high efficiency.
Co-reporter:WeiGui Chen;WenLi Chen
Photochemistry and Photobiology 2005 Volume 81(Issue 6) pp:1361-1365
Publication Date(Web):30 APR 2007
DOI:10.1562/2005-05-23-RC-538
Employing a luminescence spectrometer the usefulness of light-induced delayed luminescence (DL) for the detection of aflatoxinB1 (AfB1) contamination in whole peanut was studied. Peanut was artificially contaminated with Aspergillus flavus (7.5 × 105conidia/mL) and incubated for 0–72 h. The DL and fluorescence spectra of contaminated peanut were obtained by a luminescence spectrometer. The correlation between the spectra and contamination levels was established. The DL and fluorescence intensity has a negative correlation with the AfB1 concentration. Our results suggested that the DL technique might be useful for the rapid and noninvasive evaluation of AfB1 contamination levels in whole peanut.
Co-reporter:Da Xing;Shiming Luo;Yanfang Qin;Xueyun Zhong;Jing Zhou;Qun Chen
Photochemistry and Photobiology 2005 Volume 81(Issue 6) pp:1534-1538
Publication Date(Web):30 APR 2007
DOI:10.1562/2005-05-20-RA-536
Singlet oxygen (1O2) is the most important cytotoxic agent in photodynamic therapy (PDT). The feasibility of using a chemiluminescence (CL) probe, 3,7-dihydro-6-[4-(2-(N′-(5-fluoresceinyl)thioureido)ethoxy)phenyl]-2-methylimidazo{1,2-a}-pyrazin-3-one sodium salt (fluoresceinyl Cypridina luciferin analog, FCLA), to monitor 1O2 production during PDT is evaluated in vitro. Lymphoma cells were treated with various protocols of PDT. The results show that the FCLA-CL production during PDT is linearly related to the corresponding cytotoxicity, regardless of the treatment protocol. With minimum cytotoxicity and interference to the PDT treatment outcome, the FCLA-CL system is an effective means to quantify PDT 1O2 production and may provide an alternative real-time dosimeter.
Co-reporter:Da Xing;Lizhang Zeng;Qun Chen;Chenglong Wang;Chunfeng Ding
Luminescence 2005 Volume 20(Issue 1) pp:51-56
Publication Date(Web):31 JAN 2005
DOI:10.1002/bio.806
The structure and function of chloroplast in plant leaves can be affected by acid rain and air pollution. The photosystem II in a plant is considered the primary site where light-induced delayed light emission (DLE) is produced. With the lamina of zijinghua (Bauhinia variegata L.) and soybean (Glycine max (L.) Merr.) as testing models, we studied the effects of artificial acid rain and SO2 on characteristics of DLE by using a home-made weak luminescence detection system. The results show that the changes in DLE intensity of green plants can reflect the changes in chloroplast intactness and function. With proper calibration, DLE may provide an alternative means of evaluating environmental acid stress on plants. The changes in DLE intensity may provide a new approach for the detection of environmental pollution and its impact on the ecosystem. Copyright © 2005 John Wiley & Sons, Ltd.
Co-reporter:Yadong Wei;Xian Li;Debin Zhu;Bo Gao
Luminescence 2004 Volume 19(Issue 5) pp:278-282
Publication Date(Web):19 AUG 2004
DOI:10.1002/bio.782
A highly sensitive chemiluminescence (CL) method for evaluation of medical radiation damage degree is presented. According to the principle of cell stress response to ionizing radiation, lymphocytes will produce reactive oxygen species (ROS) after irradiation. The ROS produced can react with 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α] pyrazin-3-one (MCLA), a specific CL probe for superoxide anion (O·-2) and singlet oxygen (1O2), to emit light at 465 nm. The CL intensity is positively related to the amount of generated ROS detected 30 min after irradiation. Cell viability, which is inversely related to cell mortality, was determined by MTT assay after 3 days' culture. The results show that both CL intensity and cell mortality of lymphocytes increase with the increase of the radiation dose when the dosage is no more than 3 Gy, suggesting a positive relationship between the degree of lymphocyte cell damage and the amount of ROS generated. In addition, the effects of catalase, Cu–Zn superoxide dismutase (SOD), mannitol, sodium azide (NaN3), and D2O on MCLA-dependent CL of lymphocytes are discussed. We believe that the MCLA-dependent CL method would potentially provide an easy way for evaluating the degree of lymphocyte damage induced by radiation. Copyright © 2004 John Wiley & Sons, Ltd.
Co-reporter:Da Xing;Yonghong He;Juan Wang;Wenli Chen
Luminescence 2003 Volume 18(Issue 1) pp:19-24
Publication Date(Web):11 DEC 2002
DOI:10.1002/bio.695
Using a highly sensitive single photon counter, a spontaneous chemiluminescence (CL) study on rice (Oryza sativa L.) seeds stored in different years was carried out. We first observed that the degree of ageing in rice seeds was related to the intensity of spontaneous CL during early imbibition (0–30 min). Rice seeds stored for a shorter time had a stronger intensity of CL in early imbibition. The germination rate of rice seeds showed an obvious positive correlation with the intensity of spontaneous CL. Singlet oxygen (1O2) in rice seeds during early imbibition was investigated by a CL method using a cypridina luciferin analogue, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2α] pyrazin-3-one (MCLA), as a selective CL probe. Additional experimental evidence for the formation of 1O2 came from the quenching effect of sodium azide (NaN3) on MCLA-mediated rice seeds' CL. Analysis based on the experimental results demonstrated that spontaneous CL in rice seeds during early imbibition was mainly contributed by singlet oxygen (1O2). Copyright © 2002 John Wiley & Sons, Ltd.
Co-reporter:Shaodong Zhai, Xianglong Hu, Yongjun Hu, Baoyan Wu, Da Xing
Biomaterials (March 2017) Volume 121() pp:
Publication Date(Web):March 2017
DOI:10.1016/j.biomaterials.2017.01.002
Undesired physiological instability of nanocarriers and premature drug leakage during blood circulation result in compromised therapeutic efficacy and severe side effects, which have significantly impeded the development of nanomedicine. Facile crosslinking of drug-loaded nanocarriers while keeping the potency of site-specific degradation and drug release has emerged as a viable strategy to overcome these drawbacks. Additionally, combination therapy has already shown advantages in inhibiting advanced tumors and life extension than single drug therapy. Herein, three kinds of diselenide-rich polymers were fabricated with distinct hydrophobic side chains. The component effect was interrogated to screen out PEG-b-PBSe diblock copolymer due to its favorable self-assembly controllability and high drug loading of camptothecin (CPT) and doxorubicin (DOX) that had synergistic antitumor property. Facile visible light-induced diselenide metathesis and regeneration was employed to crosslink nanocarriers for the first time. The dual drug-loaded crosslinked micelles (CPT/DOX-CCM) were stable in physiological conditions with minimal drug leakage, possessing extended blood circulation, whereas hand-in-hand dual drug release was significantly accelerated in tumor's redox microenvironments. In vitro cytotoxicity evaluation and in vivo tumor suppression with low dosage drugs further demonstrated the favorable potency of the redox-responsive nanoplatform in tumor combination chemotherapy.
Co-reporter:Zebin Ma, Huan Qin, Hongjiang Chen, Hailong Yang, Jiankun Xu, Sihua Yang, Jun Hu, Da Xing
Nanomedicine: Nanotechnology, Biology and Medicine (January 2017) Volume 13(Issue 1) pp:
Publication Date(Web):January 2017
DOI:10.1016/j.nano.2016.09.002
Specific detection of various tumor types remains crucial for designing effective treatment strategies. We demonstrate photoacoustic imaging (PAI) using high-affinity and high-specificity peptide-based probes for accurate and specific diagnosis of osteosarcoma. Herein, two new tumor-specific oligopeptides, termed PT6 and PT7, were identified using phage display-based screening on an osteosarcoma cell line (UMR-106). The identified oligopeptides were able to detect clinical osteosarcoma samples on tissue microarrays. Oligopeptide-conjugated PEGylated gold nanorods (PGNR) were designed to specifically target UMR-106 cells. More importantly, PAI revealed that both PGNR-PT6 and PGNR-PT7 could bind selectively to subcutaneous UMR-106 xenografts after systemic administration and enhance the contrast of osteosarcoma images by 170% and 230%, respectively, compared to tumor-bearing mice injected with PGNRs conjugated to scrambled oligopeptides. PAI employing PGNRs conjugated to specifically designed nanoprobes may provide a new method for tumor type-specific diagnosis of osteosarcoma.We identified two oligopeptides (PT6 and PT7) that could specifically bind to osteosarcoma cells (UMR-106) using phage display-based screening. And the oligopeptides appeared to be selective for clinical samples recognized. Due to its high and specific binding to UMR-106, PGNR-PT6 and PGNR-PT7 nanoprobe-mediated photoacoustic imaging was proved to be an efficient modality for tumor type-specific diagnosis of osteosarcoma.
Co-reporter:Wenzheng Ding, Cunguang Lou, Jieshan Qiu, Zongbin Zhao, Quan Zhou, Minjie Liang, Zhong Ji, Sihua Yang, Da Xing
Nanomedicine: Nanotechnology, Biology and Medicine (January 2016) Volume 12(Issue 1) pp:235-244
Publication Date(Web):January 2016
DOI:10.1016/j.nano.2015.08.008
Co-reporter:Wenzheng Ding, Cunguang Lou, Jieshan Qiu, Zongbin Zhao, Quan Zhou, Minjie Liang, Zhong Ji, Sihua Yang, Da Xing
Nanomedicine: Nanotechnology, Biology and Medicine (January 2016) Volume 12(Issue 1) pp:235-244
Publication Date(Web):January 2016
DOI:10.1016/j.nano.2015.08.008
Co-reporter:Heng Zhang, Shengnan Wu, Da Xing
Cellular Signalling (January 2012) Volume 24(Issue 1) pp:224-232
Publication Date(Web):1 January 2012
DOI:10.1016/j.cellsig.2011.09.004
Deposition of amyloid-β-peptide (Aβ) in the brain is considered a pathological hallmark of Alzheimer's disease (AD). Our previous studies show that Yes-associated protein (YAP) is involved in the regulation of apoptosis induced by Aβ25–35 through YAP nuclear translocation and its pro-apoptotic function is mediated by its interaction with p73. In the present study, we first found that Low-power laser irradiation (LPLI) promoted YAP cytoplasmic translocation and inhibited Aβ25–35-induced YAP nuclear translocation. Moreover, the cytoplasmic translocation was in an Akt-dependent manner. Activated Akt by LPLI phosphorylated YAP on ser127 (S127) and resulted in decreasing the interaction between YAP and p73, and in suppressing the proapoptotic gene bax expression following Aβ25–35 treatment. Inhibition of Akt expression by siRNA significantly abolished the effect of LPLI. More importantly, LPLI could inhibit Aβ25–35-induced cell apoptosis through activation of Akt/YAP/p73 signaling pathway. Therefore, our findings first suggest that YAP may be a therapeutic target and these results directly point to a potential therapeutic strategy for the treatment of AD through Akt/YAP/p73 signaling pathway with LPLI.Highlights► LPLI promotes YAP cytoplasmic translocation in an Akt-dependent manner. ► LPLI protects cell from Aβ25–35-induced apoptosis through Akt activation. ► Activated Akt by LPLI promotes YAP phosphorylation on S127. ► Phosphorylated YAP separates from p73 and inhibits Bax expression. ► LPLI inhibits Aβ25–35-induced apoptosis through Akt/YAP/p73 pathway.
Co-reporter:Jie Feng, Chengbo Meng, Da Xing
Neurobiology of Aging (February 2015) Volume 36(Issue 2) pp:789-800
Publication Date(Web):1 February 2015
DOI:10.1016/j.neurobiolaging.2014.10.007
p53 upregulated modulator of apoptosis (PUMA) is a promising tumor therapy target because it elicits apoptosis and profound sensitivity to radiation and chemotherapy. However, inhibition of PUMA may be beneficial for curbing excessive apoptosis associated with neurodegenerative disorders. Alzheimer's disease (AD) is a representative neurodegenerative disease in which amyloid-β (Aβ) deposition causes neurotoxicity. The regulation of PUMA during Aβ-induced neuronal apoptosis remains poorly understood. Here, we reported that PUMA expression was significantly increased in the hippocampus of transgenic mice models of AD and hippocampal neurons in response to Aβ. PUMA knockdown protected the neurons against Aβ-induced apoptosis. Furthermore, besides p53, PUMA transactivation was also regulated by forkhead box O3a through p53-independent manner following Aβ treatment. Notably, PUMA contributed to neuronal apoptosis through competitive binding of apoptosis repressor with caspase recruitment domain to activate caspase-8 that cleaved Bid into tBid to accelerate Bax mitochondrial translocation, revealing a novel pathway of Bax activation by PUMA to mediate Aβ-induced neuronal apoptosis. Together, we demonstrated that PUMA activation involved in Aβ-induced apoptosis, representing a drug target to antagonize AD progression.Download high-res image (113KB)Download full-size image
Co-reporter:Jian Zhang, Sihua Yang, Xuanrong Ji, Quan Zhou, Da Xing
Journal of the American College of Cardiology (29 July 2014) Volume 64(Issue 4) pp:385-390
Publication Date(Web):29 July 2014
DOI:10.1016/j.jacc.2014.04.053
BackgroundHistologic studies have demonstrated that lipid content and its spatial distribution is related to plaque vulnerability. However, in vivo imaging is still limited. Photoacoustic imaging may provide novel in vivo insights into these lipid-rich plaques.ObjectivesThis study sought to examine whether intravascular photoacoustic tomography (IVPAT) allows localization and quantification of lipid content in atherosclerotic plaques.MethodsRabbits fed with a high-fat/high-cholesterol diet served as the atherosclerotic model. Catheter-based IVPAT was used to evaluate pixel-based lipid relative concentration (LRC) of the vessel wall. The aorta of 4 groups of rabbits (n = 12) were examined ex vivo with IVPAT after 0, 5, 10, and 15 weeks of a high-fat diet, respectively. Six rabbits underwent 3-dimensional (3D) IVPAT after 20 weeks of the high-fat diet. Three rabbits were examined in vivo using IVPAT without interruption of blood flow. Concentration-based lipid map and quantitative index were calculated. For subsequent histologic correlation, all specimens were evaluated with Oil Red O staining.ResultsCross-sectional LRC maps allowed visualization of concentration and depth information of lipid content in the atherosclerotic plaques. Lipid accumulation within plaque, assessed by the maximum LRC, mean LRC, and high lipid content area correlated to duration of a high-fat diet. Three-dimensional LRC maps enabled overall evaluation of focal plaques in an intact explanted aorta including spatial and structural features. In vivo–obtained LRC maps accurately showed the structure of lipid core with high contrast. Ex vivo and in vivo IVPAT results were highly consistent with histological results.ConclusionsIn an animal model, IVPAT allowed characterization of spatial and quantitative features of lipid-rich plaques.
Co-reporter:Hui Li, Lei Liu, Da Xing, Wei R. Chen
FEBS Letters (19 November 2010) Volume 584(Issue 22) pp:4672-4678
Publication Date(Web):19 November 2010
DOI:10.1016/j.febslet.2010.10.050
Here we studied the mechanism by which heat shock protein 70 (Hsp70) prevents Bax activation during ultraviolet (UV)-induced apoptosis. UV treatment led to c-Jun N-terminal kinase (JNK) phosphorylation, Bim redistribution and subsequent Bax activation. Bim depletion caused a smaller reduction in apoptosis than that by JNK inhibition, indicating that Bim activation is not entirely responsible for induction of apoptosis and other mechanisms are involved. Hsp70 knockdown resulted in high levels of activated JNK and Bax, while Hsp70 overexpression inhibited these processes. These findings demonstrate that Hsp70 prevented Bax activation via inhibiting the JNK/Bim pathway. Simultaneously, increased binding of Hsp70 to Bax was observed. Collectively, our results for the first time demonstrate that Hsp70 prevents Bax activation both by inhibiting the JNK/Bim pathway and by interacting with Bax in UV-induced apoptosis.
Co-reporter:Xianwang Wang, Da Xing, Lei Liu, Wei R. Chen
FEBS Letters (18 June 2009) Volume 583(Issue 12) pp:1873-1879
Publication Date(Web):18 June 2009
DOI:10.1016/j.febslet.2009.04.045
Bcl-2-interacting mediator of cell death (Bim) has been considered to initiate intrinsic apoptotic pathway through Bax activation. Previous studies indicated that BimL was involved in UV-induced apoptosis, but it remains unclear whether Bim activates Bax by directly engaging it or by releasing it from pro-survival relatives such as Bcl-xL. In this study, we attempt to determine the interactions between BimL and Bax/Bcl-xL during Ultraviolet (UV)-induced apoptosis. BimL activation appeared to be an important event in our experiments, as demonstrated by the significant inhibition of cell death, caspase-3 activity, and Bax translocation in cells with knockdown of endogenous BimL by RNAi approach. Both fluorescence resonance energy transfer (FRET) and Co-immunoprecipitation (CO-IP) assays indicated that Bcl-xL directly bound to Bax to inhibit its activation, while BimL directly neutralized Bcl-xL, followed by Bax release and activation upon UV irradiation. Not detected in our experiment was the interaction between BimL and Bax either using FRET approach in living cells or endogenous CO-IP assay. Thus, our findings provide strong evidence in living cells for the first time that BimL initiates apoptosis by abrogating Bcl-xL and promoting Bax activation under UV irradiation.Structured summaryMINT-7034091: BIML (uniprotkb:O43521) physically interacts (MI:0218) with Bcl2-Xl (uniprotkb:Q92934) by anti bait coimmunoprecipitation (MI:0006)MINT-7034079: Bcl2-Xl (uniprotkb:Q92934) physically interacts (MI:0218) with BAX (uniprotkb:Q07812) and BIML (uniprotkb:O43521) by anti bait coimmunoprecipitation (MI:0006)MINT-7034069: BAX (uniprotkb:Q07812) physically interacts (MI:0218) with BIML (uniprotkb:O43521) by anti bait coimmunoprecipitation (MI:0006)MINT-7034114: BIML (uniprotkb:O43521) and BAX (uniprotkb:Q07812) physically interact (MI:0218) by fluorescent resonance energy transfer (MI:0055)
Co-reporter:Xiaoke Yang, Xiaoming Zhou, Minjun Zhu, Da Xing
Biosensors and Bioelectronics (15 May 2017) Volume 91() pp:
Publication Date(Web):15 May 2017
DOI:10.1016/j.bios.2016.11.044
•A multivalent brush-like magnetic nano-platform was constructed.•Brush-like magnetic nanoprobes offer high efficiently enrichment and satisfactory purity for pathogens.•Listeria monocytogenes can be rapidly and accurately detected at levels as low as 10 cfu mL-1.•PCR-ECL detection can guarantee sensitive and specific pathogen detection in complex samples based on the enrichment platform.Pathogens pose a significant threat to public health worldwide. Despite many technological advances in the rapid diagnosis of pathogens, sensitive pathogen detection remains challenging because target pathogenic bacteria usually exist in complex samples at very low concentrations. Here, the construction of multivalent brush-like magnetic nanoprobes and their application for the efficient enriching of pathogens are demonstrated. Brush-like magnetic nanoprobes were constructed by modification with poly-L-lysine (PLL) onto amino-modified magnetic beads, followed by coupling of PEG (amine-PEG5000-COOH) to the amine sites of PLL. Subsequently, vancomycin (Van), a small-molecule antibiotic with affinity to the terminal peptide (D-alanyl-D-alanine) on the cell wall of Gram-positive bacteria, was conjugated to the carboxyl of the PEG. The use of multivalent brush-like magnetic nanoprobes (Van-PEG-PLL-MNPs) results in a high enrichment efficiency (>94%) and satisfactory purity for Listeria monocytogenes (employed as a model) within 20 min, even at bacterial concentrations of only 102 cfu mL-1. Integrated with the enrichment of the Van-PEG-PLL-MNP nano-platform and electrochemiluminescence (ECL) detection, Listeria monocytogenes can be rapidly and accurately detected at levels as low as 10 cfu mL-1. The approach described herein holds great potential for realizing rapid and sensitive pathogen detection in clinical samples.
Co-reporter:Zhongyu Fu, Xiaoming Zhou, Da Xing
Methods (15 December 2013) Volume 64(Issue 3) pp:260-266
Publication Date(Web):15 December 2013
DOI:10.1016/j.ymeth.2013.08.003
•HRCA based GNP colorimetric method is novel and useful to detect L. monocytogenes.•HRCA could avoid complicated thermal cycling steps.•This method leads to a limit of detection of 100 aM hly gene with high specificity.•The proposed method could detect food pathogenic bacteria even by naked eyes.Listeria monocytogenes (L. monocytogenes), one of most problematic food-borne bacteria, is mainly transmitted through the food chain and may cause listeriosis. Therefore, the development of rapid and sensitive L. monocytogenes detection technique has become an urgent task. In this study, we proposed a method using hyperbranching rolling circle amplification (HRCA) combined with gold nanoparticle (GNP) based colorimetric strategy to offer an isothermal, highly sensitive and specific assay for the detection of L. monocytogenes. First, a linear padlock probe targeting a specific sequence in the hly gene was designed and followed with a ligation by Taq DNA ligase. After ligation, further amplification by HRCA with a thiolated primer and an unlabeled primer is performed. The resulting thiolated HRCA products were then captured onto GNP surface and made GNP more salt-tolerant. Detection of the bacteria can be achieved by a facilitated GNP based colorimetric testing using naked eyes. Through this approach, as low as 100 aM synthetic hly gene targets and about 75 copies of L. monocytogenes can be detected. The specificity is evaluated by distinguishing target L. monocytogenes from other bacteria. The artificial contaminated food samples were also detected for its potential applications in real food detection. This method described here is ideal for bacteria detection due to its simplicity and high sensitivity.Taking the advantages of the high amplification efficiency of hyperbranching rolling circle amplification, the speediness and convenience of colorimetric assay, a colorimetric gene-sensing assay for high sensitive detection of Listeria monocytogenes has been developed.Download full-size image
Co-reporter:Fuqiang Xing, Zhe Li, Aizhen Sun, Da Xing
FEBS Letters (11 July 2013) Volume 587(Issue 14) pp:2164-2172
Publication Date(Web):11 July 2013
DOI:10.1016/j.febslet.2013.05.034
•Light-dependent ROS production is associated with FB1-induced cell death.•ROS cause chloroplast dysfunction in response to FB1 treatment.•ROS promote SA synthesis via PAL-mediated signaling pathway in FB1-induced cell death.We report a novel regulatory mechanism by which reactive oxygen species (ROS) regulate fumonisin B1 (FB1)-induced cell death. We found that FB1 induction of light-dependent ROS production promoted the degradation of GFP-labeled chloroplast proteins and increased phenylalanine ammonia lyase (PAL) activity, PAL1 gene expression and SA content, while pretreatment with ROS manipulators reversed these trends. Moreover, treatment with H2O2 or 3-amino-1,2,4-triazole increased PAL activity, PAL1 gene expression and SA content. PAL inhibitor significantly blocked FB1-induced lesion formation and SA increase. Our results demonstrate that light-dependent ROS accumulation stimulates the degradation of chloroplastic proteins and up-regulates PAL-mediated SA synthesis, thus promoting FB1-induced light-dependent cell death.
Co-reporter:Ting Zhou, Baoyan Wu and Da Xing
Journal of Materials Chemistry A 2012 - vol. 22(Issue 2) pp:
Publication Date(Web):
DOI:10.1039/C1JM13692E
Co-reporter:Xiaoming Zhou and Da Xing
Chemical Society Reviews 2012 - vol. 41(Issue 13) pp:NaN4656-4656
Publication Date(Web):2012/05/01
DOI:10.1039/C2CS35045A
Human telomerase is a ribonucleoprotein complex that functions as a telomere terminal transferase by adding multiple TTAGGG hexamer repeats using its integral RNA as the template. There is a very strong association between telomerase activity and malignancy in nearly all types of cancer, suggesting that telomerase could be used not only as a diagnostic and prognostic marker but also as a therapeutic target for managing cancer. The significant progress in biomedical telomerase research has necessitated the development of new bioanalytical methods for the rapid, sensitive, and reliable detection of telomerase activity in a particular cell or clinical tissue and body fluids. In this review, we highlight some of the latest methods for identifying telomerase activity and inhibition and discuss some of the challenges for designing innovative telomerase assays. We also summarise the current technologies and speculate on future directions for telomerase testing.
Co-reporter:Liyun Xue, Xiaoming Zhou and Da Xing
Chemical Communications 2010 - vol. 46(Issue 39) pp:NaN7375-7375
Publication Date(Web):2010/09/06
DOI:10.1039/C0CC02038A
An isothermal and sensitive fluorescence assay for protein detection using aptamer–protein–aptamer conjugates based on nicking enzyme amplification has been developed, which was combined with magnetic microparticles separation, making this assay suitable for protein detection in biological samples.
Co-reporter:Guojia Huang, Zhan Si, Sihua Yang, Cong Li and Da Xing
Journal of Materials Chemistry A 2012 - vol. 22(Issue 42) pp:NaN22581-22581
Publication Date(Web):2012/09/10
DOI:10.1039/C2JM33990K
A dextran based pH-sensitive near-infrared nanoprobe was prepared and applied as a contrast agent for photoacoustic imaging to identify a tumor in vivo. The nanoprobe has two resonance absorption peaks in the near-infrared region and the cleavage of hydrazone bonds in the low pH value environment can cause their coefficients to change, making a different ratio of photoacoustic signal output. By comparing the photoacoustic signal ratio under the two wavelengths, in vitro photoacoustic images show that the nanoprobe has the capacity to distinguish cancer cells and normal cells. Moreover, an in vivo mouse breast tumor model (EMT-6 cells) can be identified from peripheral normal tissues by dual-wavelength photoacoustic imaging. These results suggest the potential application of this pH-sensitive near-infrared nanoprobe as a contrast agent to noninvasively detect tumors in their early stage with high optical contrast by dual-wavelength photoacoustic imaging based on the tunable absorbance ratio in normal physiological and tumor acidic microenvironments.
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