Co-reporter:Ya Yan, Lingyan Meng, Wenqiang Zhang, Yan Zheng, Shuo Wang, Bin Ren, Zhilin Yang, and Xiaomei Yan
ACS Sensors September 22, 2017 Volume 2(Issue 9) pp:1369-1369
Publication Date(Web):August 24, 2017
DOI:10.1021/acssensors.7b00522
Metal-enhanced fluorescence (MEF) based on localized surface plasmon resonance (LSPR) is an effective strategy to increase the detection sensitivity in biotechnology and biomedicine. Because plasmonic nanoparticles are intrinsically heterogeneous, high-throughput single-particle analysis of MEF in free solution are highly demanded for the mechanistic understanding and control of this nanoscale process. Here, we report the application of a laboratory-built high-sensitivity flow cytometer (HSFCM) to investigate the fluorescence-enhancing effect of individual plasmonic nanoparticles on nearby fluorophore molecules. Ag@SiO2 core–shell nanoparticles were used as the model system which comprised a silver core, a silica shell, and an FITC-doped thin layer of silica shell. FITC-doped silica nanoparticles of the same particle size but without silver core were used as the counterparts. Both the side scattering and fluorescence signals of single nanoparticles in suspension were measured simultaneously by the HSFCM at a speed of thousands of particles per minute. The roles of silver core size (40–100 nm) and fluorophore–metal distance (5–30 nm) were systematically examined. Fluorescence enhancement factor exceeding 30 was observed at silver core size of 70 nm and silica shell thickness of 5 nm. Compared with ensemble-averaged spectrofluorometric measurements, our experimental observation at the single-particle level was well supported by the finite difference time domain (FDTD) calculation. It allows us to achieve a fundamental understanding of MEF, which is important to the design and control of plasmonic nanostructures for efficient fluorescence enhancement.Keywords: Ag@SiO2 nanostructure; flow cytometry; localized surface plasmon resonance; metal-enhanced fluorescence; single-particle analysis;
Co-reporter:Shuo Wang, Lihong Li, Shenghao Jin, Weifeng Li, Wei Hang, and Xiaomei Yan
Analytical Chemistry September 19, 2017 Volume 89(Issue 18) pp:9857-9857
Publication Date(Web):August 18, 2017
DOI:10.1021/acs.analchem.7b01885
Semiconducting quantum dots (QDs) are finding a wide range of biomedical applications due to their intense fluorescence brightness and long-term photostability. Here, we report precise quantification of the fluorescence intensity of single QDs on a laboratory-built high-sensitivity flow cytometer (HSFCM). The nearly uniform illumination of the particles at the intense portions of the radiation field resulted in narrowly distributed signals with high signal-to-noise ratios. By analysis of thousands of QDs individually in as little time as 1 min, intrinsic polydispersity was quickly revealed in a statistically robust manner. Applications of this technique in QD quality assessment, study of metal ion influence, and evaluation of aggregation upon biomolecule coupling are presented. Moreover, an accurate measurement of the QD particle concentration was achieved via single-particle enumeration. HSFCM is believed to provide a powerful characterization tool for QD synthesis and application development.
Co-reporter:Chaoxiang Chen, Shaobin Zhu, Shuo Wang, Wenqiang Zhang, Yu Cheng, and Xiaomei Yan
ACS Applied Materials & Interfaces April 26, 2017 Volume 9(Issue 16) pp:13913-13913
Publication Date(Web):April 4, 2017
DOI:10.1021/acsami.7b01867
Drug-encapsulated liposomes have been considered the most clinically acceptable drug-delivery systems. However, current methods fall short in the quantitative characterization of individual nanoliposomes because of their small sizes and large heterogeneity. Here, we report a high-throughput method for the absolute quantification of particle size, drug content, fraction of drug encapsulation, and particle concentration of liposomal nanomedicines at the single-particle level. A laboratory-built high-sensitivity flow cytometer was used to simultaneously detect the side-scatter and fluorescence signals generated by individual nanomedicine particles at a speed up to 10 000 nanoparticles/min. To cope with the size dependence of the refractive index of liposomal nanomedicines, different sizes of doxorubicin-loaded liposomes were fabricated and characterized to serve as the calibration standards for the measurement of both particle size and drug content. This method provides a highly practical platform for the characterization of liposomal nanomedicines, and broad applications can be envisioned.Keywords: doxorubicin-encapsulated liposomes; flow cytometry; nanomedicine; nanoparticle characterization; single-particle detection;
Co-reporter:Lina Wu, Xu Wang, Jianqiang Zhang, Tian Luan, Emmanuelle Bouveret, and Xiaomei Yan
Analytical Chemistry 2017 Volume 89(Issue 5) pp:
Publication Date(Web):February 7, 2017
DOI:10.1021/acs.analchem.6b03603
Cell-based two-hybrid assays have been key players in identifying pairwise interactions, yet quantitative measurement of protein–protein interactions in vivo remains challenging. Here, we show that by using relative reporter protein expression (RRPE), defined as the level of reporter expression normalized to that of the interacting protein, quantitative analysis of protein interactions in a bacterial adenylate cyclase two-hybrid (BACTH) system can be achieved. A multicolor flow cytometer was used to measure simultaneously the expression levels of one of the two putative interacting proteins and the β-galactosidase (β-gal) reporter protein upon dual immunofluorescence staining. Single-cell analysis revealed that there exists bistability in the BACTH system and the RRPE is an intrinsic characteristic associated with the binding strength between the two interacting proteins. The RRPE-BACTH method provides an efficient tool to confirm interacting pairs of proteins, investigate determinant residues in protein–protein interaction, and compare interaction strength of different pairs.
Co-reporter:Lina Wu, Yiyi Song, Tian Luan, Ling Ma, Liuqin Su, Shuo Wang, Xiaomei Yan
Biosensors and Bioelectronics 2016 Volume 86() pp:102-108
Publication Date(Web):15 December 2016
DOI:10.1016/j.bios.2016.06.041
•Using tetracysteine-tagged PP01 bacteriophage, a rapid and sensitive method was developed for the specific detection of live E. coli O157:H7.•PP01-TC phage in viable E. coli O157:H7 host cell can be fluorescently labeled by a membrane permeable biarsenical dye.•The bright green fluorescence of single E. coli O157:H7 cells can be readily detected by flow cytometery and fluorescence microscopy.•Trace amount of E. coli O157:H7 in artificially contaminated 100% apple juice can be detected down to 1cfu/mL via fluorescence microscopic measurement.Sensitive and rapid detection of Escherichia coli O157:H7, one of the most notorious bacterial pathogens, is urgently needed for public health protection. Yet, the existing methods are either lack of speed or limited in discriminating viable and dead cells. Using a recombinant bacteriophage, here we report the development of a rapid and sensitive method for live E. coli O157:H7 detection. First, the wild-type PP01 phage was engineered with a tetracysteine (TC)-tag fused with the small outer capsid (SOC) protein. Then, this PP01-TC phage was used to inoculate bacterial sample for 30 min. Specific infection and rapid replication of PP01-TC phage in viable E. coli O157:H7 host cell yields a large number of progeny phages with capsids displaying TC tags that can be fluorescently labeled by a membrane permeable biarsenical dye (FlAsH). The bright green fluorescence of single E. coli O157:H7 cells can be readily detected by flow cytometry (FCM) and fluorescence microscopy. High specificity of the assay was verified with seven other bacterial strains. Practical application in E. coli O157:H7 detection in drinks was successfully demonstrated with artificially contaminated 100% apple juice. In less than three hours (including sample preconcentration) and with 40 mL of sample volume, as low as 1 cfu/mL E. coli O157:H7 can be detected in the presence of large excess of other nontarget bacteria via fluorescence microscopic measurement. The as-developed TC-PP01-FlAsH approach shows a great potential in the safeguard of liquid food products by providing rapid, sensitive, and specific detection of live E. coli O157:H7.
Co-reporter:Shaobin Zhu, Chaoxiang Chen, Ling Ma, Shuo Wang, Wenqiang Zhang, Xiaomei Yan
Nanomedicine: Nanotechnology, Biology and Medicine 2016 Volume 12(Issue 2) pp:465-466
Publication Date(Web):February 2016
DOI:10.1016/j.nano.2015.12.058
Co-reporter:Lina Wu;Shuo Wang;Yiyi Song;Xu Wang
Science China Chemistry 2016 Volume 59( Issue 1) pp:30-39
Publication Date(Web):2016 January
DOI:10.1007/s11426-015-5518-3
Flow cytometry (FCM) is a powerful technique for single-bacteria analysis via simultaneous light-scattering and fluorescence measurements. By offering high-throughput, quantitative, and multiparameter analysis at the single-cell level, FCM has gained an increased popularity in microbiological research, food safety monitoring, water quality control, and clinical diagnosis. Here we will review the recent applications of flow cytometry in areas such as (1) total bacterial cell count, (2) bacterial viability analysis, (3) specific bacterial detection and identification, (4) characterization of physiological changes under environmental perturbations, and (5) biological function studies. Nevertheless, despite these widespread applications, challenges still remain for the detection of small sizes of bacteria and biochemical features that cannot be brightly stained via fluorescence. Recent improvement in FCM instrumentation will be discussed, and particularly the development of high sensitivity flow cytometry for advanced analysis of single bacterial cells will be highlighted.
Co-reporter:Muxin Yu, Lina Wu, Tianxun Huang, Shuo Wang and Xiaomei Yan
Analytical Methods 2015 vol. 7(Issue 7) pp:3072-3079
Publication Date(Web):16 Feb 2015
DOI:10.1039/C4AY02919D
Safe and secure supply of drinking water is an essential requirement for human health. Because many different microbiological contaminants may occur in drinking water and beverages, the total bacterial count represents one of the key parameters for quality assessment. Flow cytometry has been recognized as a rapid cultivation independent tool to assess the bacteriological quality and biological stability of water. Yet the limited detection sensitivity and the high background signal generated by impurity particles in the sheath fluid make data analysis difficult to perform, particularly for bacteria of small sizes. Using a laboratory-built high-sensitivity flow cytometer (HSFCM) with enhanced sensitivity and significantly reduced background signals of impurity particles, here we report the development of a rapid approach for the accurate quantification of total bacterial cells in drinks. Bacteria in drinking water and tea beverages were stained with PicoGreen nucleic acid dye, and both the side scatter and fluorescence signals of individual bacterial cells were detected simultaneously using the HSFCM. Using bottled drinking water and filtered tea beverage artificially contaminated with E. coli ER2738 as the model system, good correlations (R2 > 0.995) between the results measured by HSFCM enumeration and the traditional plate-counting method were obtained. The established approach was successfully applied to total bacterial quantification in barreled drinking water and Jasmine Green Tea beverages. Addition of 1 mM EDTA (chelating reagent) to tea beverage can efficiently block the interference of magnesium ions (Mg2+) on PicoGreen fluorescence staining. Compared with plate counting, HSFCM not only shortens the analysis time from days to less than 20 min, but also reveals the presence of dead and viable but non-cultivable (VBNC) bacterial cells. Therefore, HSFCM holds great potential in the rapid and accurate screening of the presence of bacteria in drinking water and tea beverages.
Co-reporter:Chaoxiang Chen, Xiang Zhang, Shuyue Zhang, Shaobin Zhu, Jingyi Xu, Yan Zheng, Jinyan Han, Jin-Zhang Zeng, Xiaomei Yan
Biosensors and Bioelectronics 2015 Volume 74() pp:476-482
Publication Date(Web):15 December 2015
DOI:10.1016/j.bios.2015.06.057
Bcl-2 family proteins, represented by antiapoptotic protein Bcl-2 and proapoptotic protein Bax, are key regulators of mitochondria-mediated apoptosis pathway. To build a quantitative model of how Bcl-2 family protein interactions control mitochondrial outer membrane permeabilization and subsequent cytochrome c release, it is essential to know the number of proteins in individual mitochondria. Here, we report an effective method to quantify the copy number and distribution of proteins in single mitochondria via immunofluorescent labeling and sensitive detection by a laboratory-built high sensitivity flow cytometer (HSFCM). Mitochondria isolated from HeLa cells were stained with Alexa Fluor 488 (AF488)-labeled monoclonal antibodies specifically targeting Bcl-2 or Bax and with nucleic acid dye. A series of fluorescent nanospheres with fluorescence intensity calibrated in the unit of molecules of equivalent soluble fluorochrome (MESF)-AF488 were used to construct a calibration curve for converting the immunofluorescence of a single mitochondrion to the number of antibodies bound to it and then to the number of proteins per mitochondrion. Under the normal condition, the measured mean copy numbers were 1300 and 220 per mitochondrion for Bcl-2 and Bax, respectively. A significant variation in protein copy number was identified, which ranged from 130 to 6000 (2.5–97.5%) for Bcl-2 and from 65 to 700 (2.5–97.5%) for Bax, respectively. We observed an approximately 4.4 fold increase of Bax copy number per mitochondrion upon 9 h of apoptosis stimulation while the abundance of Bcl-2 remained almost unchanged. To the best of our knowledge, this is the first report of Bcl-2 family protein copy number and variance in single mitochondria. Collectively, we demonstrate that the HSFCM-based immunoassay provides a rapid and sensitive method for determining protein copy number distribution in single mitochondria.
Co-reporter:Xiang Zhang, Shuyue Zhang, Shaobin Zhu, Sha Chen, Jinyan Han, Kaimin Gao, Jin-zhang Zeng, and Xiaomei Yan
Analytical Chemistry 2014 Volume 86(Issue 11) pp:5232
Publication Date(Web):May 13, 2014
DOI:10.1021/ac500918g
Mitochondria play a pivotal role in determining the point-of-no-return of the apoptotic process. Therefore, anticancer drugs that directly target mitochondria hold great potential to evade resistance mechanisms that have developed toward conventional chemotherapeutics. In this study, we report the development of an in vitro strategy to quickly identify the therapeutic agents that induce apoptosis via directly affecting mitochondria. This result is achieved by treating isolated mitochondria with potential anticancer compounds, followed by simultaneously measuring the side scatter and mitochondrial membrane potential (Δψm) fluorescence of individual mitochondria using a laboratory-built high-sensitivity flow cytometer. The feasibility of this method was tested with eight widely used anticarcinogens. Dose-dependent Δψm losses were observed for paclitaxel, antimycin A, betulinic acid, curcumin, ABT-737, and triptolide, but not for cisplatin or actinomycin D, which agrees well with their mechanisms of apoptosis induction reported in the literature. The as-developed method offers an effective approach to identify mitochondria-targeting anticancer compounds.
Co-reporter:Lina Wu, Tian Luan, Xiaoting Yang, Shuo Wang, Yan Zheng, Tianxun Huang, Shaobin Zhu, and Xiaomei Yan
Analytical Chemistry 2014 Volume 86(Issue 1) pp:907
Publication Date(Web):December 4, 2013
DOI:10.1021/ac403572z
Advanced methods are urgently needed to determine the identity and viability of trace amounts of pathogenic bacteria in a short time. Existing approaches either fall short in the accurate assessment of microbial viability or lack specificity in bacterial identification. Bacteriophages (or phages for short) are viruses that exclusively infect bacterial host cells with high specificity. As phages infect and replicate only in living bacterial hosts, here we exploit the strategy of using tetracysteine (TC)-tagged phage in combination with biarsenical dye to the discriminative detection of viable target bacteria from dead target cells and other viable but nontarget bacterial cells. Using recombinant M13KE-TC phage and Escherichia coli ER2738 as a model system, distinct differentiation between individual viable target cells from dead target cells was demonstrated by flow cytometry and fluorescence microscopy. As few as 1% viable E. coli ER2738 can be accurately quantified in a mix with dead E. coli ER2738 by flow cytometry. With fluorescence microscopic measurement, specific detection of as rare as 1 cfu/mL original viable target bacteria was achieved in the presence of a large excess of dead target cells and other viable but nontarget bacterial cells in 40 mL artificially contaminated drinking water sample in less than 3 h. This TC-phage-FlAsH approach is sensitive, specific, rapid, and simple, and thus shows great potential in water safety monitoring, health surveillance, and clinical diagnosis of which trace detection and identification of viable bacterial pathogens is highly demanded.
Co-reporter:Dr. Chengwen Yang;Sha Chen;Mi Zhou;Dr. Yan Li;Yangfeng Li;Zhengxi Zhang;Dr. Zhen Liu;Dr. Qian Ba;Dr. Jingquan Li; Hui Wang; Xiaomei Yan; Dawei Ma; Renxiao Wang
ChemMedChem 2014 Volume 9( Issue 7) pp:1436-1452
Publication Date(Web):
DOI:10.1002/cmdc.201400058
Abstract
Antiapoptotic Bcl-2 family proteins, such as Bcl-xL, Bcl-2, and Mcl-1, are often overexpressed in tumor cells, which contributes to tumor cell resistance to chemotherapies and radiotherapies. Inhibitors of these proteins thus have potential applications in cancer treatment. We discovered, through structure-based virtual screening, a lead compound with micromolar binding affinity to Mcl-1 (inhibition constant (Ki)=3 μM). It contains a phenyltetrazole and a hydrazinecarbothioamide moiety, and it represents a structural scaffold not observed among known Bcl-2 inhibitors. This work presents the structural optimization of this lead compound. By following the scaffold-hopping strategy, we have designed and synthesized a total of 82 compounds in three sets. All of the compounds were evaluated in a fluorescence-polarization binding assay to measure their binding affinities to Bcl-xL, Bcl-2, and Mcl-1. Some of the compounds with a 3-phenylthiophene-2-sulfonamide core moiety showed sub-micromolar binding affinities to Mcl-1 (Ki=0.3–0.4 μM) or Bcl-2 (Ki≈1 μM). They also showed obvious cytotoxicity on tumor cells (IC50<10 μM). Two-dimensional heteronuclear single quantum coherence NMR spectra of three selected compounds, that is, YCW-E5, YCW-E10, and YCW-E11, indicated that they bind to the BH3-binding groove on Bcl-xL in a similar mode to ABT-737. Several apoptotic assays conducted on HL-60 cells demonstrated that these compounds are able to induce cell apoptosis through the mitochondrial pathway. We propose that the compounds with the 3-phenylthiophene-2-sulfonamide core moiety are worth further optimization as effective apoptosis inducers with an interesting selectivity towards Mcl-1 and Bcl-2.
Co-reporter:Shaobin Zhu, Ling Ma, Shuo Wang, Chaoxiang Chen, Wenqiang Zhang, Lingling Yang, Wei Hang, John P. Nolan, Lina Wu, and Xiaomei Yan
ACS Nano 2014 Volume 8(Issue 10) pp:10998
Publication Date(Web):October 9, 2014
DOI:10.1021/nn505162u
Ultrasensitive detection and characterization of single nanoparticles (<100 nm) is important in nanotechnology and life sciences. Direct measurement of the elastically scattered light from individual nanoparticles represents the simplest and the most direct method for particle detection. However, the sixth-power dependence of scattering intensity on particle size renders very small particles indistinguishable from the background. Adopting strategies for single-molecule fluorescence detection in a sheathed flow, here we report the development of high sensitivity flow cytometry (HSFCM) that achieves real-time light-scattering detection of single silica and gold nanoparticles as small as 24 and 7 nm in diameter, respectively. This unprecedented sensitivity enables high-resolution sizing of single nanoparticles directly based on their scattered intensity. With a resolution comparable to that of TEM and the ease and speed of flow cytometric analysis, HSFCM is particularly suitable for nanoparticle size distribution analysis of polydisperse/heterogeneous/mixed samples. Through concurrent fluorescence detection, simultaneous insights into the size and payload variations of engineered nanoparticles are demonstrated with two forms of clinical nanomedicine. By offering quantitative multiparameter analysis of single nanoparticles in liquid suspensions at a throughput of up to 10 000 particles per minute, HSFCM represents a major advance both in light-scattering detection technology and in nanoparticle characterization.Keywords: flow cytometry; light scattering; nanomedicine; nanoparticle characterization; single-molecule detection; single-nanoparticle detection; size distribution;
Co-reporter:Chaoxiang Chen, Shaobin Zhu, Tianxun Huang, Shuo Wang and Xiaomei Yan
Analytical Methods 2013 vol. 5(Issue 9) pp:2150-2157
Publication Date(Web):07 Mar 2013
DOI:10.1039/C3AY40219C
Liposomes or phospholipid vesicles are one of the most versatile nanoparticles used to convey drugs, vaccines, genes, enzymes, or other substances to target cells and as a model to mimic biological membranes. To fulfil their roles in drug delivery and biotechnology, the physical and chemical properties of liposomes, such as size, shape, chemical composition, lamellarity, encapsulation efficiency of cargo molecules, and the density of proteins reconstituted in the membrane, need to be characterized to ensure reproducible preparation of the vesicles. Compared to bulk analysis, techniques focusing on the individual analysis of liposomes can reveal heterogeneity that is otherwise masked by ensemble averaging. Herein, we review the recent advances in techniques for single-liposome characterization.
Co-reporter:Qing Shao;Yan Zheng;Xueming Dong; Kai Tang; Xiaomei Yan; Bengang Xing
Chemistry - A European Journal 2013 Volume 19( Issue 33) pp:10903-10910
Publication Date(Web):
DOI:10.1002/chem.201301654
Abstract
Bacterial resistance to antibiotics poses a great clinical challenge in fighting serious infectious diseases due to complicated resistant mechanisms and time-consuming testing methods. Chemical reaction-directed covalent labeling of resistance-associated bacterial proteins in the context of a complicated environment offers great opportunity for the in-depth understanding of the biological basis conferring drug resistance, and for the development of effective diagnostic approaches. In the present study, three fluorogenic reagents LRBL1–3 for resistant bacteria labeling have been designed and prepared on the basis of fluorescence resonance energy transfer (FRET). The hydrolyzed probes could act as reactive electrophiles to attach the enzyme, β-lactamase, and thus facilitated the covalent labeling of drug resistant bacterial strains. SDS electrophoresis and MALDI-TOF mass spectrometry characterization confirmed that these probes were sensitive and specific to β-lactamase and could therefore serve for covalent and localized fluorescence labeling of the enzyme structure. Moreover, this β-lactamase-induced covalent labeling provides quantitative analysis of the resistant bacterial population (down to 5 %) by high resolution flow cytometry, and allows single-cell detection and direct observation of bacterial enzyme activity in resistant pathogenic species. This approach offers great promise for clinical investigations and microbiological research.
Co-reporter:Lingling Yang, Tianxun Huang, Shaobin Zhu, Yingxing Zhou, Yunbin Jiang, Shuo Wang, Yuqing Chen, Lina Wu, Xiaomei Yan
Biosensors and Bioelectronics 2013 Volume 48() pp:49-55
Publication Date(Web):15 October 2013
DOI:10.1016/j.bios.2013.03.078
•Absolute quantification of low copy number β-gal in single bacterial cells is reported.•Distributions of basal and near-basal expression of β-gal were obtained in high-throughput.•This method was developed upon a laboratory-built high sensitivity flow cytometer.•This method takes advantage of the good cell retention of fluorogenic C12FDG.Single-cell analysis is vital in providing insights into the heterogeneity in molecular content and phenotypic characteristics of complex or clonal cell populations. As many essential proteins and most transcription factors are produced at a low copy number, analytical tools with superior sensitivity to enable the analysis of low abundance proteins in single cells are in high demand. β-galactosidase (β-gal) has been the standard cellular reporter for gene expression in both prokaryotic and eukaryotic cells. Here we report the development of a high-throughput method for the single-cell analysis of low copy number β-gal proteins using a laboratory-built high-sensitivity flow cytometer (HSFCM). Upon fluorescence staining with a fluorogenic substrate, quantitative measurements of the basal and near-basal expression of β-gal in single Escherichia coli BL21(DE3) cells were demonstrated. Statistical distribution can be determined quickly by analyzing thousands of individual cells in 1–2 min, which reveals the heterogeneous expression pattern that is otherwise masked by the ensemble analysis. Combined with the quantitative fluorometric assay and the rapid bacterial enumeration by HSFCM, the β-gal expression distribution profile could be converted from arbitrary fluorescence units to protein copy numbers per cell. The sensitivity and speed of the HSFCM offers great capability in quantitative analysis of low abundance proteins in single cells, which would help gaining a deeper insight into the heterogeneity and fundamental biological processes in microbial populations.Graphical abstract
Co-reporter:Lingling Yang, Yingxing Zhou, Shaobin Zhu, Tianxun Huang, Lina Wu, and Xiaomei Yan
Analytical Chemistry 2012 Volume 84(Issue 3) pp:1526
Publication Date(Web):January 6, 2012
DOI:10.1021/ac2031332
Cellular autofluorescence can affect the sensitivity of fluorescence microscopic or flow cytometric assays by interfering with or even precluding the detection of low-level specific fluorescence. Here we developed a method to detect and quantify bacterial autofluorescence in the green region of the spectrum at the single-cell level using a laboratory-built high-sensitivity flow cytometer (HSFCM). The detection of the very weak bacterial autofluorescence was confirmed by analyzing polystyrene beads of comparable and larger size than bacteria in parallel. Dithionite reduction and air re-exposure experiments verified that the green autofluorescence mainly originates from endogenous flavins. Bacterial autofluorescence was quantified by calibrating the fluorescence intensity of nanospheres with known FITC equivalents, and autofluorescence distribution was generated by analyzing thousands of bacterial cells in 1 min. Among the eight bacterial strains tested, it was found that bacterial autofluorescence can vary from 80 to 1400 FITC equivalents per cell, depending on the bacterial species, and a relatively large cell-to-cell variation in autofluorescence intensity was observed. Quantitative measurements of bacterial autofluorescence provide a reference for the background signals that can be expected with bacteria, which is important in guiding studies of low-level gene expression and for the detection of low-abundance biological molecules in individual bacterial cells. This paper presents the first quantification of bacterial autofluorescence in FITC equivalents.
Co-reporter:Shuyue Zhang, Shaobin Zhu, Lingling Yang, Yan Zheng, Min Gao, Shuo Wang, Jin-zhang Zeng, and Xiaomei Yan
Analytical Chemistry 2012 Volume 84(Issue 15) pp:6421
Publication Date(Web):July 9, 2012
DOI:10.1021/ac301464x
Mitochondria are one of the most important organelles responsible for cellular energy metabolism and apoptosis regulation. However, single-mitochondrion analysis is challenging, because of their small sizes and the low content of organelle constituents. Here, we report the development of a sensitive and versatile platform for high-throughput multiparameter analysis of individual mitochondria. Employing specific fluorescent staining with a laboratory-built high-sensitivity flow cytometer (HSFCM), we demonstrate the simultaneous detection of side scatter, cardiolipin, and mitochondria DNA (mtDNA) of a single mitochondrion. Simultaneous measurements of side scatter, porin, and cytochrome c of individual mitochondria are reported for the first time. Correlation analysis among multiple attributes on an organelle-by-organelle basis could provide a more definitive assessment of the purity, structure integrity, and apoptosis-related proteins of isolated mitochondria than bulk measurement. This work represents a significant advancement in single-mitochondrion analysis. We believe that the HSFCM holds great potential for studying apoptotic signal transduction pathways at the single-mitochondrion level.
Co-reporter:Huan Huang;Zhijun Chen
Journal of Separation Science 2012 Volume 35( Issue 3) pp:436-444
Publication Date(Web):
DOI:10.1002/jssc.201100778
Abstract
This article describes the development of a rapid, simple, and sensitive analytical approach for the simultaneous determination of serotonin (5-hydroxytryptamine) and creatinine in urine samples by combining two ultrasound-assisted emulsification microextractions (USAEMEs) in series with on-column stacking in CE. This serial USAEME procedure comprises analytes extraction from the donor solution (urine with K2CO3 additive) to an organic solvent followed by a back-extraction from the organic phase into a small volume of hydrochloric acid. After 15 min of sample pretreatment, the acidic acceptor solution was analyzed directly on CE in the mode of capillary zone electrophoresis. The adoption of HCl as the acceptor phase not only provided effective back-extraction but also facilitated pH-mediated on-column stacking in CE analysis. About 360-fold sensitivity enhancement was achieved for serotonin detection. The limits of detection were 7.9 nM for serotonin and 13.3 μM for creatinine, respectively. Satisfactory results were obtained with respect to precision and recovery. The proposed method has been demonstrated to be convenient and effective for the analysis of real urine samples. We believe that two USAEMEs in series will find wide applications in simplified sample pretreatment prior to CE analysis.
Co-reporter:Dr. Lina Wu;Tingting Huang;Lingling Yang;Jianbo Pan;Shaobin Zhu ;Dr. Xiaomei Yan
Angewandte Chemie 2011 Volume 123( Issue 26) pp:5995-5999
Publication Date(Web):
DOI:10.1002/ange.201100334
Co-reporter:Dr. Lina Wu;Tingting Huang;Lingling Yang;Jianbo Pan;Shaobin Zhu ;Dr. Xiaomei Yan
Angewandte Chemie International Edition 2011 Volume 50( Issue 26) pp:5873-5877
Publication Date(Web):
DOI:10.1002/anie.201100334
Co-reporter:Yan Zheng, Lin Lin, Wei Hang, Xiaomei Yan, Babetta L. Marrone
Talanta 2011 Volume 85(Issue 1) pp:638-643
Publication Date(Web):15 July 2011
DOI:10.1016/j.talanta.2011.04.037
Studying metal–biomolecule interactions is critical to the elucidation of the molecular basis of the biological functions and toxicity of metals. In the present study, a competitive fluorimetric approach has been developed to measure the apparent affinity of biomolecules for Be2+ by using a Be2+-specific fluorigenic probe (10-hydroxybenzo[h]quinoline-7-sulfonate, HBQS). Under physiological conditions, HBQS coordinates with Be2+ in a molar ratio of 1:1 and results in a fluorescence shift from 580 nm for HBQS to 480 nm for the Be–HBQS complex associated with significant fluorescence enhancement. When a beryllium ligand is present in the mixture of Be2+ and HBQS, the competition of ligand against HBQS for beryllium ion binding results in dissociation and thus a fluorescence decrease of the Be–HBQS complex. By titrating ligand and monitoring the dose-dependent decrease of Be–HBQS complex fluorescence at 480 nm, the apparent affinity between ligand and Be2+ can be derived. Applying this simple approach, the apparent affinities of various nucleotides and the iron-storage protein ferritin for beryllium ion have been determined. In particular, the apparent dissociation constant of Be2+ and adenosine 5′-triphosphate (ATP) was also validated by an electrospray ionization mass spectrometric (ESI-MS) method. The general applicability of the proposed competition assay was further demonstrated using FluoZin-1, a zinc fluorescent indicator, in a binding study for Zn2+ and bovine serum albumin. This newly developed competitive fluorimetric assay provides a sensitive, simple, and generic approach for affinity estimation of metal and biomolecule binding.
Co-reporter:ShaoBin Zhu;Shuo Wang;LingLing Yang;TianXun Huang
Science China Chemistry 2011 Volume 54( Issue 8) pp:
Publication Date(Web):2011 August
DOI:10.1007/s11426-011-4313-z
Nanoparticles have recently attracted extensive attention in view of their great potential in biomedicine and bioanalytical applications. Single particle detection via light scattering offers a simple and efficient approach for the size, size distribution, and concentration analysis of nanoparticles. In particular, intrinsic heterogeneity or rare events masked by ensemble averaging can be revealed. However, the sixth power dependence of Rayleigh scattering on particle size makes it very challenging to detect individual nanoparticles of small sizes. This article is intended to provide an overview of recent progress in the development of techniques based on light scattering for the detection of single nanoparticles.
Co-reporter:Shaobin Zhu ; Lingling Yang ; Yao Long ; Min Gao ; Tianxun Huang ; Wei Hang
Journal of the American Chemical Society 2010 Volume 132(Issue 35) pp:12176-12178
Publication Date(Web):August 13, 2010
DOI:10.1021/ja104052c
Employing single nanoparticle detection with a laboratory-built high-sensitivity flow cytometer, we developed a simple and versatile platform that is capable of detecting the surface plasmon resonance scattering of gold nanoparticles (GNPs) as small as 24 nm, differentiating GNPs of different sizes, and providing accurate quantification of GNPs. Low-concentration samples (fM to pM) in small volumes (μL) can be measured in minutes with an analysis rate of up to 100−200 GNPs per second. Among these features, absolute quantification provides a distinct advantage because it does not require standard samples.
Co-reporter:Zhiling Zhang, Yao Long, Jianbo Pan and Xiaomei Yan
Journal of Materials Chemistry A 2010 vol. 20(Issue 6) pp:1179-1185
Publication Date(Web):15 Dec 2009
DOI:10.1039/B919955A
Fluorescence-encoded microspheres are widely used in the detection and analysis of biological molecules, especially in suspension arrays. Here, we report an efficient strategy for the preparation of fluorescence-encoded polystyrene microspheres with desirable optical and surface properties. The micron-sized, monodisperse polystyrene seed beads were first synthesized by dispersion polymerization. Then, dye molecules and carboxyl functional groups were copolymerized on the surface of the seed beads by forming a core–shell structure. Rhodamine 6G (R6G) was used as a model dye molecule to prepare the fluorescent beads, and the fluorescence intensity of the beads can be precisely controlled by adjusting the quantity of R6G. These fluorescent beads were characterized by environmental scanning electron microscopy, laser scanning confocal microscopy, and spectrofluorometry. The differences of the fluorescence spectra between fluorescent beads and R6G in solution were investigated. Twelve kinds of fluorescent beads encoded with different R6G fluorescence intensities were prepared, and they can be clearly distinguished on a conventional flow cytometer. Furthermore, the encoded beads are stable in water and resistant to photobleaching, which is crucial for their potential applications in diagnostic assays and imaging. Detection of human alpha fetoprotein antigen via a sandwich microsphere-based immunoassay yielded a detection limit of 80 pg mL−1, demonstrating that the fluorescence-encoded microspheres synthesized herein are efficient in serving as the microcarriers in suspension arrays. As both the encoding and functionalizing procedures are made simultaneously, the newly designed technique is extremely simple and time-saving. Moreover, it could be readily applicable to the preparation of a wide size range of fluorescent particles made by polymerization.
Co-reporter:Lingling Yang, Lina Wu, Shaobin Zhu, Yao Long, Wei Hang and Xiaomei Yan
Analytical Chemistry 2010 Volume 82(Issue 3) pp:1109
Publication Date(Web):December 29, 2009
DOI:10.1021/ac902524a
This paper describes a rapid and sensitive strategy for the absolute and simultaneous quantification of specific pathogenic strain and total bacterial cells in a mixture. A laboratory-built compact, high-sensitivity, dual channel flow cytometer (HSDCFCM) was modified to enable dual fluorescence detection. A bacterial cell mixture comprising heat-killed pathogenic Escherichia coli E. coli O157:H7 and harmless E. coli DH5α was used as a model system. Pathogenic E. coli O157:H7 cells were selectively labeled by red fluorescent probe via antibody−antigen interaction, and all bacterial cells were stained with membrane-permeable nucleic acid dye that fluoresces green. When each individual bacterium passes through the interrogating laser beam, E. coli O157:H7 emits both red and green fluorescence, while E. coli DH5α exhibits only green fluorescence. Because the fluorescence burst generated from each individual bacterial cell was easily distinguished from the background, accurate enumeration and consequently absolute quantification were achieved for both pathogenic and total bacterial cells. By using this strategy, accurate counting of bacteria at a density above 1.0 × 105 cells/mL can be accomplished with 1 min of data acquisition time after fluorescent staining. Excellent correlation between the concentrations measured by the HSDCFCM and the conventional plate-counting method were obtained for pure-cultured E. coli O157:H7 (R2 = 0.9993) and E. coli DH5α (R2 = 0.9998). Bacterial cell mixtures with varying proportions of E. coli O157:H7 and E. coli DH5α were measured with good ratio correspondence. We applied the established approach to detecting artificially contaminated drinking water samples; E. coli O157:H7 of 1.0 × 102 cells/mL were accurately quantified upon sample enrichment. It is believed that the proposed method will find wide applications in many fields demanding bacterial identification and quantification.
Co-reporter:Yao Long, Zhiling Zhang, Xiaomei Yan, Jinchun Xing, Kaiyan Zhang, Jingxiong Huang, Jiaxin Zheng, Wei Li
Analytica Chimica Acta 2010 Volume 665(Issue 1) pp:63-68
Publication Date(Web):14 April 2010
DOI:10.1016/j.aca.2010.03.009
A new suspension array built upon laboratory-prepared functional fluorescence-encoded polystyrene beads (FFPBs) was developed for multiplex immunodetection of tumor markers. The FFPBs were synthesized by copolymerizing rhodamine 6G (R6G) and carboxyl function groups on the surface of the seed beads forming a core–shell structure. The fabrication process was facile and the encoding fluorescence intensity of the beads can be precisely controlled by adjusting the quantity of R6G. In present work, we demonstrated that the quantity variation of impregnated R6G had negligible effect on the coupling efficiency of biomolecules onto the surface of the FFPBs. The R6G encoding fluorescence remained good monodispersity upon capture probe coupling and immunocomplex formation. No fluorescence resonance energy transfer was observed between the R6G doped in the bead shell and fluorophore used for antibody labeling. Under the optimal conditions, the proposed suspension array allowed simultaneous detection of α-fetoprotein, carcinoembryonic antigen, and prostate specific antigen in the ranges of 0.07–500 ng mL−1, 1–2000 ng mL−1, and 0.5–500 ng mL−1, respectively, with detection limits of 0.0626 ng mL−1, 0.554 ng mL−1, and 0.250 ng mL−1. Test on clinical serum samples demonstrated that the results obtained with suspension array were in good agreement with those of the reference electrochemiluminescence immunoassay method. We conclude that the laboratory-made FFPBs are sufficient as the microcarrier for the construction of suspension array in clinical diagnosis.
Co-reporter:Lingling Yang, Shaobin Zhu, Wei Hang, Lina Wu and Xiaomei Yan
Analytical Chemistry 2009 Volume 81(Issue 7) pp:2555
Publication Date(Web):March 4, 2009
DOI:10.1021/ac802464a
A compact, high-sensitivity, dual-channel flow cytometer (HSDCFCM) was developed for the individual analysis of nanosized particles and biomolecules. A hydrodynamic focusing technique was applied to confine the sample stream and enable small probe volume. Fluorescence bursts from single R-phycoerythrin (R-PE) molecules passing through the laser beam were well resolved from the background with signal-to-noise ratio of 17. Excellent size discrimination was demonstrated with a mixture of three sizes of polystyrene nanoparticles. Simultaneous measurement of fluorescence and light scattering signals from individual nanoparticles was demonstrated with the 100 nm fluorescent latex beads. Doxorubicin-loaded ZrO2 nanoparticles and fluorescently stained Escherichia coli ER2738 cells were analyzed successfully with dual-channel detection. Particle counting is demonstrated with the 210 nm fluorescent latex beads, and excellent correlation (R2 > 0.998) between the manufacturer-reported concentrations and those measured by HSDCFCM enumeration was obtained. The measured sample detection efficiency was ∼90% on average for particle concentrations ranging from 1.62 × 105 to 3.93 × 107 particles/mL. Sample mixtures with varying proportions of fluorescently labeled and unlabeled nanoparticles were also analyzed with good ratio correspondence. By providing rapid, quantitative, and multiparameter characterization of nanoparticles, it is believed that the HSDCFCM will find many applications in the fields of bionanotechnology, bioanalytical chemistry, and biomedicine.
Co-reporter:Jian Ding, Lin Lin, Wei Hang and Xiaomei Yan
Metallomics 2009 vol. 1(Issue 6) pp:471-478
Publication Date(Web):14 Sep 2009
DOI:10.1039/B913265A
Investigation of cellular uptake of metal compounds is important in understanding metal-related toxicity and diseases. Inhalation of beryllium aerosols can cause chronic beryllium disease, a progressive, granulomatous fibrosis of the lung. Studies in laboratory animals and cultured animal cells indicate that alveolar macrophages take up beryllium compounds and participate in a hypersensitivityimmune response to a beryllium-containing antigen. In the present work, human monocytecell line THP-1 was induced with phorbol myristate acetate to differentiate into a macrophage. This cell with characteristics of human alveolar macrophages was employed to study cellular beryllium uptake and related biological effects. Morphological changes, phagocytosis of fluorescent latex beads, and cell surface CD14 expression were used to verify the successful differentiation of THP-1 monocytes into macrophages. An improved mass spectrometry method for quantitative analysis of intracellular beryllium as opposed to the traditional radioisotopic approach was developed using ICP-MS. The influence of the solubility of beryllium compounds, exposure duration, and beryllium concentration on the incorporation of beryllium was studied. Our data indicated that the uptake of particulate BeO was much more significant than that of soluble BeSO4, suggesting the major cellular uptake pathway is phagocytosis. Nevertheless, subsequent DAPI nuclear staining and PARP cleavage study indicated that beryllium uptake had a negligible effect on the apoptosis of THP-1 macrophages compared to the unstimulated macrophage control. Meanwhile, no substantial variation of tumour necrosis factor-alpha production was observed for THP-1 macrophages upon beryllium exposure. These data imply alveolar macrophages could have some level of tolerance to beryllium and this may explain why most Be-exposed individuals remain healthy throughout life.
Co-reporter:Zhijun Chen and Xiaomei Yan
Journal of Agricultural and Food Chemistry 2009 Volume 57(Issue 19) pp:8742-8747
Publication Date(Web):September 17, 2009
DOI:10.1021/jf9021916
This article describes the development of a simple analytical approach for the simultaneous determination of melamine and 5-hydroxymethylfurfural (HMF) in milk samples using capillary electrophoresis (CE) with diode array detection (DAD) for the first time. Ultraviolet absorption at wavelengths of 214 and 280 nm was applied for the detection of melamine and HMF, respectively. Milk samples were extracted with 1% trichloroacetic acid using a high-speed blender and ultrasonication. After centrifugation and filtration, the extract was analyzed by CE-DAD directly. Micellar electrokinetic capillary chromatography was employed as the separation mode by adding sodium dodecyl sulfate (SDS) to the electrolyte. Under optimal separation conditions, melamine, HMF, and interferents were well resolved. The linear dynamic ranges were 0.05−100 μg/mL for melamine (R2 = 0.9996) and 0.1−100 μg/mL for HMF (R2 = 0.9997). The assay detection limits were 0.047 μg/mL and 0.067 μg/mL for melamine and HMF, respectively. Satisfactory results were obtained for the assay recovery rate and repeatability. The proposed method was successfully applied for the analysis of melamine and HMF in real milk samples, and the results of melamine were comparable to those obtained using HPLC-UV reference method.
Co-reporter:
Analytical Methods (2009-Present) 2013 - vol. 5(Issue 9) pp:
Publication Date(Web):
DOI:10.1039/C3AY40219C
Liposomes or phospholipid vesicles are one of the most versatile nanoparticles used to convey drugs, vaccines, genes, enzymes, or other substances to target cells and as a model to mimic biological membranes. To fulfil their roles in drug delivery and biotechnology, the physical and chemical properties of liposomes, such as size, shape, chemical composition, lamellarity, encapsulation efficiency of cargo molecules, and the density of proteins reconstituted in the membrane, need to be characterized to ensure reproducible preparation of the vesicles. Compared to bulk analysis, techniques focusing on the individual analysis of liposomes can reveal heterogeneity that is otherwise masked by ensemble averaging. Herein, we review the recent advances in techniques for single-liposome characterization.
Co-reporter:Zhiling Zhang, Yao Long, Jianbo Pan and Xiaomei Yan
Journal of Materials Chemistry A 2010 - vol. 20(Issue 6) pp:NaN1185-1185
Publication Date(Web):2009/12/15
DOI:10.1039/B919955A
Fluorescence-encoded microspheres are widely used in the detection and analysis of biological molecules, especially in suspension arrays. Here, we report an efficient strategy for the preparation of fluorescence-encoded polystyrene microspheres with desirable optical and surface properties. The micron-sized, monodisperse polystyrene seed beads were first synthesized by dispersion polymerization. Then, dye molecules and carboxyl functional groups were copolymerized on the surface of the seed beads by forming a core–shell structure. Rhodamine 6G (R6G) was used as a model dye molecule to prepare the fluorescent beads, and the fluorescence intensity of the beads can be precisely controlled by adjusting the quantity of R6G. These fluorescent beads were characterized by environmental scanning electron microscopy, laser scanning confocal microscopy, and spectrofluorometry. The differences of the fluorescence spectra between fluorescent beads and R6G in solution were investigated. Twelve kinds of fluorescent beads encoded with different R6G fluorescence intensities were prepared, and they can be clearly distinguished on a conventional flow cytometer. Furthermore, the encoded beads are stable in water and resistant to photobleaching, which is crucial for their potential applications in diagnostic assays and imaging. Detection of human alpha fetoprotein antigen via a sandwich microsphere-based immunoassay yielded a detection limit of 80 pg mL−1, demonstrating that the fluorescence-encoded microspheres synthesized herein are efficient in serving as the microcarriers in suspension arrays. As both the encoding and functionalizing procedures are made simultaneously, the newly designed technique is extremely simple and time-saving. Moreover, it could be readily applicable to the preparation of a wide size range of fluorescent particles made by polymerization.
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