As an emerging fascinating fluorescent nanomaterial, carbon nanodots (CDs) have attracted much attention owing of their unique properties such as small size, antiphotobleaching, and biocompatibility. However, its use in biomedical analysis is limited because of its low quantum yield. Herein, we constructed a dual amplification fluorescence sensor by incorporating immunohybridization chain reaction (immuno-HCR) and metal-enhanced fluorescence (MEF) of CDs for the detection of alpha fetal protein (AFP). The immunoplasmonic slide and detection antibodies-conjugated oligonucleotide initiator are served to capture and probe AFP molecules, respectively. Then, CD-tagged hairpin nucleic acids were introduced to trigger the HCR, in which the hairpin nucleic acid and oligonucleotide initiator are complementary. The interaction between CDs and the gold nanoisland film greatly improves the radiative decay rate, increases the quantum yield, and enhances the fluorescence emission of the CDs. Furthermore, the HCR provides secondary amplification of fluorescence intensity. Therefore, the MEF-capable immunohybridization reactions provide obvious advantages and result in exceptional sensitivity. In addition, the sandwich immunoassay method offers high specificity. The results show a wide linearity between the fluorescence intensity and AFP concentration over 5 orders of magnitude (0.0005–5 ng/mL), and the detection limit reaches as low as 94.3 fg/mL. This method offers advantages of high sensitivity and reliability, wide detection range, and versatile plasmonic chips, thus presenting an alternative for the technologies in biomedical analysis and clinical applications.Keywords: AFP; CDs; HCR; MEF; sandwich immunoassays;

Patients with prostate cancer and systemic lupus erythematosus exhibit reduced DNase I activity, and patients with myocardial infarction exhibit increased DNase I activity. So the assay of DNase I is of high importance. A colorimetric assay is described here for the determination of the activity of DNase. It is based on strand scission of dsDNA as catalyzed by DNase I. The products of digestion (nucleoside monophosphates) can better stabilize citrate capped AuNPs than dsDNA. In the absence of DNase I, the AuNPs aggregate in presence of NaCl and then display a blue color. In the presence of the analyte (DNase I), AuNPs do not aggregate but rather remain dispersed and display a red color. These findings were exploited to design a DNase I activity assay that is based on the measurement of ratio of absorbances at 520 nm (red) to 650 nm (blue). The detection limit for DNase I activity is found to be 7.1 U⋅L−1. In our perception, this assay has a large potential with respect to diagnoses of DNase I activity-related diseases and in drug screening.

We report a single-microsphere based imaging assay method by integrating up-converting luminescence with optical tweezers for detecting microRNA-21 sequences. This method achieves a competitive detection limit of 12 fM with good selectivity and no dedicated signal amplification designs.

A method for fluorescent sensing of thrombin was developed by using fluorescent silica nanoparticles as the fluorescence signal probes and magnetic beads as target enriching platform. RuBpy-doped silica nanoparticles are highly photostable and provide significant fluorescent signal amplification as compared with single dye molecules. TBA1 is a 15-mer DNA aptamer which binds exosite I of thrombin (Fibrinogen Binding Site), while TBA2 is a 29-mer DNA aptamer binding to exosite II of thrombin (Heparin Binding Domain). In this work, 15-mer thrombin-binding aptamer (15-A) was immobilized onto the surface of RuBpy-doped silica nanoparticles, and 29-mer thrombin-binding aptamer (29-A) was immobilized onto the surface of magnetic beads, and sandwich aptamer complexes were formed upon the addition of target thrombin molecules with the two kinds of aptamer-modified particles, and therefore quantitative analysis of thrombin was performed by detecting the fluorescence intensity of the complexes after magnetic separation of the targets. The results show that the detection limit of thrombin is 0.70 nM, and this method has some advantages such as speed, high sensitivity, high specificity and low cost.

•An analytical setup based on NIR optical trapping and two-photon fluorescence was constructed.•Quantitative detection was realized by trapping single beads with fluorescence imaging.•One-step separation-free detection of biomarker CEA in human whole serum was conducted.•The assay for real samples is concordant with the results by standard chemiluminescence method.Direct analysis of biomolecules in complex biological samples remains a major challenge for fluorescence-based approaches due to the interference of background signals. Herein, we report an analytical methodology by exploiting a single low-cost near-infrared sub-nanosecond pulse laser to synchronously actualize optical trapping and two-photon excitation fluorescence for senstive detection of carcinoembryonic antigen (CEA) in buffer solution and human whole serum with no separation steps. The assay is performed by simultaneously trapping and exciting the same immune-conjugated microsphere fabricated with a sandwich immunization strategy. Since the signal is strictly limited in the region of a three-dimensional focal volume where the microsphere is trapped, no obvious background signal is found to contribute the detected signals and thus high signal-to-background data are obtained. As a proof-of-concept study, the constructed platform exhibits good specificity for CEA and the detection limit reaches as low as 8 pg/mL (45 fM) with a wide linear range from 0.01 to 60 ng/mL in the both cases. To investigate the potential application of this platform in clinical diagnosis, 15 cases of serum samples were analyzed with satisfactory results, which further confirm the applicability of this method.

We present an analytical platform by combining near-infrared optical tweezers with two-photon excitation for fluorescence detection of H5N1 virus gene sequences. A heterogeneous enrichment strategy, which involved polystyrene (PS) microsphere and quantum dots (QDs), was adopted. The final hybrid-conjugate microspheres were prepared by a facile one-step hybridization procedure by using PS microspheres capturing target DNA and QDs tagging, respectively. Quantitative detection was achieved by the optical tweezers setup with a low-cost 1064 nm nanosecond pulse laser for both optical trapping and two-photon excitation for the same hybrid-conjugate microsphere. The detection limits for both neuraminidase (NA) gene sequences and hemagglutinin (HA) gene sequences are 16–19 pM with good selectivity for one-base mismatch, which is approximately 1 order of magnitude lower than the most existing fluorescence-based analysis method. Besides, because of the fact that only signal from the trapped particle is detected upon two-photon excitation, this approach showed extremely low background in fluorescence detection and was successfully applied to directly detect target DNA in human whole serum without any separation steps and the corresponding results are very close to that in buffer solution, indicating the strong anti-interference ability of this method. Therefore, it can be expected to be an emerging alternative for straightforward detecting target species in complex samples with a simple procedure and high-throughput.

We explore the effect of sufficient GO on the property of a dye labeled adenosine 5′-triphosphate (ATP) aptamer (P) which shows similar affinity and specificity for ATP and its analogues including adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), and adenosine (AD). It is found that ATP and its analogues give rise to fluorescence recovery of GO-quenched P to a different extent (in the order of ATP > AD > ADP > AMP), and the difference becomes larger when increasing the concentration of GO in a certain range, implying an improvement of specificity of the ATP aptamer. Based on this finding, a fluorescence turn-on assay for alkaline phosphatase (ALP) and creatine kinase (CK) is proposed, by using AMP and ADP as the substrate, respectively. Specifically, the GO-quenched P system containing substrate shows low fluorescence intensity. In the presence of target enzyme, the substrate is converted into either AD or ATP which have higher affinity with P, resulting in stronger fluorescence of the mixture of P and GO. The entire assay is sensitive and selective. More importantly, the ability of GO with suitable concentration to improve the specificity of aptamers not only offers an exciting new way to detect protease, but also is valuable for developing the application of GO and aptamers in the biosensing field and is expected to be used in aptamer screening systems, to improve the specificity of screened aptamers.

•A label-free aptamer-based biosensor was developed.•This approach employs dsDNA/graphene oxide (GO) as the indicator probe.•This method is expected to promote the application of dsDNA/GO complex in biomedicine.We developed a fluorescent aptasensor based on the making use of double-stranded DNA (dsDNA)/graphene oxide (GO) as the signal probe and the activities of exonuclease I (Exo I). This method takes advantage of the stronger affinity of the aptamer to its target rather than to its complementary sequence (competitor), and the different interaction intensity of dsDNA, mononucleotides with GO. Specifically, in the absence of target, the competitor hybridizes with the aptamer, preventing the digestion of the competitor by Exo I, and thus the formed dsDNA is adsorbed on GO surface, allowing fluorescence quenching. When the target is introduced, the aptamer preferentially binds with its target. Thereby, the corresponding nuclease reaction takes place, and slight fluorescence change is obtained after the introduction of GO due to the weak affinity of the generated mononucleotides to GO. Adenosine (AD) was chosen as a model system and tested in detail. Under the optimized conditions, smaller dissociation constant (Kd, 311.0 µM) and lower detection limit (LOD, 3.1 µM) were obtained in contrast with traditional dye-labeled aptamer/GO based platform (Kd=688.8 µM, LOD=21.2 µM). Satisfying results were still obtained in the evaluation of the specificity and the detection of AD in human serum, making it a promising tool for the diagnosis of AD-relevant diseases. Moreover, we demonstrated the effect of the competitor on the LOD, and the results reveal that the sensitivity could be enhanced by using the rational competitor. The present design not only constructs a label-free aptamer based platform but also extends the application of dsDNA/GO complex in biochemical and biomedical studies.

A novel strategy for the fabrication of a colorimetric aptasensor using label free gold nanoparticles (AuNPs) is proposed in this work, and the strategy has been employed for the assay of adenosine deaminase (ADA) activity. The aptasensor consists of adenosine (AD) aptamer, AD and AuNPs. The design of the biosensor takes advantage of the special optical properties of AuNPs and the interaction between AuNPs and single-strand DNA. In the absence of ADA, the AuNPs are aggregated and are blue in color under appropriate salt concentration because of the grid structure of an AD aptamer when binding to AD, while in the presence of the analyte, AuNPs remain dispersed with red color under the same concentration of salt owing to ADA converting AD into inosine which has no affinity with the AD aptamer, thus allowing quantitative investigation of ADA activity. The present strategy is simple, cost-effective, selective and sensitive for ADA with a detection limit of 1.526 U L−1, which is about one order of magnitude lower than that previously reported. In addition, a very low concentration of the inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) could generate a distinguishable response. Therefore, the AuNP-based colorimetric biosensor has great potential in the diagnosis of ADA-relevant diseases and drug screening.

A novel fluorescence “turn-on” strategy, which is based on the formation of Hg2+-mediated molecular-beacons (MBs), the preferable cleavage capacity of exonuclease III to double-stranded DNA compared with single-stranded one, and the remarkable difference in the binding ability of graphene oxide (GO) with single-stranded DNA and the mononucleotides, is designed for Hg2+ assay. The Hg2+-mediated base pairs facilitate the dye labeled MBs to fold into a hairpin structure, which is more likely to be digested by exonuclease III, and an obvious increase in the fluorescence intensity is observed after incubating with GO due to the weak affinity of the product-mononucleotides to GO. A fluorescent “turn-on” method based on graphene oxide and exonuclease III was designed for Hg2+ assay. The introduction of GO greatly increases the signal-to-background ratio, and the sensitivity is significantly improved due to the amplified capability of exonuclease III. Under the optimal conditions, Hg2+ is specifically and sensitively detected with a detection limit of 0.83 nM. Compared with the reported Hg2+ assay methods, the proposed strategy is simple, cost effective and selective, which might provide a new platform for developing a sensitive Hg2+ biosensor. Mercury level in the blood is an important indicator of mercury poisoning in clinical study. To testify the possibility of the use of this method for the assay of Hg2+ in real samples, detection of Hg2+ in 1% human serum was investigated and satisfactory results were obtained, which suggests that this method has great potential for bioanalysis.

•Fluorescent silica nanoprobes for rapid detection of pathogenic Escherichia coli O157:H7 were prepared.•Protein A was immobilized on the surface of the nanoparticles to sensitively recognize the pathogens.•Indirect immunofluorescence microscopy and flow cytometry were applied to detect the pathogens using the same nanoprobes.•The detection of E. Coli O157:H7 in buffer and bacterial mixture shows high sensitivity and specificity.A method of fluorescent nanoparticle-based indirect immunofluorescence assay using either fluorescence microscopy or flow cytometry for the rapid detection of pathogenic Escherichia coli O157:H7 was developed. The dye-doped silica nanoparticles (NPs) were synthesized using W/O microemulsion methods with the combination of 3-aminopropyltriethoxysilane (APTES) and fluorescein isothiocyanate (FITC) and polymerization reaction with carboxyethylsilanetriol sodium salt (CEOS). Protein A was immobilized at the surface of the NPs by covalent binding to the carboxyl linkers and the surface coverage of Protein A on NPs was determined by the Bradford method. Rabbit anti-E. Coli O157:H7 antibody was used as primary antibody to recognize E. coli O157:H7 and then antibody binding protein (Protein A) labeled with FITC-doped silica NPs (FSiNPs) was used to generate fluorescent signal. With this method, E. Coli O157:H7 in buffer and bacterial mixture was detected. In addition, E. coli O157:H7 in several spiked background beef samples were measured with satisfactory results. Therefore, the FSiNPs are applicable in signal-amplified bioassay of pathogens due to their excellent capabilities such as brighter fluorescence and higher photostability than the direct use of conventional fluorescent dyes.

Two different functional groups modified RuBpy-doped silica fluorescent nanoprobes Probe A and B that conjugated with avidin were prepared for the recognition of liver cancer cells. Firstly RuBpy-doped silica nanoparticles were synthesized by reverse microemulsion and then modified with different functional groups. Probe A was prepared by the conjugation of avidin with carboxyl modified nanoparticles through covalent binding using EDC/sulfo-NHS, whereas Probe B was prepared by the conjugation of avidin with the PEG linkers on the surface nanoparticles using cyanogen bromide method. Therefore, different from Probe A, Probe B was obtained by coupling avidin to the nanoparticles through long-chain PEG molecules. The two probes were incubated with liver cancer cells respectively, and microscopic fluorescence imaging showed that Probe B which contained PEG molecules was more effectively applied for the recognition of tumor marker carcinoembryonic antigen (CEA) in liver cancer cells.Two different functional groups modified RuBpy-doped silica fluorescent nanoprobes that conjugated with avidin were prepared for the recognition of liver cancer cells. The results showed that the probe which contained PEG molecules was more effectively applied for the recognition of tumor marker carcinoembryonic antigen (CEA) in liver cancer cells.

•A simple and sensitive method for multiplexed DNA detection using silica NPs as the platform was developed.•The binding capacity of the avidin-covered NPs for ligand biotin was quantified.•Capture DNA strands on the NPs for targets were measured using fluorescent dye hoechst33258.•The method is quite sensitive for one-base mismatched DNA sequences.We present a simple and sensitive method for multiplexed DNA detection by simultaneously capturing two different DNA sequences with a same silica nanoparticle (NP) through a sandwich mode. This biobarcode assay method was demonstrated by using oligonucleotide sequences of 64 bases associated with human papillomavirus (HPV) 16 and 18 L1 genes as model systems. The nonfluorescent carboxyl-modified silica NPs were prepared using water-in-oil (W/O) microemulsion methods. Avidin was immobilized on the surface of the NPs by covalent binding to the carboxyl linkers. The binding capacity of the avidin-covered NPs for ligand biotin was quantified and the results show that about 8 avidin molecules are bound to one nanoparticle. The silica nano-platforms were prepared through the biotin–avidin interaction and the amounts of capture DNA strands for HPV-16 and HPV-18 (C-16 and C-18, respectively) conjugated to the surface of the same NPs were measured using fluorescent dye hoechst33258. The calculated result shows that the amounts of conjugated C-16 and C-18 on 1 mg of NPs (9.2 pmol) are about 13.5 pmol and 15.5 pmol, respectively. A one-step hybridization reaction was performed by mixing the silica nano-platforms, HPV-16 and HPV-18 target DNA (T-16 and T-18), fluorescein amidite (FAM) or 6-carboxyl-X-rhodamine (Rox) labeled HPV-16 and HPV-18 probes. The hybrid-conjugated NPs were separated by centrifugation, and T-16 and T-18 were detected by measuring fluorescence signals of FAM and Rox respectively. The results show linear dependence of the fluorescence intensity on target DNA concentration in the range from 0.5 to 9 nM, and the detection limit (3σ) of T-16 and T-18 is 0.17 nM and 0.78 nM, respectively.

We report an indirect method for cancer cell recognition using photostable fluorescent silica nanoprobes as biological labels. The dye-doped fluorescent silica nanoparticles were synthesized using the water-in-oil (W/O) reverse microemulsion method. The silica matrix was produced by the controlled hydrolysis of tetraethylorthosilicate (TEOS) in water nanodroplets with the initiation of ammonia (NH3·H2O). Fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate conjugated with dextran (RBITC-Dextran) was doped in silica nanoparticles (NPs) with a size of 60 ± 5 nm as a fluorescent signal element by covalent bonding and steric hindrance, respectively. The secondary antibody, goat anti-rabbit IgG, was conjugated on the surface of the PEG-terminated modified FITC-doped or RBITC-Dextran-doped silica nanoparticles (PFSiNPs or PBSiNPs) by covalent binding to the PEG linkers using the cyanogen bromide method. The concentrations of goat anti-rabbit IgG covering the nanoprobes were quantified via the Bradford method. In the proof-of-concept experiment, an epithelial cell adhesion molecule (EpCAM) on the human breast cancer SK-Br-3 cell surface was used as the tumor marker, and the nanoparticle functionalized with rabbit anti-EpCAM antibody was employed as the nanoprobe for cancer cell recognition. Compared with fluorescent dye labeled IgG (FITC-IgG and RBITC-IgG), the designed nanoprobes display dramatically increased stability of fluorescence as well as photostability under continuous irradiation.

We present a low background, highly selective and amplified fluorescent sensor for potassium ions using graphene oxide (GO) and a cationic conjugated polymer (CCP). This method takes advantage of the phenomenon that the addition of CCP cannot release the dye labeled guanine-rich DNA from the GO surface, and the conformational switch of the guanine-rich DNA from random coil to G-quadruplex induced by the target.

We explore the interactions between a fluorescein (FAM)-labeled single-stranded DNA (P), graphene oxide (GO), and a cationic conjugated polymer, poly [(9,9-bis(6′-N,N,N-trimethylammonium)hexyl)-fluorenylene phenylene dibromide] (PFP). It is found that the fluorescence change of P-GO-PFP system is dependent on the addition order of P and PFP. When adding PFP into P/GO complex, the fluorescence resonance energy transfer (FRET) from PFP to P is inefficient. If P is added to PFP/GO complex, efficient FRET is obtained. This may be attributed to the equal binding ability for P and PFP to GO. The results of time-resolved fluorescence and fluorescence anisotropy support the different fluorescent response under different addition order of P and PFP to GO. Based on the above phenomenon, we demonstrate a method to reduce the high background signal of a traditional PFP-based DNA sensor by introducing GO. In comparison to the use of single PFP, the combination of PFP with GO-based method shows enhanced sensitivity with a detection limit as low as 40 pM for target DNA detection.

Micrococcal nuclease (MNase) is the extracellular nuclease of Staphylococcus aureus (S. aureus). It preferentially digests single-stranded nucleic acids. The existence of MNase can be the standard to identify S. aureus and the content of MNase can be used to evaluate the pathogenicity of S. aureus. Herein, an ultra-high sensitive and selective fluorescent sensing platform for MNase is developed based on MNase-induced DNA strand scission and the difference in affinity of graphene oxide (GO) for single-stranded DNA containing different numbers of bases in length. In the absence of MNase, the adsorption of the dye-labeled ssDNA on GO makes the dyes close proximity to GO surface resulting in high efficiency quenching of fluorescence of the dyes. Conversely, and very importantly, in the presence of MNase, it cleaves the dye-labeled ssDNA into small fragments. The introduction of GO into the sensing solution results in weak quenching of the fluorescence of the dyes due to the weak affinity of the short dye-labeled oligonuleotide fragment to GO, and the fluorescence intensity gradually increases with increasing concentration of MNase. MNase can be detected in a range of 8×10−5 to 1.6×10−3 unit/mL with a detection limit of 2.7×10−5 unit/mL and good selectivity. The detection limit is of two orders of magnitude lower than those reported fluorescence MNase assays. Moreover, when the GO-based biosensor is used in S. aureus sample assays, preeminent fluorescence signals are obtained, thus the platform of the GO-based biosensor can be used to detect MNase in real-world samples.Highlights► An ultra-high sensitive strategy for micrococcal nuclease detection was developed. ► A low detection limit (2.7×10−5 unit/mL) was obtained. ► This method can be extended to the assay of the MNase secreted by S. aureus.

•A method is proposed for Hg2+ detection.•This method utilizes the advantages of aptamer and AuNPs.•Hg2+ can be detected in a range of 0.02–1.0 µM with a detection limit of 16 nM.•This assay shows excellent selectivity for Hg2+ over other metal cations.A method is proposed for the detection of Hg2+ using Hg2+ specific DNA (MSD) functionalized gold nanoparticles (AuNPs) based on the formation of T–Hg2+–T complex and the excellent quenching fluorescence property of AuNPs. The MSD is rich in thymine (T) and readily forms T–Hg2+–T configuration in the presence of Hg2+. The MSD which is labeled with a fluorescein (FAM) at the 3′-end and a thiol at the 5′-end is bounded to the AuNPs through Au–S covalent bonds to form the probes (AuNPs–MSD). Hg2+ detection can be easily realized by monitoring the change of fluorescence signal of AuNPs–MSD probes. Hg2+ can be detected in a range of 0.02–1.0 µM with a detection limit of 16 nM. Besides, the assay shows excellent selectivity for Hg2+ over other metal cations such as Fe3+, Ca2+, Mg2+, Mn2+, Cr3+, Ni2+, Cu2+, Co2+ and Pb2+. The major advantages of this Hg2+ assay are its water-solubility, simplicity, low cost and high sensitivity. Moreover, this method provides a potentially useful method for the Hg2+ detection in aqueous solution.

Mucin 1 (MUC1) which presents in epithelial malignancies, is a well-known tumor biomarker. In this paper, a highly sensitive and selective fluorescent aptasensor for Mucin 1 (MUC1) detection is constructed, utilizing graphene oxide (GO) as a quencher which can quench the fluorescence of single-stranded dye-labeled MUC1 specific aptamer. In the absence of MUC1, the adsorption of the dye-labeled aptamer on GO brings the dyes in close proximity to the GO surface resulting in high efficiency quenching of dye fluorescence. Therefore, the fluorescence of the designed aptasensor is completely quenched by GO, and the system shows very low background fluorescence. Conversely, and very importantly, upon the adding of MUC1, the quenched fluorescence is recovered significantly, and MUC1 can be detected in a wide range of 0.04–10 μM with a detection limit of 28 nM and good selectivity. Moreover, the results have also been verified for real sample application by testing 2% serum containing buffer solution spiked with a series of concentrations of MUC1.

Progress in biomedical imaging depends on the development of bioprobes with a high sensitivity and stability. Fluorescent silica nanoparticles (NPs) covalent conjugation of avidin has been proposed for cancer cells imaging by fluorescence microscopy. Uniform silica NPs were prepared using water-in-oil (W/O) microemulsion methods and primary amine groups were introduced onto the surface of the NPs by condensation of tetraethyl orthosilicate (TEOS). Optically stable organic dyes, tris(2,2'-bipyridyl) dichlororuthenium(II) hexahydrate (Rubpy), were doped inside the silica NPs. The amine functions were transferred to carboxyl groups coupled with a linker elongation. Avidin was immobilized at the surface of the NPs by covalent binding to the carboxyl linkers. The binding capacity of the avidin-covered NPs for ligand biotin was quantified by titration with biotin(5-fluorescein) conjugate to 1.25 biotin binding sites/100 nm2. We used biotinylated antibody and cell recognition by fluorescence microscopy imaging technique. The lung carcinoma cells were identified easily with high efficiency using these antibody-coated NPs. By comparison with fluorescein isothiocyanate (FITC), dye-doped silica NPs display dramatically increased stability of fluorescence as well as photostability, as compared to the common organic dye, when under continuous irradiation.Highlights► A new method to prepare luminescent and photostable based on dye-doped silica nanopaticles was developed. ► This approach was based on dye-doped silica nanopaticles and avidin was used as the substrate. ► Binding capacity of the avidin-covered NPs for ligand biotin was quantified to be 1.25/100 nm2. ► Lung carcinoma cells were identified with high efficiency using these antibody-coated NPs. ► Dye-doped silica NPs display increased photostability under continuous irradiation.

We present a novel fluorescent aptasensor for simple and accurate detection of adenosine deaminase (ADA) activity and inhibition on the basis of graphene oxide (GO) using adenosine (AD) as the substrate. This aptasensor consists of a dye-labeled single-stranded AD specific aptamer, GO and AD. The fluorescence intensity of the dye-labeled AD specific aptamer is quenched very efficiently by GO as a result of strong π–π stacking interaction and excellent electronic transference of GO. In the presence of AD, the fluorescence of the GO-based probe is recovered since the competitive binding of AD and GO with the dye-labeled aptamer prevents the adsorption of dye-labeled aptamer on GO. When ADA was introduced to this GO-based probe solution, the fluorescence of the probe was quenched owing to ADA can convert AD into inosine which has no affinity to the dye-labeled aptamer, thus allowing quantitative investigation of ADA activity. The as-proposed sensor is highly selective and sensitive for the assay of ADA activity with a detection limit of 0.0129 U/mL in clean buffer, which is more than one order of magnitude lower than the previous reports. Meanwhile, a good linear relationship with the correlation coefficient of R=0.9922 was obtained by testing 5% human serum containing a series of concentrations of ADA. Additionally, the inhibition effect of erythro-9-(2-hydroxy-3-nonyl) adenine on ADA activity was investigated in this design. The GO-based fluorescence aptasensor not only provides a simple, cost-effective and sensitive platform for the detection of ADA and its inhibitor but also shows great potential in the diagnosis of ADA-relevant diseases and drug development.Highlights► A novel fluorescence method for adenosine deaminase (ADA) and its inhibitor assay was developed. ► This approach was based on graphene oxide and adenosine was used as the substrate. ► This assay only requires one labeled DNA probe, making the assay more cost-effective. ► A low detection limit (0.0129 U/mL) was obtained. ► The approach was applied to determine ADA in real human serum sample.

In this work, we have developed a simple and sensitive method for ATP detection using silica nanoparticles (NPs) as the platform and hoechst33258 as the signal reporter. The ATP-binding aptamers hybridize with the probe DNA (DNAp) immobilized NPs to form the aptamer/DNAp duplex on the NPs surface. The conformational change of the aptamer leads to the decrease of the aptamer/DNAp duplex on the NPs due to the ATP-binding aptamer switches its structure from the aptamer/DNAp duplex to the aptamer/target complex in the presence of ATP. ATP detection can be easily realized by separating the silica nanoparticles and adding the hoechst33258 of intercalating to aptamer/DNAp (dsDNA). Good selectivity between ATP and CTP, GTP or UTP has been demonstrated, which is due to the specific recognition between ATP aptamer and ATP. The Kd was estimated to be ∼1 mM from 0 to 4 mM and a liner response was observed from 0 to 0.2 mM with a detection limit of ∼20 μM. Compared with other methods, the carboxyl-modified silica nanoparticles (∼60 nm) prepared by the reverse microemulsion method can serve as a stable and sensitive sensor platform because of their smaller size and facile conjugation with amine-containing molecules. In addition, the high sensitivity and selectivity of hoechst33258 was employed for the ssDNA and dsDNA determination, which takes advantage of the label-free aptamer and lower cost.

A novel molecular aptamer beacon (MAB) was designed by integrating a single-labeled hairpin-shaped aptamer and graphene oxide (GO). The hairpin-shaped aptamer was constructed with anti-ATP aptamer and another five nucleotides added to the 5′-end of the aptamer which are complementary to nucleotides at the 3′-end of the aptamer to form a hairpin-shaped probe. This newly designed MAB which acts as a low background signal platform was used for the ATP detection based on long-range resonance energy transfer (LrRET). In the absence of ATP, the adsorption of the dye-labeled hairpin-shaped aptamer on GO makes the dyes close proximity to GO surface resulting in high efficiency quenching of fluorescence of the dyes. Therefore, the fluorescence of the designed MAB is completely quenched by GO, and the system shows very low background. Conversely, and very importantly, upon the adding of ATP, the quenched fluorescence is recovered significantly, and ATP can be detected in a wide range of 5–2500 μM with a detection limit of 2 μM and good selectivity. Moreover, when the GO-based MAB was used in cellular ATP assays, preeminent fluorescence signals were obtained, thus the platform of GO-based MAB could be used to detect ATP in real-world samples.

This study presents the investigation of bioconjugating ability of near-infrared (NIR) CdSeTe/ZnS quantum dots (QDs) (710 nm) and visible CdSe QDs (595 nm) in immunofluorescent staining for cancer biomarkers in gastric cancer tissues probed with the homemade Hadamard transform (HT) spectral imaging microscope and a commercial multispectral imaging system. The results show that imunostaining ability of NIR QDs probes is stronger than that of visible QDs when the two kinds of QDs are simultaneously used to probe the cancer biomarkers such as cytokeratin 20 (CK20) and proliferating cell nuclear antigen (PCNA) in gastric cancer tissues. Moreover, when the two QDs probes are used for immunostaining successively for the same target molecules, staining order has great influences on the final results due to their different conjugating ability to the marker proteins. The results imply that NIR QDs hold more promise for real-time imaging of tumor tissues due to its higher sensitivity and contrast. In addition, the results also demonstrate the potential of Hadamard transform spectral imaging as a useful tool in biomedical analysis and quantitative evaluation for tumor tissues.

Chemiluminescence (CL) has been a useful tool for analytical applications. Fluorescein can be used to enhance CL emission of luminol. With three-dimensional (3D) dynamic CL spectrum obtained from a linear CCD (charge-coupled device) flow-injection CL spectrometer, the fluorescein enhanced CL analysis was studied. The enhanced process can be described with chemiluminescence resonance energy transfer (CRET) at certain fluorescein concentrations. In the process of CRET, fluorescein enlarges CL intensity but does not join the CL reaction. Compared with classic luminol–Co2+ CL system, the fluorescein-enhanced system exhibits stronger CL signal with the linear range and detection limit for traces of Co2+ unchanged.

By coupling flow-injection with laser-induced fluorescence detection, a setup was developed and a novel method combining fluorescence resonance energy transfer (FRET) and flow-injection analysis (FIA) was proposed for the determination of vitamin B12 (VB12) based on its fluorescence quenching on the system of acridine orange (AO)/rhodamine 6G (R6G). The effective energy transfer could occur between AO and R6G in the dodecyl benzene sodium sulfonate (DBS) while 454 nm argon laser was used as the excitation source, and as a result, the fluorescence emission of R6G has been increased significantly. It was found that the fluorescence of the above system could be sharply diminished by VB12. By using the mixed solution AO–R6G–DBS and the same solution containing VB12 as the carrier and sample, respectively, a series of negative peaks which could be applied for the quantification of VB12 were obtained. The detection limit for VB12 was 1.65 × 10−6 mol/L. The linear range for determining VB12 was 4 × 10−4 to 2 × 10−6 mol/L (correlation coefficient, r = 0.9923). The method was applied to measure VB12 injections with satisfactory results.

A novel system of Hadamard transform microscopic fluorescence imaging for single cells is presented, based on which the DNA ploidy of rat hepatocyte was quantitatively measured. The result shows that diploid rat hepatocyte has a stable DNA content, thus diploid rat hepatocyte was used to investigate the binding of five clinical anticancer agents, vincristine, cyclophosphamide, nitrogen mustard, cis-diamminedichloroplatinum(II) (CDDP) and mitomycin-C, with cellular DNA when acridine orange (AO) was used as the competitive fluorescence probe. Based on this model, some Schiff base complexes–cellular DNA interactions were investigated. The results indicate that all the twenty-two compounds, including Schiff base ligands of N-2-hydroxy-naphthaldehyde with D-glucoamine (NG) and the complexes of 3d-transitional metals ions with NG and with D-glucoamine (Glu) and the mixed complexes of NG and Glu series with α-glycine (GNG), have the ability to enter the cell membrane and interact with cellular DNA. Four of the compounds, CuGlu, Fe(II)NG, Fe(III)NG and CuGluG can intercalate with DNA like AO does and depress AO–DNA fluorescence to 70% or lower. An in intro UV-visible spectroscopic study on the compound–DNA spectra testified the above results and suggests that diverse interaction mechanisms coexist for all these complexes except intercalating mode. This study presents a new in vitro method for initial screening of anticancer compounds.

We report a single-microsphere based imaging assay method by integrating up-converting luminescence with optical tweezers for detecting microRNA-21 sequences. This method achieves a competitive detection limit of 12 fM with good selectivity and no dedicated signal amplification designs.