Ultrasensitive Fluorescence Research Articles

Ultrasensitive detection of 3′-5′ exonuclease enzymatic activity using molecular beacons

An ultrasensitive and rapid turn-on fluorescence assay has been developed for the detection of 3'-5' exonuclease activity of exonuclease III (Exo III) using molecular beacons (MBs). This method has a linear detection range from 0.04 to 8.00 U mL(-1) with a limit of detection of 0.01 U mL(-1). In order to improve the selectivity of the method, a dual-MB system has been developed to distinguish between different exonucleases. With the introduction of two differently designed MBs which respond to different exonucleases, the T5 exonuclease, Exo III and RecJf exonucleases can be easily distinguished from each other. Furthermore, fetal bovine serum and fresh mouse serum were used as complex samples to investigate the feasibility of the dual-MB system for the detection of the enzymatic activity of Exo III. As a result, the dual-MB system showed a similar calibration curve for the detection of Exo III as in the ideal buffer solution. The designed MB probe could be a potential sensor for the detection of Exo III in biological samples.

Analytical tools for single-molecule fluorescence imaging in cellulo

Recent technological advances in cutting-edge ultrasensitive fluorescence microscopy have allowed single-molecule imaging experiments in living cells across all three domains of life to become commonplace. Single-molecule live-cell data is typically obtained in a low signal-to-noise ratio (SNR) regime sometimes only marginally in excess of 1, in which a combination of detector shot noise, sub-optimal probe photophysics, native cell autofluorescence and intrinsically underlying stochasticity of molecules result in highly noisy datasets for which underlying true molecular behaviour is non-trivial to discern. The ability to elucidate real molecular phenomena is essential in relating experimental single-molecule observations to both the biological system under study as well as offering insight into the fine details of the physical and chemical environments of the living cell. To confront this problem of faithful signal extraction and analysis in a noise-dominated regime, the 'needle in a haystack' challenge, such experiments benefit enormously from a suite of objective, automated, high-throughput analysis tools that can home in on the underlying 'molecular signature' and generate meaningful statistics across a large population of individual cells and molecules. Here, I discuss the development and application of several analytical methods applied to real case studies, including objective methods of segmenting cellular images from light microscopy data, tools to robustly localize and track single fluorescently-labelled molecules, algorithms to objectively interpret molecular mobility, analysis protocols to reliably estimate molecular stoichiometry and turnover, and methods to objectively render distributions of molecular parameters.

Synthesis of gold nanoclusters/glucose oxidase/graphene oxide multifunctional catalyst with surprisingly enhanced activity and stability and its application for glucose detection

The paper reports the synthesis of gold nanoclusters/glucose oxidase/graphene oxide (GNC/GOD/GO) multifunctional catalyst and its application for glucose detection. First, an amine group was introduced into the GO sheet through the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide mediated reaction between carboxylic acid and ethylenediamine to form a positively charged GO sheet (GO-NH3+). Then, negatively charged GNC and GOD were assembled on the surface of GO-NH3+ with electrostatic interaction. Finally, the hybrid was dried by freeze drying. The resulting hybrid gives high fluorescence intensity and optical stability. More importantly, the hybrid exhibits largely enhanced activities towards the decomposition of hydrogen peroxide and oxidation of glucose at neutral pH. Owing to the greatly synergistic catalytic effect between GNC and GOD, the nanosensor based on the hybrid displays an ultrasensitive fluorescence response to glucose. The fluorescence peak intensity linearly decreases with the increase of glucose concentration in the range of 1.1 × 10−2 to 1.6 × 10−7 M with a detection limit of 6.8 × 10−8 M (S/N = 3). The method provides the advantage of sensitivity, repeatability and stability compared with present glucose sensors. It has been successfully applied in detection of glucose in real serum samples. The study also opens a new avenue for the fabrication of GO-based catalytic materials that holds great promise in potential applications such as biocatalyst, nanosensors and molecular carriers.

Design and Synthetic Scheme of Water Dispersible Graphene Oxide-Coumarin Complex for Ultra-Sensitive Fluorescence Based Detection of Copper (Cu2+) Ion in Aqueous Environment

Copper oxides and its salts are now widely used as pesticides to control fungal and bacterial diseases of field crops. Copper toxicity is often a major contributor of human health problems caused through accumulation of excess copper ions in various organs via drinking water, fruits and vegetables. So, detection and estimation of cupric ions in biological organs, drinking water, fruits and vegetables are extremely important. Recently, a fluorescence based sensor using coumarin dye (high quantum yield) has been proposed to detect micromolar Cu++ ion in biological organs. But major problem with coumarin dye is that it is insoluble in water and undergoes dye-dye aggregation in organic solvents. We proposed here a synthetic scheme of preparation of graphene oxide conjugated coumarin dye derivative which would be water dispersible and expected to be an ideal candidate for Cu2+ ion estimation in biological organs and drinking water.

Label-free and ultrasensitive fluorescence detection of cocaine based on a strategy that utilizes DNA-templated silver nanoclusters and the nicking endonuclease-assisted signal amplification method

A general and reliable strategy for the detection of cocaine was proposed utilizing DNA-templated silver nanoclusters as signal indicators and the nicking endonuclease-assisted signal amplification method. This strategy can detect cocaine specifically with a detection limit as low as 2 nM by using a small volume of 5 μL.

Ultrasensitive fluorescence detection of heparin based on quantum dots and a functional ruthenium polypyridyl complex

A new strategy for the detection of heparin is developed by utilizing quantum dots (QDs) and a functional ruthenium polypyridyl complex [Ru(phen)2(dppz-idzo)]2+ (phen=1,10-phenanthroline, dppz-idzo=dipyrido-[3,2-a:2',3'-c] phenazine-imidazolone). The emission of CdTe QDs is found to be quenched by Ru complex due to electron transfer. Upon addition of the polyanionic heparin, it could remove the quencher (Ru complex) from the surface of QDs owing to the electrostatic and/or hydrogen bonding interactions between heparin and Ru complex, which led to significant fluorescence recovery of CdTe QDs. The fluorescence intensity enhanced with the increase of heparin and a linear relationship was observed in the range of 21–77nM for heparin detection in buffer solution and the limit of detection (LOD) is 0.38nM. Moreover, the strategy was successfully applied to detect heparin as low as 0.68nM with a linear range of 35–98nM in fetal bovine serum samples. The selectivity results of the fluorescence assay revealed that our system displayed excellent fluorescence selectivity towards heparin over its analogues, such as chondroitin 4-sulfate (Chs) or hyaluronic acid (Hya). This fluorescence “switch on” assay for heparin is label-free, convenient, sensitive and selective, which can be used to detect heparin in biological systems even with the naked eyes.

An ultrasensitive homogeneous aptasensor for kanamycin based on upconversion fluorescence resonance energy transfer

We developed an ultrasensitive fluorescence resonance energy transfer (FRET) aptasensor for kanamycin detection, using upconversion nanoparticles (UCNPs) as the energy donor and graphene as the energy acceptor. Oleic acid modified upconversion nanoparticles were synthesized through a hydrothermal process followed by a ligand exchange with hexanedioic acid. The kanamycin aptamer (5'-NH2-AGATGGGGGTTGAGGCTAAGCCGA-3') was tagged to UCNPs through an EDC–NHS protocol. The π–π stacking interaction between the aptamer and graphene brought UCNPs and graphene in close proximity and hence initiated the FRET process resulting in quenching of UCNPs fluorescence. The addition of kanamycin to the UCNPs–aptamer–graphene complex caused the fluorescence recovery because of the blocking of the energy transfer, which was induced by the conformation change of aptamer into a hairpin structure. A linear calibration was obtained between the fluorescence intensity and the logarithm of kanamycin concentration in the range from 0.01nM to 3nM in aqueous buffer solution, with a detection limit of 9pM. The aptasensor was also applicable in diluted human serum sample with a linear range from 0.03nM to 3nM and a detection limit of 18pM. The aptasensor showed good specificity towards kanamycin without being disturbed by other antibiotics. The ultrahigh sensitivity and pronounced robustness in complicated sample matrix suggested promising prospect of the aptasensor in practical applications.

Colorimetric and ultra-sensitive fluorescence resonance energy transfer determination of H2O2 and glucose by multi-functional Au nanoclusters

Ultra-sensitive colorimetric determination of H2O2 is accomplished based on the intrinsic peroxidase-like activity of Au nanoclusters (AuNCs) stabilized by glutathione (GSH). The color change of 3,3,5,5-tetramethylbenzidine (TMB) catalyzed by AuNCs offers an indirect method to measure glucose. This sensing platform makes use of a dual optical signal change, including the color change in an aqueous solution under visible light illumination and an ultra-sensitive fluorescent assay arising from efficient fluorescence resonance energy transfer (FRET) between the AuNCs and oxidized TMB. The detection limits of H2O2 and glucose are 4.9 × 10(-13) M and 1.0 × 10(-11) M, respectively. In addition, enhanced fluorescence is observed from the AuNCs due to the use of ethanol which produces clear changes in the quantum yield and lifetime of the AuNCs. The quantum yield of AuNCs is enhanced from ∼12.5% as an isolated fluorophore to 38.9% in an AuNCs-ethanol complex. The enhanced fluorescence lowers the detection limits of H2O2 and glucose by 2 orders of magnitude compared to those attained from the original AuNCs.

Carbon nanotube signal amplification for ultrasensitive fluorescence polarization detection of DNA methyltransferase activity and inhibition

A versatile sensing platform based on multiwalled carbon nanotube (MWCNT) signal amplification and fluorescence polarization (FP) is developed for the simple and ultrasensitive monitoring of DNA methyltransferase (MTase) activity and inhibition in homogeneous solution. This method uses a dye-labeled DNA probe that possess a doubled-stranded DNA (dsDNA) part for Mtase and its corresponding restriction endonuclease recognition, and a single-stranded DNA part for binding MWCNTs. In the absence of MTase, the dye-labeled DNA is cleaved by restriction endonuclease, and releases very short DNA carrying the dye that cannot bind to MWCNTs, which has relatively small FP value. However, in the presence of MTase, the specific recognition sequence in the dye-labeled DNA probe is methylated and not cleaved by restriction endonuclease. Thus, the dye-labeled methylated DNA product is adsorbed onto MWCNTs via strong π–π stacking interactions, which leads to a significant increase in the FP value due to the enlargement of the molecular volume of the dye-labeled methylated DNA/MWCNTs complex. This provides the basic of a quantitative measurement of MTase activity. By using the MWCNT signal amplification approach, the detection sensitivity can be significantly improved by two orders of magnitude over the previously reported methods. Moreover, this method also has high specificity and a wide dynamic range of over five orders of magnitude. Additionally, the suitability of this sensing platform for MTase inhibitor screening has also been demonstrated. This approach may serve as a general detection platform for sensitive assay of a variety of DNA MTases and screening potential drugs.

Fluorescence-based immunoassay for human chorionic gonadotropin based on polyfluorene-coated silica nanoparticles and polyaniline-coated Fe3O4 nanoparticles

We report on an ultrasensitive fluorescence immunoassay for human chorionic gonadotrophin antigen (hCG). It is based on the use of silica nanoparticles coated with a copolymer (prepared from a fluorene, a phenylenediamine, and divinylbenzene; PF@SiO2) that acts as a fluorescent label for the secondary monoclonal antibody to β-hCG antigen. In parallel, Fe3O4 nanoparticles were coated with polyaniline, and these magnetic particles (Fe3O4@PANI) served as a solid support for the primary monoclonal antibody to β-hCG antigen. The PF@SiO2 exhibited strong fluorescence and good dispersibility in water. A fluorescence sandwich immunoassay was developed that enables hCG concentrations to be determined in the 0.01–100 ng·mL−1 concentration range, with a detection limit of 3 pg·mL−1.

An ultrasensitive fluorescence assay for protein detection by hybridization chain reaction-based DNA nanotags

An ultrasensitive fluorescence method for determination of protein is developed based on hybridization chain reaction (HCR). In this assay, the streptavidin-magnetic nanobeads were conjugated to biotinylated initiators and biotinylated anti-IgG. In the presence of human IgG, the magnetic nanobeads were fixed on the substrate and the carried initiators propagated the chain reaction of hybridization to form the nicked polymers. Because the nanobead probe carries with a large number of oligonucleotides per protein binding event, there is obvious amplification in the nicked polymers. Then, numerous SYBR Green I molecules were intercalated into the grooves of the long dsDNA polymers, generating a substantially apparent increase in the corresponding fluorescence intensity. With HCR amplification and magenetic nanobead to preamplify the fluorescence signal and reduce the background signal, the detection limit of this assay was 14aM. Compared with the reported protein detection methods, our method exhibited ultrahigh sensitivity. In addition, the proposed method possessed excellent selectivity and low matrix effect. What is more, the assay was also studied for clinical application in human serum with a satisfactory and reliable result.

Ultrasensitive Detection of Proteins on Western Blots with Semiconducting Polymer Dots

Ultrasensitive fluorescence imaging of proteins on western blots using a bright, compact, and orange-emitting semiconducting polymer dot (CN-PPV) is demonstrated. A detection limit at the single-picogram level in dot blots is achieved; with conventional western blotting, 50 pg of transferrin and trypsin inhibitor after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer onto a polyvinylidene fluoride membrane are detected. This method does not require any additional equipment or time compared with the conventional procedure with traditional fluorescent probes.

653 Towards Instant Detection of (Pre)Malignant Lesions by Ultrasensitive Near-Infrared Fluorescence Endoscopy, Using Molecular Targeted Fluorescent Antibodies

Introduction and aim: Colonoscopy is considered to be the most sensitive tool to detect colorectal neoplasms, including small and flat lesions. Nevertheless, detection of such lesions is still incomplete, giving rise to interval cancers, particularly in patients with Lynch syndrome and other hereditary disorders. To improve the detection of (pre)malignant lesions nearinfrared endoscopy, guided bymolecular targeted fluorescent tracers, may hold great promise. Therefore, we developed a combined white-light and near-infrared (WLNI) endoscopic system, as well as two clinically approved fluorescent labeled antibodies, targeting vascular endothelial growth factor-A (VEGF-A) and epithelial growth factor receptor (EGFR). In the present study we investigated VEGF-A and EGFR expression in adenomas and CRC of Lynch patients, as potential targets for (pre)malignant detection. Subsequently we validated the newly developed WLNI endoscopic system, together with intravenously injected fluorescent anti-VEGF-A and anti-EGFR antibodies, in human CRC mouse models. Materials and Methods: VEGF-A and EGFR expression was analyzed by immunohistochemical staining of 34 CRC, 64 high-grade dysplastic (HGD) adenomas, and adjacent normal colon crypts of Lynch patients. The endoscopic system consisted of a custom made clinically approved WLNI camera, comprising a color camera and an ultra-sensitive camera for concurrent white-light and NIR fluorescence acquisition, attached to either a clinical fiberscope or a multi-modal fiber-optic bundle. The WLNI system was evaluated in vivo in CRC subcutaneous (sc) and intraperitoneal (ip) mouse models of bioluminescent CRC cell lines (HCT116-luc, HT29luc2), using bevacizumab-800CW (anti-VEGF-A) and cetuximab-800CW (anti-EGFR). Results: VEGF-A and EGFR were significantly overexpressed in 95% and 74% of HGD adenomas and in 100% and 85% of CRC, compared to adjacent normal crypts (resp. P,0.001/P,0.05 and P,0.05/P,0.001). Tumor-to-background ratio was high (3.2±0.9 for bevacizumab-800CW and 5.7±2.9 for cetuximab-800CW in the HCT116-luc sc model). WLNI endoscopy demonstrated excellent instant visualization of the sc and ip tumors (diameter ≥1 mm), with clear tumor boundaries and a low background fluorescence, demonstrating very high sensitivity and specificity of our WLNI endoscopic system. Conclusion: VEGF-A and EGFR are attractive targets for molecular targeted fluorescence endoscopy in Lynch patients based on their expression patterns. The newly developed WLNI endoscopic system using clinically approved molecular targeted fluorescent antibodies, enables instant visualization of very small tumor lesions in CRC mice models, with an excellent sensitivity and specificity. These results support clinical evaluation of WLNI endoscopy, in order to enhance early detection of colorectal (pre)malignancies and improve potentially outcome in patients.

A highly sensitive fluorescence resonance energy transfer aptasensor for staphylococcal enterotoxin B detection based on exonuclease-catalyzed target recycling strategy

An ultrasensitive fluorescence resonance energy transfer (FRET) bioassay was developed to detect staphylococcal enterotoxin B (SEB), a low molecular exotoxin, using an aptamer-affinity method coupled with upconversion nanoparticles (UCNPs)-sensing, and the fluorescence intensity was prominently enhanced using an exonuclease-catalyzed target recycling strategy. To construct this aptasensor, both fluorescence donor probes (complementary DNA1–UCNPs) and fluorescence quencher probes (complementary DNA2–Black Hole Quencher3 (BHQ3)) were hybridized to an SEB aptamer, and double-strand oligonucleotides were fabricated, which quenched the fluorescence of the UCNPs via FRET. The formation of an aptamer–SEB complex in the presence of the SEB analyte resulted in not only the dissociation of aptamer from the double-strand DNA but also both the disruption of the FRET system and the restoration of the UCNPs fluorescence. In addition, the SEB was liberated from the aptamer–SEB complex using exonuclease I, an exonuclease specific to single-stranded DNA, for analyte recycling by selectively digesting a particular DNA (SEB aptamer). Based on this exonuclease-catalyzed target recycling strategy, an amplified fluorescence intensity could be produced using different SEB concentrations. Using optimized experimental conditions produced an ultrasensitive aptasensor for the detection of SEB, with a wide linear range of 0.001–1ngmL−1 and a lower detection limit (LOD) of 0.3pgmL−1 SEB (at 3σ). The fabricated aptasensor was used to measure SEB in a real milk samples and validated using the ELISA method. Furthermore, a novel aptasensor FRET assay was established for the first time using 30mol% Mn2+ ions doped NaYF4:Yb/Er (20/2mol%) UCNPs as the donor probes, which suggests that UCNPs are superior fluorescence labeling materials for food safety analysis.

Manipulating ionic strength to improve single cell electrophoretic separations

A capillary electrophoresis system with ultrasensitive two-color laser-induced fluorescence detection was used to probe the effect of ionic strength on single cell separations of glycosphingolipids. Differentiated PC12 cells were incubated with two ganglioside substrates tagged with different fluorophores within the BODIPY family such that two distinct metabolic patterns could be simultaneously monitored. Aspiration of single differentiated PC12 cells suspended in a phosphate-buffered saline solution showed excessive peak dispersion, poor resolution, and peak efficiencies below 100,000 theoretical plates. Aspiration of single differentiated PC12 cells suspended in deionized water corrected peak dispersion. Average peak efficiencies ranged between 400,000 and 600,000 theoretical plates. Improved performance was due to the dilution of the high salt concentrations inside of single neuronal-like cells to produce field amplified sample stacking. Single cell separations showed the highest resolution when aspiration of single differentiated PC12 cells suspended in deionized water were separated using a running buffer of high ionic strength. The improvement in resolution allowed for the identification of analytes not previously detected in single cell metabolism studies.

High temporal resolution fluorescence measurements of a mitochondrial dye for detection of early stage apoptosis

In the present study, early stage apoptosis is explored with high temporal resolution. In addition to monitoring early apoptosis induction in single cells by ultrasensitive confocal fluorescence microscopy (UCFM), the mitochondrial protein release kinetics was explored. The current study shows development and optimization of a novel, rapid apoptosis assay to explore the earliest changes in cells by the intrinsic apoptosis pathway. We show that early apoptotic changes in the mitochondria begin nearly simultaneously with the addition of an apoptosis-inducing drug, such as staurosporine. With a temporal resolution of five minutes, this non-invasive analytical technique can elucidate the earliest apoptotic events in living cells. Moreover, our results show that the mitochondrial inter-membrane proteins are not involved in the extrinsic pathway of Ramos cells mediated by an anti-CD95 antibody. Additional techniques such as light microscopy and flow cytometry were employed to confirm the results obtained by ultrasensitive confocal fluorescence microscopy. The results of this study help to understand the earliest mechanisms of apoptosis induction in cells, enabling new methods of drug testing and dose-response analyses.

Capillary electrophoresis with three-color fluorescence detection for the analysis of glycosphingolipid metabolism

A capillary electrophoresis system with an ultrasensitive three-color laser-induced fluorescence detector was constructed for the simultaneous measurement of glycosphingolipids conjugated with a family of BODIPY fluorophores. The compounds were separated by capillary electrophoresis and detected by laser-induced fluorescence excited within a sheath-flow cuvette. Diode-pumped solid-state lasers operating at 473 nm and 532 nm, and a diode laser operating at 633 nm were used to excite glycosphingolipids tagged with BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 fluorophores. Detection limits were 34 ± 1 molecules, 67 ± 7 molecules, and 36 ± 13 molecules of BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 labeled glycosphingolipids. Separation efficiencies were typically one million theoretical plates. To test the ability of the system to analyze cellular contents in an in vitro biological model, differentiated PC12 cells were co-incubated with BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 labeled lactosylceramide substrates. Cells were homogenized. The metabolic products originating from the glycosphingolipid substrates were simultaneously analyzed using the system.

Ultrasensitive fluorescence detection of bleomycin via exonuclease III-aided DNA recycling amplification

A "signal on" approach for ultrasensitive detection of bleomycin was developed by bleomycin-Fe(II) induced hairpin DNA scission to release its loop, which was subsequently recycled with the aid of exonuclease III and a probe to amplify the detectable fluorescent signal.

On-chip selective capture of cancer cells and ultrasensitive fluorescence detection of survivin mRNA in a single living cell

The rapid recognition of cancer cells and detection of tumor biomarker survivin mRNA plays a critical role in the early diagnosis of many cancers. Based on the integration of specific cancer cell capture and intracellular survivin mRNA detection, this work presents a novel and sensitive on-chip approach for the bioanalysis of survivin mRNA in a single living cell. The microchannel surface was firstly modified with a prostate stem cell antigen (PSCA) monoclonal antibody as the recognition element for prostate cancer cells (PC-3). As a result of the antigen-antibody specific affinity interactions, PC-3 cells could be selectively captured on the microchannel surface. After cell capture, nano-sized graphene oxide-poly(ethylene glycol) bis(amine) (NGO-PEG) was employed as a quencher and carrier of a signal tag, fluorescein isothiocyanate (FITC)-labeled antisense oligonucleotide (F-S1), which is complementary to part of survivin mRNA (target survivin mRNA), to transfect into the captured PC-3 cells. Upon the selective binding of S1 to intracellular survivin mRNA, F-S1 will be released from the NGO-PEG, inducing the fluorescence recovery of FITC. This antibody-based microfluidic device enables simple and inexpensive monitoring of the amount of survivin mRNA in single captured cell without the need for sample pretreatment. The survivin mRNA content in each PC-3 cell was estimated to be (4.8 ± 1.8) × 10(6) copies. This strategy opens a different perspective for ultrasensitive survivin mRNA detection, which may facilitate the early screening for malignancy.

An ultrasensitive and selective fluorescence assay for Sudan I and III against the influence of Sudan II andIV

We report on an ultrasensitive and selective fluorescence assay for Sudan I and III against the influence of Sudan II and IV based on ligand exchange mechanism. Calcein as a fluorescence indicator and Sudan I–IV as model analytes were employed to investigate the analytical feature of this assay platform. Results show that the fluorescence of calcein can be efficiently quenched by Cu(II). When the ligand exchange reaction proceeds, calcein is deprived of Cu(II) by Sudan I and III, resulting in the fluorescence recovery of calcein. However, the ligand exchange reaction does not happen in the presence of Sudan II or IV due to the 2-methyl steric effects, which is favorable for selective determination of Sudan I and III against the influence of Sudan II and IV. It was found that the fluorescence enhancement efficiency (FEE) against the concentration of Sudan (cSudan, nmol L−1) shows a linear relationship. The calibration equations are FEESudan I=0.0032 cSudan I−0.02613, and FEESudan III=0.0033 cSudan III−0.02467 over the corresponding linear range of 11.25–2078.29 and 9.44–1035.78nmolL−1 with the correlation coefficients (R2) of 0.9984 and 0.9955, respectively. And the detection limits (3σ/slope) are calculated to be 211.3 and 208.5pmolL−1 for Sudan I and III, respectively, showing ultralow detection limit. The Sudan dye in a commercial chilli powder sample was assayed with satisfactory results.