Label-free Fluorescence Assay Research Articles

A label-free fluorescent assay for free chlorine in drinking water based on protein-stabilized gold nanoclusters

Bovine serum albumin stabilized Au nanoclusters (BSA–AuNCs) were demonstrated as a novel fluorescence probe for sensitive and selective detection of free chlorine in drinking water. The fluorescence of BSA–AuNCs was found to be quenched effectively by the free chlorine, and the decrease in fluorescence intensity of BSA–AuNCs allowed the sensitive detection of free chlorine in the range of 0.8–800μM. The detection limit is 0.50μM at a signal-to-noise ratio of 3. The present fluorescent assay for free chlorine possesses low detection limit, wide linear range and good selectivity. Real tap water samples were analyzed with satisfactory results, which suggested its potential for water quality analysis.

Label-free fluorescence assay for protein kinase based on peptide biomineralized gold nanoclusters as signal sensing probe

A label-free, sensitive and simple method to detect protein kinase based on the selective aggregation of phosphorylated peptide-gold nanoclusters (peptide-AuNCs) triggered by Zr4+ ion coordination is developed. The AuNCs were synthesized by peptide without any strong reducing agents, which prevent peptides from being disrupted. Under optimal conditions, a linear relationship between the decreased PL intensity of peptide-AuNCs and the concentration of casein kinase II (CK2) in the range of 0.08–2.0unitmL−1 with a detection limit of 0.027unitmL−1 (3σ) was obtained. The feasibility of this AuNCs-based sensor was further demonstrated by the assessment of kinase inhibition by ellagic acid, 5,6-dichlorobenzimidazole-1-β-d-ribofuranoside, emodin, and quercetin in human serum. As expected, the PL intensity increased with increasing inhibitor efficiency in the presence of inhibitors. The IC50 value (inhibitor concentration producing 50% inhibition) for ellagic acid was estimated to be 0.045μM. With more sophisticated design of the peptide substrate sequences, the detection of other enzymes will be realized. With characteristics of homogeneous, facile, universal, label-free, and applicable for kinase assay, the proposed sensor provides potential application in kinase-related biochemical fundamental research and inhibitor screening.

An exonuclease I-based label-free fluorometric aptasensor for adenosine triphosphate (ATP) detection with a wide concentration range

A novel aptamer-based label-free assay for sensitive and selective detection of ATP was developed. This assay employs a new aptamer/fluorescent probe system that shows resistance to exonuclease I (Exo I) digestion upon binding to ATP molecules. In the absence of ATP, the complex between the ATP-binding aptamer (ATP–aptamer) and a DNA binding dye, berberine, is digested upon the addition of exonuclease I, leading to the release of berberine into solution and consequently, quenched berberine fluorescence. In the presence of ATP, the ATP-binding aptamer folds into a G-quadruplex structure that is resistant to Exo I digestion. Accordingly, berberine is protected in the G-quadruplex structure and high fluorescence intensity is observed. As such, based on the fluorescence signal change, a label-free fluorescence assay for ATP was developed. Factors affecting the analysis of ATP including the concentration of ATP-binding aptamer, reaction time, temperature and the concentration of Exo I were comprehensively investigated. Under optimal conditions, the fluorescence intensity of the sensing system displayed a response for ATP in a wide range up to 17.5mM with a detection limit of 140nM.

A label-free fluorescence assay for trypsin based on the electron transfer between oligonucleotide-stabilized Ag nanoclusters and cytochrome c.

A label-free fluorescent assay for the detection of trypsin by using oligonucleotide-templated silver nanoclusters (Ag NCs) and cytochrome c (Cyt c) has been demonstrated. When negatively charged Ag NCs and positively charged Cyt c are mixed, they tend to form a hybrid complex, and then lead the fluorescence of Ag NCs to be quenched significantly due to electron transfer between Ag NCs and the heme cofactor of Cyt c. In the presence of trypsin, it catalyzes the hydrolytic cleavage of Cyt c to small peptide fragments, and releases the heme moiety from the Ag NCs/Cyt c complex; the quenched fluorescence restores therewith. By virtue of this specific response, the fluorescent biosensor has a linear range of from 0.7 to 4 μg mL(-1) and from 9 to 120 μg mL(-1) with a detection limit of 58.7 ng mL(-1). Aside from the easy manufacture aspect, our method also possesses a high signal-to-background ratio (~11), excellent selectivity and good biocompatibility, which makes it a promising bioanalysis for a trypsin activity assay.

A versatile biosensing system for DNA-related enzyme activity assay via the synthesis of silver nanoclusters using enzymatically-generated DNA as template

In the present day, oligonucleotide-encapsulated silver clusters (DNA–AgNCs) have been widely applied into bio-analysis as a signal producer. Herein, we developed a novel method to synthesize DNA–AgNCs encapsulated by long-chain cytosine (C)-rich DNA. Such DNA was polymerized in a template-free way by terminal deoxynucleotidyl transferase (TdT). We demonstrated that TdT-polymerized long chain C-rich DNA can serve as an excellent template for AgNCs synthesis. Based on this novel synthesis strategy, we developed a label-free and turn-on fluorescence assay to detect TdT activity with ultralow limit of detection (LOD) of 0.0318U and ultrahigh signal to background (S/B) of 46.7. Furthermore, our proposed method was extended to a versatile biosensing strategy for turn-on nucleases activity assay based on the enzyme-activated TdT polymerization. Two nucleases, EcoRI and ExoIII as model of endonuclease and exonuclease, respectively, have been detected with high selectivity and competitive low LOD of 0.0629U and 0.00867U, respectively. Our work demonstrates the feasibility of TdT polymerization-based DNA–AgNCs synthesis strategy as a versatile and potent biosensing platform to detect the activity of DNA-related enzymes.

A "turn-on" and label-free fluorescent assay for the rapid detection of exonuclease III activity based on Tb(3+)-induced G-quadruplex conjugates.

A "turn-on" and label-free fluorescent assay for the specific, rapid, and sensitive detection of 3' → 5' exonuclease III activity is reported in this study. The assay is based on the Tb(3+)-promoted G-quadruplex, which lead to the enhancement of Tb(3+) fluorescence due to the energy transfer from guanines. The proposed assay is highly simple, rapid, and cost-effective, and does not require sophisticated experimental techniques such as gel-based equipment or radioactive labels. It can be used for the rapid detection of exonuclease III activity with a detection limit of 0.8 U and a RSD (n = 6) <5 %. Notably, no dye was covalently conjugated to the DNA strands, which offers the advantages of low-cost and being interference-free.

Rapid fluorescence assay for Sudan dyes using polyethyleneimine-coated copper nanoclusters

We report that the intensity of the blue fluorescence of copper nanoclusters coated with polyethyleneimine (PEI) is strongly reduced in the presence of the food dyestuffs Sudan I-IV. This finding was exploited in a label-free fluorescence assay for these Sudan dyes both in ethanol and aqueous solutions. The PEI-capped nanoclusters have an average diameter of 1.8 nm and are displaying, under 355 nm excitation, a blue emission at 480 nm that matches the absorption bands of the Sudan dyes. The clusters are stable in solution for at least 1 month. Under optimum conditions, this assay can be applied to the quantification of the dyes Sudan I, II, III, and IV, respectively, in the 0.1−30, 0.1–30, 0.1–25, and 0.1–25 μM concentration ranges, and the detection limits (3σ/slope) are 65, 70, 45, and 50 nM, respectively. The capability of reducing the fluorescence of the PEI-capped copper nanoclusters is directly related to the number of the functional groups in that Sudan III and IV give lower detection limits. This analytical scheme exhibits a remarkably high selectivity for the Sudan dyes over potentially interfering substances. The method was successfully applied to determine Sudan I, II, III, and IV in hot chilli powder.

Label-free fluorescent assay of ATP based on an aptamer-assisted light-up of Hoechst dyes

A label-free assay was developed for the visual detection of ATP based on the aptamer-directing fluorescence of Hoechst dyes.

A label-free fluorescence assay for thrombin based on aptamer exonuclease protection and exonuclease III-assisted recycling amplification-responsive cascade zinc(ii)-protoporphyrin IX/G-quadruplex supramolecular fluorescent labels

A simple, label-free and sensitive fluorescence protein assay has been developed on the basis of aptamer exonuclease protection and exonuclease III (Exo III)-assisted recycling amplification-responsive cascade ZnPPIX/G-quadruplex supramolecular fluorescent labels. In the sensing system, a special aptamer probe containing the aptamer sequence at the 3'-terminus and the DNAzyme sequence at the 5'-terminus was applied, which has the capacity to recognize a protein target with high affinity and specificity. Exonuclease I (Exo I) can efficiently catalyze the degradation of free single stranded DNA probes in the 3' to 5' direction. In the presence of the target protein, the strong binding between the target protein and its aptamer can protect aptamer probes from degradation. Subsequently, the protected aptamer probes act as catalysators to trigger hybridization with the hairpin DNA probe that contains a partially "caged" G-quadruplex sequence. Upon interaction with the protected aptamer probes, the hairpin opens to yield the active G-quadruplex structure. In the presence of exonuclease III (Exo III), Exo III-assisted recycling amplification occurs generating numerous G-quadruplex supramolecular structures. The zinc(ii)-protoporphyrin IX (ZnPPIX) fluorophore binds to the G-quadruplexes and this results in the enhanced fluorescence of the fluorophore. The resulting fluorescence of the ZnPPIX/G-quadruplex provides the readout signal for the sensing event. Thrombin is used as the model analyte in the current proof-of-concept. The developed method was demonstrated to have very high sensitivity for the detection of proteins with a limit of detection of 0.2 pM without using washes or separations. In addition, this new method for protein detection is simple and inherits all the advantages of aptamers. The mechanism, moreover, may be generalized and used for other forms of protein analysis.

Insight into aggregation-induced emission characteristics of red-emissive quinoline-malononitrile by cell tracking and real-time trypsin detection

Water-soluble, long wavelength fluorescent aggregation-induced emission (AIE)-active materials are in great demand for high contrast biosensing and bioimaging. The substitution position effects of the sulfonate group on the basis of two quinoline-malononitrile (QM) derivatives (EDS and EDPS) provide insight into efficient modulation in the hydrophilicity, emitting color, and specific AIE characteristics. EDS shows a unique AIE behaviour in aqueous solution, but EDPS does not. The abnormal non-fluorescence aggregation for EDS in pure water is capsule-like with loose packing characteristics, but still has enough cavities or free volume to consume the radiative energy, resulting in nearly no fluorescence. When binding with the protein BSA, the sulfonate unit as a conformation function group (CFG) plays a vital role in altering its initial loose ensemble into tightly compact aggregation with light-up AIE characteristics. By cell tracking, dynamic light scattering (DLS) and transmission electron microscopy (TEM), the key role of sulfonate groups in the conformation alteration has been well demonstrated for the first time. Moreover, EDS is successfully exploited in a label-free real time AIE fluorescent assay for trypsin detection and inhibitor screening. The hydrophilic sulfonate group from the different substitution position in the AIE-active QM building blocks provides an effective way to tailor the intermolecular aggregation associated with molecular stacking, especially for in situ cell tracking and real-time trypsin detection.

Label-free fluorescence assay for thrombin based on unmodified quantum dots

Rapid and sensitive assay of thrombin and its inhibition in a high-throughput manner is of great significance in the diagnostic and pharmaceutical fields. In this article, we developed a novel biosensor for the detection of thrombin and its inhibition based on the aggregation behavior of the unmodified CdTe QDs. A cationic substrate peptide of thrombin (GGLVPRGSCC-NH2, S-peptide) can attach to the surface of CdTe QDs, partly balance their surface negative charge, and induce the aggregation of QDs, which results in the fluorescence quenching of QDs. After hydrolysis of S-peptide by thrombin, two kinds of shorter peptides (P1-peptide, GGLVPR, and P2-peptide, GSCC) are produced. The uncharged P2-peptide rather than the cationic P1-peptide would bind to QDs. Hence, the CdTe QDs were kept stable in the solution with the fluorescence being maintained. The change of fluorescence intensity would sensitively respond to thrombin activity and its inhibition. Fluorescence spectroscopy, transmission electron microscopy and dynamic light scattering were performed to discuss the quenching mechanism. Under optimized conditions, this method enables measurement of thrombin in the range of 10–100μU/mL with the detection limit of 1.5μU/mL. Not only in buffer, but also in blood serum, such sensor exhibited extraordinarily high sensitivity and excellent specificity. In addition, the typical inhibitor of thrombin, hirudin, was also successfully assayed by this method (from 2μU/mL to 30μU/mL with the LOD of 0.21μU/mL). Furthermore, the present approach could also be potentially extended to other proteases and their inhibitors detection with unmodified CdTe QDs.

A label-free and signal-on supersandwich fluorescent platform for Hg2+ sensing

A label-free supersandwich fluorescent assay was demonstrated for the first time by taking Hg2+ as a detection candidate. The principle of the proposed supersandwich fluorescent platform is based on the formation of supersandwich structure by T-Hg2+-T coordination and the fluorescence enhancement of the intercalated Genefinder (GF) in double strand DNA (dsDNA). Such supersandwich fluorescent DNA sensor exhibits a linear range of 10–300nM for the detection of Hg2+, with a detection limit of 2.5nM on the basis of the 3σ/slope (σ represents the standard deviation of the blank samples), which is well below the permit of the U.S. Environmental Protection Agency (<10nM). The detection can be fulfilled in less than 10min. The proposed mix-and-detect fluorescent platform exhibits excellent sensitivity, selectivity, and convenient manipulation. The assay was successfully used to detect Hg2+ in the lake water samples, which suggested its potential in practical samples.

Label-free fluorescent detection of thrombin activity based on a recombinant enhanced green fluorescence protein and nickel ions immobilized nitrilotriacetic acid-coated magnetic nanoparticles

Herein, a novel label-free fluorescent assay has been developed to detect the activity of thrombin and its inhibitor, based on a recombinant enhanced green fluorescence protein (EGFP) and Ni2+ ions immobilized nitrilotriacetic acid-coated magnetic nanoparticles (Ni2+–NTA MNPs). The EGFP, containing a thrombin cleavage site and a hexahistidine sequence (His-tag) at its N-terminal, was adsorbed onto Ni2+-NTA MNPs through Ni2+-hexahistidine interaction, and dragged out of the solution by magnetic separation. Thrombin can selectively digest EGFP accompanied by His-tag peptide sequence leaving, and the resulting EGFP cannot be captured by Ni2+-NTA MNPs and kept in supernatant. Hence the fluorescence change of supernatant can clearly represent the activity of thrombin. Under optimized conditions, such assay showed a relatively low detection limit (3.0×10−4UmL−1), and was also used to detect the thrombin inhibitor, Hirudin, and further applied to detect thrombin activity in serum. Combined with the satisfactory reusability of Ni2+-NTA MNPs, our method presents a promising candidate for simple, sensitive, and cost-saving protease activity detecting and inhibitor screening.

A Ligation Triggered Label-Free Fluorescent Assay for Adenosine-Triphosphate Based on Nicking Endonuclease Signal Amplification and Ligand Responsive G-Quadruplex Formation

A novel label-free fluorescent assay strategy was developed for sensitive and selective detection of adenosine-triphosphate (ATP) based on nicking endonuclease signal amplification (NESA) and N-Methyl mesoporphyrin (NMM)-responsive G-quadruplex formation. In this assay, the nick in the specially designed DNA duplex can be sealed by T4 DNA ligase in the presence of ATP. The following replication and strand displacement yield the duplex that contains the full recognition site for nicking endonuclease. Nicking enzyme's cutting the replicated DNA duplex results in a new replication site for polymerase and the continuous displacement of the nicked G-rich strand. The nicked strand can fold into G-quadruplex structure in the presence of K+ and selectively binds with NMM, yielding a significant fluorescence signal. Under the optimal conditions, the fluorescent assay strategy showed a dynamic response to ATP in the concentration range from 10 nM to 1000 nM and a detection limit of 0.8 nM, which compared favorably with most of the previous ATP assay methods. In addition, the proposed strategy exhibited superior selectivity toward ATP over other nucleosides and its analogues. The fluorescent assay was also applied in detecting ATP in serum with satisfactory results.

A label-free near-infrared fluorescent assay for the determination of deoxyribonuclease I activity based on malachite green/G-quadruplexes

Owing to the biological and clinical significance of deoxyribonuclease I (DNase I), it is highly desirable to develop near-infrared (NIR) fluorescent assays for the determination of DNase I activity. Here we report a label-free NIR fluorescent assay for selective determination of DNase I activity based on malachite green (MG)/G-quadruplexes. In the presence of Na(+) or K(+), single stranded DNA (ssDNA) is able to form a G-quadruplex structure, thus to increase the rigidity of MG structure and result in a remarkable NIR fluorescence. As DNase I is capable of cleaving all types of DNA indiscriminately to release nucleotide products, the G-quadruplexes are cleaved into oligonucleotides in the presence of DNase I. As a result, the rigidity of MG structure is reduced, and the NIR fluorescence of the solution decreases with increase of DNase I activity, providing a useful platform for low-cost, label-free and convenient detection of DNase I activity. Under the optimum conditions, the proposed label-free NIR fluorescent assay gave a detection limit of 1 u mL(-1), and a relative standard deviation of 3.2% for eleven replicate detections of 50 u mL(-1) DNase I. The proposed assay was applied to the determination of DNase I activity in spiked human urine samples with recoveries from 99.1 to 109.0%.

Fluorescent graphene quantum dots with a boronic acid appended bipyridinium salt to sense monosaccharides in aqueous solution

Using graphene quantum dots (GQDs) with a boronic acid-substituted bipyridinium salt (BBV), a label-free fluorescence assay for glucose detection is presented.

Immune-independent and label-free fluorescent assay for Cystatin C detection based on protein-stabilized Au nanoclusters

Cystatin C (Cys C) is a significant cysteine protease inhibitor in human bodies, and is proposed as a fascinating novel marker of glomerular filtration rate for kidney injury detection. Almost all traditional methods for Cys C measurement are immunoassays. In this article, we report a simple, immune-independent (no need to rely on immunoassay) and label-free method for Cystatin C detection using BSA-stabilized Au nanoclusters (Au NCs) as a fluorescent probe. This method relies on the BSA scaffold degradation caused by the cysteine protease activity of papain and the specific inhibition of papain activity by Cys C. The fluorescence of BSA-Au NCs can be effectively quenched by papain, and restored by the coexistence of Cys C. Under optimized conditions, this method enables sensitive and selective measurement of Cys C concentration in the range of 25ng/mL–2.0μg/mL with the detection limit of 4.0ng/mL, which is above 40 fold lower than that of commercial immune-based methods. SDS-PAGE, the absorption spectroscopy, transmission electron microscope, dynamic light scattering, and X-ray photoelectron spectroscopy were performed to discuss the quenching mechanism. In addition, percentage recoveries of Cys C in the spiked urine samples were ranged from 102.2% to 114.9% with the relative standard deviation ranging from 0.9–1.8%, demonstrating the applicability of the developed method in clinical samples. Furthermore, the present approach would be potentially extended to other proteases and their inhibitors detection with different protein-stabilized Au NCs.

Hybridization-triggered isothermal signal amplification coupled with MutS for label-free and sensitive fluorescent assay of SNPs

Combining the specific recognization of MutS protein for mismatched DNA sequences with the target-driven molecular switch that acts as both the template and primer of the polymerization reaction, a new label-free and sensitive fluorescent assay strategy for specific single-stranded DNA sequences or SNPs is proposed.

A carbon nanoparticle-based low-background biosensing platform for sensitive and label-free fluorescent assay of DNA methylation

Combining a DNA intercalator, SYBR Green I, and enzyme-linkage reactions with carbon nanoparticles, a low-background biosensing platform for label-free and sensitive fluorescent assay of DNA methylation is reported.

A sensitive strategy for label-free and time-resolved fluorescence assay of thrombin using Tb-complex and unmodified gold nanoparticles

Gold nanoparticles (GNPs) can effectively differentiate the unfolded and folded aptamer, and quench the fluorescence of terbium ternary complexes (Tb-complexes), thus the authors herein report a sensitive strategy for protein detection, using label-free aptamer, Tb-complexes and GNPs. In the presence of thrombin, the aptamer is inclined to form G-quartet, and the folded aptamer cannot adsorb on the surface of GNPs, inducing the GNPs aggregation in the presence of 0.5 mol L(-1) salt. After centrifugation at low speed to remove the aggregated GNPs, the quenching capability of the supernatant for Tb-complexes is decreased. The fluorescence intensity of Tb-complexes increases as the concentration of thrombin increases. Due to the highly specific recognition ability of the aptamer for thrombin and the strong quenching property of GNPs for Tb-complexes, the proposed protocol has good selectivity and high sensitivity for thrombin. Under the optimum conditions, a linear range from 1.0 × 10(-9) M to 1.0 × 10(-8) M is obtained with a detection limit of 0.14 nM, which is much lower than those commonly obtained for colorimetric sensors and some fluorescent sensors. The signal of Tb-complexes can be measured by time-resolved manner which made most of the unspecific fluorescent background signals be eliminated. The proposed sensor has been successfully applied in complicated biological samples for thrombin detection, and it can provide a promising potential for label-free aptamer-based protein detection.