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Prostate-specific antigen (PSA) is the only biomarker for the diagnosis of prostate cancer. So the PSA screening test is very important due to the high occurrence of prostate cancer in men. In this work, a label-free fluorescent method was developed based on terminal deoxynucleotidyl transferase (TdT) and G-quadruplex-thioflavin T complex for detecting PSA. In the absence of PSA, the PSA aptamer can be used as the primer for TdT extension reactions, resulting in the formation of G-quadruplexes and generation of strong fluorescent signals. After the addition of PSA, the PSA-aptamer complex prevented the TdT extension reaction due to steric hindrance, thus resulting in a poor fluorescent signal. The assay showed a wide linear range (0.1 to 80 pg/mL) and a detection limit of 0.086 pg/mL (S/N = 3). It also has good specificity for PSA determination and gives satisfactory results when applied to biological samples. Conceivably, its merits such as good selectivity and high sensitivity indicate that the proposed method has a promising application potential in the clinical diagnosis and treatment of prostate cancer. Graphical abstract.

In this report, we propose a fast, reliable and convenient approach to determine the alkaline phosphatase (ALP) activity based on a label-free fluorescence strategy. Upon catalysis of ALP, dephosphorylated dsDNA hampers the λ exonuclease (λexo) cleavage, shows high affinity to SYBR Green I (SG I), resulting in a strong fluorescence emission peak at 520 nm. In the absence of ALP, the dsDNA with 5′-phosphoryl-termini could be employed as a substrate of λexo. After cleavage, a weak fluorescence emission peaks at 520 nm could be observed. The assay was both selective and sensitive, and the detection limit was found to be as low as 3 U/L. This method was utilized to evaluate Na3VO4 as ALP inhibitor. The method was successfully applied to the determination of the activity of ALP in spiked human serum samples.

In this paper, we propose a fluorometric approach for the highly sensitive detection of inorganic pyrophosphatase (PPase) based on G-quadruplex-thioflavin T (ThT). In the absence of PPase, Cu2+ can coordinate with pyrophosphate (PPi) to generate a Cu2+/PPi complex. Then the G-rich sequence folds into the G-quadruplex structure, which can combine with ThT to generate a remarkable fluorescent signal. In the presence of PPase, the coordinated compound can be destroyed by the PPase catalyzed hydrolysis of PPi into inorganic phosphate (Pi). The subsequent release of Cu2+ can compete with ThT to induce a tighter G-quadruplex structure, causing the release of ThT and a sharp fluorescence decrease. Based on this mechanism, a facile and quantitative strategy for PPase detection was developed. The fluorescence intensity of the system shows a linear relationship with the PPase activities in the range of 0.5–30 U/L with a detection limit as low as 0.48 U/L. The proposed strategy for fluorescence spectrometric PPase detection is convenient, cost effective, and sensitive. This can be utilized to evaluate the inhibition effect of NaF on PPase as well as diagnose PPase-related diseases.

Actin is the most abundant protein in almost all eukaryotic cells. Due to the important biological properties of actin, it is necessary to develop a specific and sensitive method for actin detection. Herein, a label-free fluorescence assay for the detection of actin based on the digestion ability of deoxyribonuclease I (DNase I) and the formation of DNA-templated silver nanoclusters (DNA-AgNCs) is reported. In this strategy, two particular DNA sequences (Ag-DNA and G-rich DNA) were designed, synthesized, and used for the formation of DNA-AgNCs. Upon addition of DNase I, double-stranded DNA (dsDNA) degraded to form a shorter double-strand or mono-nucleotide that could not be further utilized to synthesize DNA-AgNCs. As a result, the reaction system generated a very low fluorescence signal. However, in the presence of actin, enzymatic digestion could be prevented due to the formation of a stable complex between actin and DNase I, ultimately resulting in an unbroken dsDNA that could be further used as a template for the fluorescent DNA-AgNCs (λex = 570 nm, λem = 620 nm). As a consequence, various actin concentrations could be detected by monitoring the fluorescence intensity variations. Because of good water solubility and excellent fluorescence properties of DNA-AgNCs, this novel fluorescence strategy exhibits several advantages such as being facile, sensitive and environment-friendly. The detection method featured a wide linear range from 0.1 to 20 μg mL−1 and a detection limit of 0.03 μg mL−1 (S/N = 3) under optimized conditions. Besides, this novel fluorescence strategy exhibited a good specificity and gives satisfactory results for biological samples. Overall, the proposed method has promising application potential in the quantification and detection of actin.

A sensitive and label-free fluorometric method has been developed for the determination of polynucleotide kinase (PNK) activity, by employing exonuclease III (Exo III)-assisted cyclic signal amplification and poly(thymine)-templated copper nanoparticles (polyT-CuNPs). In the presence of PNK, cDNA with 5'-hydroxyl termini was phosphorylated and then hybridized with tDNA to form the cDNA/tDNA duplex, which subsequently triggered the λ exonuclease cleavage reaction, eventually resulting in the release of tDNA. The released tDNA could unfold the hairpin structure of HP DNA to generate partially complementary duplex (tDNA/HP DNA), wherein the HP DNA possessed T-rich sequences (T30) and tDNA recognition sequence. With the help of Exo III digestion, the tDNA was able to initiate the cycle for the generation of T-rich sequences, the template for the formation of fluorescent CuNPs. Conversely, the cDNA could not be cleaved by λ exonuclease without PNK and individual HP DNA could not be hydrolyzed by Exo III. The T-rich sequence was caged in HP DNA, resulting in a weak fluorescence signal. Under optimized conditions, the fluorescence intensity was linearly correlated to a concentration range of 0.001 to 1 U mL-1 with a low detection limit of 2 × 10-4 U mL-1. Considering the intriguing analytical performance, this approach could be explored to screen T4 PNK inhibitors and hold promising applications in drug discovery and disease therapy.

This study describes a novel fluorometric method for the determination of methyltransferase (MTase) activity in DNA adenine methylation (Dam) by using graphene oxide (GO) and ribonuclease H (RNase H)-assisted signal amplification. In the presence of DNA adenine methylation methyltransferase (Dam MTase) and DpnI, the hairpin probe containing 5′-GATC-3′ as the recognition site is methylated by Dam MTase. Then, it is cleaved by methylation-sensitive restriction enzyme DpnI, and a single-stranded DNA (ssDNA) is released. In the next step, the released ssDNA hybridizes with FAM-labeled RNA (F-RNA) to generate DNA/RNA duplexes. Due to the specific ability of RNase H to hydrolyze RNA in RNA/DNA double strands, the F-RNA is digested by RNase H and the FAM fluorophore is released. Because this molecule is adsorbed on the GO surface, the fluorescence signal remains high. RNase H-assisted cyclic signal amplification is achieved by repeating the hybridization of ssDNA/F-RNA and digestion of F-RNA, a strategy allowing the fluorescence signal to be significantly amplified. The hairpin probe cannot be cleaved by DpnI and individual F-RNA cannot be degraded by RNase H without Dam MTase. As a result, the F-RNA is adsorbed by GO, resulting in quenching the fluorescence. Under the optimum conditions, the fluorescence intensity is linearly correlated in a concentration range of 0.1 to 10 U mL−1 with a low detection limit of 0.06 U mL−1. In addition, this approach proved to be trustworthy based on a successful application in inhibitor screening and determination of Dam MTase activity in human serum samples.

Herein we propose a label-free and sensitive detection method for coralyne and heparin, based on utilizing the complex of adenosine16 (A16) and coralyne to induce the formation of a G-quadruplex scaffold. In the present work, two specific oligonucleotide strands were designed. Upon addition of coralyne, the two oligonucleotide strands formed a double chain through A2–coralyne–A2 coordination, which induced the formation of an intermolecular G-quadruplex accompanied by the proximity of two G-rich sequences. An increase in system fluorescence was observed after the addition of N-methylmorpholine (NMM). In the presence of heparin, the formation of the heparin–coralyne complex caused coralyne to be removed from the A16–coralyne complex, leading to a weaker fluorescence output. At optimal concentrations, a linear coralyne response could be observed from 0.01–5 μM and the detection limit was estimated to be 5.8 nM. The detection limit of the heparin probe was as low as 1 nM, with a linear range from 1–100 nM. Overall, this study offers a sensitive and practical assay for coralyne and heparin detection.

In this paper, a label-free fluorescent method for glutathione (GSH) detection based on a thioflavin T/G-quadruplex conformational switch is developed. The sensing assay is fabricated depending on the virtue of mercury ions to form a thymine–thymine mismatch, which collapses the distance between two ssDNA and directs the guanine-rich part to form an intra-strand asymmetric split G-quadruplex. The newly formed G-quadruplex efficiently reacts with thioflavin T and enhances the fluorescent intensity. In the presence of GSH, Hg2+ is absorbed, destroying the G-quadruplex formation with a significant decrease in fluorescence emission. The proposed fluorescent assay exhibits a linear range between 0.03–5 μM of GSH with a detection limit of 9.8 nM. Furthermore, the efficacy of this method is examined using human serum samples to detect GSH. Besides GSH, other amino acids are also investigated in standard samples, which display satisfactory sensitivity and selectivity. Above all, we develop a method with features including potentiality, facility, sensitivity, and selectivity for analyzing GSH for clinical diagnostics.

MicroRNA (miRNA) expression is perturbed in various diseases. Herein, we have aimed to develop a novel and rapid fluorescence-based assay for detecting microRNA-21 (miR-21) activity based on FAM molecular signal amplification and graphene oxide (GO) quenching. In this system, a single stranded DNA (ssDNA) with a phosphate group at the 5'-end is labeled with a FAM molecular label at the 3'-end. In the presence of miR-21, this ssDNA forms a DNA/RNA duplex, which is cleaved by λ exonuclease (λ-exo), releasing FAM and resulting in fluorescence signal amplification at 530 nm. However, the DNA/RNA duplex is not generated in the absence of miR-21, which impedes λ-exo cleavage; subsequently, GO quenches the fluorescence intensity. The results show a detection limit of 0.02 nM and a wide linear range of 0.02-5 nM. The high sensitivity and easy operability of this assay can be applied for detecting miR-21 during clinical diagnosis of certain diseases and in biological research.

The authors describe a novel, facile, and sensitive fluorometric strategy based on a Cu2+-thiamine (Cu2+-TH) system for the detection of alkaline phosphatase (ALP) activity and inhibition. The principle of the method is as follows. Under a basic conditions, TH, which does not exhibit a fluorescence signal, is oxidized into fluorescent thiochrome (TC) by Cu2+. Ascorbic acid 2-phosphate (AAP), which is the enzyme substrate, is hydrolyzed to produce ascorbic acid (AA) by ALP. The newly formed AA then reduces Cu2+ to Cu+, which prevents the oxidation of TH by Cu2+; as a result, the fluorescent signal becomes weaker. On the contrary, in the absence of ALP, AAP cannot reduce Cu2+; additions of Cu2+ and TH result in a dramatic increase of the fluorescent signal. The sensing strategy displays brilliant sensitivity with a detection limit of 0.08 U/L, and the detection is linear in the concentration range of 0.1 to 100 U/L. This approach was successfully applied to ALP activity in human serum samples, indicating that it is reliable and may be applied to the clinical diagnosis of ALP-related diseases.

Thioflavin T (ThT), a water-soluble fluorescence probe and the conventional dye for the detection of amyloid fibrils has recently been demonstrated to recognize and bind nucleic acids. It induces the formation of G-quadruplex (G4) structure and appears as a sensor by emitting enhanced fluorescence. Incorporation of G-rich sequences in the nucleic acid amplification procedures assists the observation of the final products. In this manner, ThT with the “light-up” nature has emerged as a well-liked dye being devised in several biosensing applications. It offers sensitive and straightforward detection of biosubstrates including nucleic acids, proteins, small molecules as well as measurement of enzyme activities with the advantages of specificity, reduced background signal, and minimal cost. In this review, we aim to present a thorough overview of the ThT-based biosensing applications along with ThT/G-quadruplex complex formation and ThT-associated amplification techniques.

We proposed a colorimetric method for l -histidine detection based on Cu2+-mediated DNAzyme and G-quadruplex-hemin complex catalyzed oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS). In this system, after the addition of l -histidine, the formation of G-quadruplex-hemin complex will be disturbed, thus the colorimetric signal intensity conversely corresponds to the concentration of histidine. In this assay, a lower detection limit of l -histidine (50 nM) is addressed comparing to previously reported colorimetric methods. The cost is extremely low as the proposed design is both label-free and enzyme-free. All the more vitally, the colorimetric detection procedure is substantially straightforward with no further modification processes. By and large, the sensor can provide a promising plan for the detection of l -histidine.

In this paper, we have developed a label-free and rapid fluorescence assay for the detection of exonuclease III (exo III) activity via thioflavin T (ThT) as the G-quadruplex inducer. In this assay, a hairpin probe (HP) with a 5'-guanine-rich (G-rich) sequence is employed as the substrate for exo III. In the presence of exo III, HP can be digested at 3'-OH termini releasing 5'-G-rich sequence. Then, the 5'-G-rich sequence folds into a G-quadruplex, which can be recognized quickly by the ThT dye resulting in an increase in fluorescence emission. This strategy can detect exo III activity as low as 0.5 U/mL. This assay is simple and of low cost without the requirement of labeling with a fluorophore-quencher pair.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulated diverse cellular processes including differentiation, proliferation, apoptosis, metabolism and signal transduction pathways. An increasing number of data suggested that miRNA-21 could be identified as diagnostic and therapeutic biomarker for breast cancer. Meanwhile, inhibiting the function of miRNA-21, resulting in cells growth inhibition and apoptotic cells death. To realize miRNA-21detection and inhibition to diagnostic and therapeutic breast cancer cells, we developed gold nanoparticle-based 2'-O-methyl modified DNA probes (AuNP-2'-OMe-DNA probes) for diagnostic and therapeutic breast cancer. Gold nanoparticles were functionalized with chemically modified miRNA-21 inhibitor to suppress the function of miRNA-21 for the therapeutic breast cancer, at the same time, fluorophore-labeled DNA molecules were hybridized with antimiRNA-21 for diagnostic breast cancer. The results showed that the 2'-O-methyl modified DNA can improve stability, increase binding affinity to target strands and enhance the therapeutic effects. The experimental results also demonstrated that antimiR-21 were efficiently introduced into the cells and knocked down miRNA-21 to inhibit its function, leading to growth inhibition and apoptotic cells death. We prospected that chemically modified miRNA-21 inhibitor based on gold nanoparticles would be as a promising diagnostic and therapeutic platform for breast cancer clinically.

In this paper, we describe a novel, label-free and nicking enzyme-assisted fluorescence signal amplification strategy that demonstrates to be cost efficient, sensitive, and unique for assaying the RNase H activity and inhibition based on G-quadruplex formation using a thioflavin T (ThT) dye. This novel assay method is able to detect RNase H with a detection limit of 0.03 U /mL and further exhibits a good linearity R2 = 0.9923 at a concentration range of 0.03-1 U/mL under optimized conditions. Moreover, the inhibition effect of gentamycin on the RNase H activity is also studied. This strategy provides a potential tool for the biochemical enzyme analysis and inhibitor screening.

Recently, small molecule and targeted protein interactions have been applied frequently in quantitative detection, with the streptavidin (SA)–biotin model attracting most attention. We address a novel SA detection method based on the terminal protection of a small-molecule-linked DNA probe from degradation by exonuclease I and G-quadruplex-thioflavin T complex fluorescence signal amplification. With the signal amplification strategy, a limit of detection as low as 0.065 nM was reached. The developed strategy provides a new route to a low cost and highly sensitive and selective method of detection of SA in real samples.

Assays of alkaline phosphatase (ALP) activity play a critical role in clinical diagnostics and drug screening. In this work, we present a novel, sensitive and cost-effective analytical method for the detection of ALP activity based on the design of a G-rich DNA light-up DNA–silver nanocluster (AgNCs) probe and λ exonuclease (exo) cleavage reaction. Upon addition of λ exo, the G-rich DNA was cleaved, resulting in the inhibition of AgNCs to move closer to G-rich DNA sequences and, accordingly, only a low fluorescence signal was observed. Upon treatment of ALP, the 5′-phosphoryl end of pG-rich DNA was hydrolyzed and the λ exo cleavage reaction was impeded. The AgNCs were then enabled to move closer to the G-rich DNA sequences, resulting in an increase in fluorescence. Under optimized conditions, the fluorescence change was determined to be linear with the ALP concentration ranging between 1 U L−1 and 800 U L−1 (detection limit: 1 U L−1). Taken in concert, this strategy may provide a basis for a screening platform for ALP inhibitors.

As a tumor marker, carcinoembryonic antigen (CEA) is an effective indicator for the evaluation of the response to treatments of cancers. Therefore, it is necessary to find some sensitive and effective methods for CEA detection. Herein, we describe a facile method of CEA determination based on dsDNA-templated copper nanoparticles (CuNPs) coupled with an aptamer. In this fluorescence bioassay, the dsDNA residue contains a CEA-specific aptamer that seizes the formation of fluorescent CuNPs in the presence of CEA, resulting in weak fluorescence emission. In the absence of CEA, the dsDNA-templated CuNPs can generate strong fluorescence with emission peaks at 565 nm. This assay enables a sensitive and precise determination of CEA with a detection limit of 0.0065 ng mL−1 (S/N = 3) and functions successfully in biological samples.

The C-terminal domains of proteases play crucial roles in hydrolysis, substrate adsorption and targeted binding. Identifying and characterizing interactions between C-terminal domains and biomacromolecules can help to examine the diversity as well as the substrate-binding ability of C-terminal domains and to explore novel functions. The bacterial pre-peptidase C-terminal (PPC) domain is a typical C-terminal domain normally found at the C-terminus of bacterial secreted proteases. In this work, we successfully demonstrated that 8-anilinonaphthalene-1-sulfonic acid (ANS) could be used to rapidly determine the interactions between this C-terminal domain and biomacromolecules. The time-resolved ANS fluorescence of PPC and collagen interaction could be used for quantitative analysis of the collagen-binding capability based on the slope of the time-scanning curve. Using this method, we found that PPC domains had an obvious affinity to fibrillar proteins but had little or no capacity to bind polysaccharides or linear DNAs. Docking studies proved that collagen bound to the same hydrophobic site of PPC as the ANS probe, causing a decrease in the emission intensity. This method is simple and cost effective and provides an effective detection technique to analyze the interaction between this C-terminal domain and biomolecules.

A new class of intracellular nanoprobe, termed AuNP loaded split-DNAzyme probe, was developed to sense miRNA in living cells. Briefly, it consists of an AuNP and substrates hybridized with two half of split DNAzymes. In the absence of target miRNA, the split DNAzymes form an inactive DNAzyme motif with their substrate through partial paring at the end of each strand, and the fluorescence is quenched. Inside the cells, the target miRNA binds with both of the two half of split DNAzymes, forming the active secondary structure in the catalytic cores, which can cleave the substrates, resulting in the rupture of the substrate and recovery of the fluorescence. Meanwhile, the target is released and binds to another inactive DNAzyme motif to drive another cycle of activation. During the cyclic process, a very small number of target miRNAs can initiate the cleavage of many fluorophore-labeled substrate strands from AuNP surface, providing an amplified fluorescent signal of the target miRNA and, thus, offering high detection sensitivity.

In this assay, a label-free fluorescent sensing platform based on triple-helix molecular switch (THMS) and G-quadruplex was developed for the detection of tetracycline. We demonstrated this approach by using THMS, which consists of a central section with a shortened 8-mer aptamer sequence with high affinity to tetracycline and flanked by two arm segments. G-rich oligonucleotide can specifically bind to thioflavin T (ThT) as a signal transduction probe (STP). In the absence of tetracycline, THMS remains stable, the fluorescence of background is low. By the addition of target tetracycline, the aptamer-target binding results in the formation of a structured aptamer-target complex, which disassembles the THMS and releases the STP. The free STP self-assembles into G-quadruplex and specifically binds to ThT which generates a obvious fluorescence enhancement. Using the triple-helix molecular switch, the developed aptamer-based fluorescent sensing platform showed a linear relationship with the concentration of tetracycline ranging from 0.2 to 20.0 nmol/L. The detection limit of tetracycline was determined to be 970.0 pmol/L. The assay avoids complicated modifications or chemical labeling, making it simple and cost-effective. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.

The authors describe a method for the determination of the activity of alkaline phosphatase (ALP) that utilizes dsDNA-templated copper nanoparticles (CuNPs) coupled to enzymatic amplification via λ exonuclease. A hybrid of a DNA modified with a phosphate moiety at the 5′-end (P-DNA) and a P-DNA complementary sequence (cP-DNA) is employed as the dsDNA substrate for ALP. In the absence of ALP, the dsDNA is cleaved by the λ exonuclease, which hinders the formation of CuNPs which display fluorescence with excitation/emission peaks at 340/565 nm. However, ALP-mediated hydrolysis of the 5′-phosphoryl end impedes the cleavage of dsDNA by the λ exonuclease, and this promotes the formation of fluorescent dsDNA-templated CuNPs via ascorbate-mediated reduction. Under the optimized experimental conditions, this method exhibits a high specificity to ALP and has a 0.1 U⋅L−1 limit of detection. The strategy also provides the basis for a screening platform for inhibitors of ALP.

Ribonuclease H (RNase H), a highly conserved damage-repair protein, hydrolyzes RNA in the DNA:RNA hybrid duplex and breaks RNA/DNA junctions. In this study, we demonstrated a unique Tb3+-based fluorescence assay that is low cost, facile, and label-free for assaying RNase H activity and inhibitions by using a G-quadruplex formation strategy. This novel assay method can detect RNase H at the lowest detection limit of 2 U mL−1 under optimal conditions. We demonstrated the utility of the assay by antibiotics screening and identified ethidium bromide (EB) and Gentamycin as RNase H inhibitors. This approach demonstrated that Tb3+ could be used as a functional tool in specific fields in the future.

This work described a new method for the detection of humic acid (HA) based on the poly(thymine) (poly T)-templated copper nanoparticles (CuNPs). Without the presence of HA, the formation of poly T-templated CuNPs could take place, resulting in strong fluorescence emission peaks at 610 nm (upon excitation at 340 nm). On the other hand, when HA was present, strong interaction between HA and Cu 2+ took place, which then hampered the effective formation of fluorescent CuNPs, leading to the decrease in fluorescence intensity. Furthermore, under the optimal experimental conditions, the method exhibited a high specificity to HA with a detection limit of 0.4 mg/L. This work has demonstrated a low-cost and convenient method that could be accomplished within 10 min. The method could provide a simple, rapid, and sensitive fluorescent platform for the detection of HA.

Endonucleases, one of the basic tool enzymes of modern molecular biology and medical genetics, have also been clarified as the potential targets for antimicrobial and antiviral drugs screening. However, traditional assays to monitor endonuclease activity can be expensive, time-consuming, or laborious. In order to provide new detective platform, we proposed a novel label-free one-step fluorescent method for the detection of endonuclease activity based on cleavage-induced G-quadruplex formation. In this detection system, a simple DNA probe can spontaneously form a duplex structure with recognition sites of EcoRI and prevent the generation of the G-quadruplex. Once EcoRI is present, the recognition sites in the duplex DNA are cleavage, producing a free guanine-rich sequence to form G-quadruplex. When thioflavin T (ThT) is added, a strong fluorescence signal is given by ThT/G-quadruplex, and therefore the EcoRI activity can be detected. After systematic investigation and optimization, this method has gained a sensitive limit of detection at 0.75 U/mL, and a wide detection range between 0.75 U/mL and 120 U/mL. Furthermore, the inhibitory effect of 5-fluorouracil on EcoRI activity was verified and IC50 was calculated. Taken together, these experimental results have proven that this turn-on fluorescent method has considerable analytical performances. As far as we are concerned, this method is the first reported EcoRI assay based on ThT/G-quadruplex, and only one kind of probe and one kind of dye are involved, providing one of the simplest detective strategies on EcoRI. More importantly, the convenience and cost make this one-step method quite attractive for application transformation. Therefore, we hope this method could be a hopeful option for EcoRI activity determination, and further to help monitoring the quality of tool enzymes and promote the development of high-through automatic drug screening system.

The bacterial prepeptidase C-terminal (PPC) domain can be found in the C termini of a wide variety of proteases that are secreted by marine bacteria. However, the functions of these PPC domains remain unknown due to a lack of systematic research. Here, the binding and swelling abilities of eight PPC domains from six different proteases were compared systematically via scanning electron microscopy (SEM), enzyme assays, and fluorescence spectroscopy. These PPC domains all possess the ability to bind and swell insoluble collagen. PPC domains can expose collagen monomers but cannot disrupt the pyridinoline cross-links or unwind the collagen triple helix. This ability can play a synergistic role alongside collagenase in collagen hydrolysis. Site-directed mutagenesis of the PPC domain from Vibrio anguillarum showed that the conserved polar and aromatic residues Y6, D26, D28, Y30, W42, E53, C55, and Y65 and the hydrophobic residues V10, V18, and I57 played key roles in substrate binding. Molecular dynamic simulations were conducted to investigate the interactions between PPC domains and collagen. Most PPC domains have a similar mechanism for binding collagen, and the hydrophobic binding pocket of PPC domains may play an important role in collagen binding. This study sheds light on the substrate binding mechanisms of PPC domains and reveals a new function for the PPC domains of bacterial proteases in substrate degradation. IMPORTANCE Prepeptidase C-terminal (PPC) domains commonly exist in the C termini of marine bacterial proteases. Reports examining PPC have been limited, and its functions remain unclear. In this study, eight PPCs from six different bacteria were examined. Most of the PPCs possessed the ability to bind collagen, feathers, and chitin, and all PPCs could significantly swell insoluble collagen. PPCs can expose collagen monomers but cannot disrupt pyridinoline cross-links or unwind the collagen triple helix. This swelling ability may also play synergistic roles in collagen hydrolysis. Comparative structural analyses and the examination of PPC mutants revealed that the hydrophobic binding pockets of PPCs may play important roles in collagen binding. This study provides new insights into the functions and ecological significance of PPCs, and the molecular mechanism of the collagen binding of PPCs was clarified, which is beneficial for the protein engineering of highly active PPCs and collagenase in the pharmaceutical industry and of artificial biological materials.

Prostate specific antigen (PSA) is one of the most common biomarkers for the management of prostate cancer. However, it still remains urgent to develop highly sensitive, cost-effective and selective strategies for PSA assay. In this paper, we developed a low-cost, highly sensitive and specific analytical strategy for the detection of PSA by using a fluorescence sensor based on Pb2+-dependent DNAzyme. We designed a DNA sequence called cmMB with a hairpin structure, containing PSA-specific aptamers and Pb2+-dependent DNAzyme chains. Also, a fluorophore-labelled DNA sequence called Sub-FAM, which contains a cleavage site of Pb2+-dependent DNAzyme and serves as substrate, is also designed for the signal generation. In the presence of PSA, interaction between aptamer and PSA blocks the hairpin structure of cmMB, resulting in the formation of Pb2+-dependent DNAzyme with Pb2+. Then, Pb2+-dependent DNAzyme can cleavage Sub-FAM and produce a high fluorescence. In the absence of PSA, since Sub-FAM remains to be ssDNA and can be absorbed by GO, only low fluorescence can be detected. Under optimal experimental conditions, a good linear relationship in the range of 1–100 pg mL−1 was exhibited, with a limit of detection (LOD) of 0.76 pg mL−1. In addition, the proposed method has potential value in the diagnosis and monitoring of prostate cancer because of its good selectivity and practical application in biological samples.

A reliable fluorometric assay is described for the determination carcinoembryonic antigen (CEA) using exonuclease III (Exo III) and a 2-aminopurine binding aptamer. In the absence of CEA, dsDNA is degraded by Exo III, and free 2-AP (which has a blue fluorescence with excitation/emission maxima of 310/365 nm) is released. Strong fluorescence is generated after addition of graphene oxide (GO) to the solution. However, the 2-AP modified DNA (T2) cannot be degraded in the presence of CEA by Exo III due to the interaction between CEA and aptamer T1. Hence, only weak fluorescence can be detected after addition of GO. In this system, CEA can be quantified in the 0.05 - 2 ng·mL-1 concentration range with a detection limit of 30 pg·mL-1 (at S/N = 3). The method was successfully applied to analyze serum samples for CEA.

In this work, a novel, simple, and time-saving fluorescence approach for the detection of biothiols (glutathione and cysteine) was developed by employing a DNA probe labeled with 2-aminopurine. As an adenine analogue, 2-aminopurine exhibits high fluorescence intensity that can be rapidly quenched in the presence of DNA. In the presence of Ag+, the fluorescence increased significantly, which was a result of the formation of cytosine&ndash, Ag+&ndash, cytosine base pairs and the release of 2-aminopurine. Upon addition of either glutathione or cysteine, the structure of cytosine&ndash, cytosine was disrupted, a product of the stronger affinity between biothiols and Ag+. As a result, the 2-aminopurine-labeled DNA probe returned to its former structure, and the fluorescence signal was quenched accordingly. The detection limit for glutathione and cysteine was 3 nM and 5 nM, respectively. Furthermore, the determination of biothiols in human blood serum provided a potential application for the probe as a diagnostic tool in clinical practice.

The base-excision repair enzyme uracil-DNA glycosylase (UDG) plays a crucial role in the maintenance of genome integrity. The authors describe a fluorometric method for the detection of the activity of UDG. It is making use of (a) a 3’-FAM-labeled hairpin DNA probe with two uracil deoxyribonucleotides in the self-complementary duplex region of its hairpin structure, (b) exonuclease I (Exo I) that catalyzes the release of FAM from the UDG-induced stretched ssDNA probe, and (c) graphene oxide that quenches the green FAM fluorescence of the intact hairpin DNA probe in the absence of UDG. If Exo I causes the release of FAM from the hairpin DNA probe, the fluorescence peaking at 517 nm is turned off in the absence of UDG but turned on in its presence. The resulting assay has a wide linear range (0.008 to 1 U·mL−1) and a detection limit as low as 0.005 U·mL−1. It has good specificity for UDG over potentially interfering enzymes and gave satisfactory results when applied to biological samples. Conceivably, the method may be used in a wide range of applications such as in diagnosis, drug screening, and in studying the repair of DNA lesions.

This study describes a novel quencher-free fluorescent method for ochratoxin A (OTA) detection based on the photoinduced electron transfer (PIET) between guanine and fluorophore. In the absence of OTA, carboxyfluorescein (FAM)-labeled aptamer can partly hybridize with the complementary strand of OTA aptamer (OTA-cAPT), which contains four guanines at its 3&prime, end. As a result, the fluorescence of FAM is quenched due to PIET and stacked guanines. In the presence of OTA, FAM-labeled OTA aptamer can bind specifically to OTA, and thereby the high fluorescence intensity of the dye can be maintained. Under the optimal conditions, the method had a detection limit of 1.3 nM. In addition, the method we proposed is highly sensitive and specific for OTA. Furthermore, the method was proven to be reliable based on its successful application in the detection of OTA in red wine samples. Therefore, this promising, facile, and quencher-free method may be applied to detect other toxins by using other appropriate aptamers.

G-quadruplex is a three-dimensional secondary structure of nucleic acids formed by the Hoogsteen hydrogen pairing of four guanines. Diverse topologies of G-quadruplex could be employed in biosensing and bioimaging. By intercalating fluorescence dyes into G-quadruplex or forming a horseradish peroxidase (HRP)-mimicking G-quadruplex/hemin DNAzyme, G-quadruplexes based biosensors realized the sensitive and selective detection of nucleic acids, protein, enzyme activity, ions, small molecules, exosomes, cells, and microorganisms. The vital role that cellular G-quadruplexes played in genome further facilitated the application of G-quadruplex stabilizing on cancer therapy. Combined with G-quadruplex aptamer, which is an efficient therapeutic tool, a current landscape of the application potential of this fascinate nucleic acids structure from clinical diagnosis to cancer therapy is summarized here.

As an important DNA 3′-phosphatase, alkaline phosphatase can repair damaged DNA caused by replication and recombination. It is essential to measure the level of alkaline phosphatase to indicate some potential diseases, such as cancer, related to alkaline phosphatase. Here, we designed a simple and fast method to detect alkaline phosphatase quantitively. When alkaline phosphatase is present, the resulting poly T-DNA with a 3′-hydroxyl end was cleaved by exonuclease I, prohibiting the formation of fluorescent copper nanoparticles. However, the fluorescent copper nanoparticles can be monitored with the absence of alkaline phosphatase. Hence, we can detect alkaline phosphatase with this turn-off strategy. The proposed method is able to quantify the concentration of alkaline phosphatase with the LOD of 0.0098 U/L. Furthermore, we utilized this method to measure the effects of inhibitor Na3VO4 on alkaline phosphatase. In addition, it was successfully applied to quantify the level of alkaline phosphatase in human serum. The proposed strategy is sensitive, selective, cost effective, and timesaving, having a great potential to detect alkaline phosphatase quantitatively in clinical diagnosis.

In this paper, a fast and simple strategy for sensitive detection of streptavidin (SA) was proposed based on terminal protection of small molecule-linked DNA and cationic conjugated polymer-mediated fluorescence resonance energy transfer (FRET). In principle, we designed a biotin-labelled DNA probe (P1) as the recognitive probe of SA, along with a complementary DNA probe (P2) to form double-stranded DNA (dsDNA) with P1. SYBR Green I (SG I) as a fluorescent dye was further used to specifically bind to dsDNA to emit stronger fluorescence. The cationic poly[(9,9-bis(6′-N,N,N-triethy-lammonium)hexyl) fluorenylene phenylene dibromide] (PFP) acted as the donor to participate in the FRET and transfer energy to the recipient SG I. In the absence of SA, P1 could not hybridize with P2 to form dsDNA and was digested by exonuclease I (Exo I), thus, only a weak FRET signal would be observed. In the presence of SA, biotin could specifically bind to SA, which protected P1 from Exo I cleavage. Then, P1 and P2 were hybridized into dsDNA. Therefore, the addition of SG I and PFP led to obvious FRET signal due to strong electrostatic interactions. Then, SA can be quantitatively detected by monitoring FRET changes. As the whole reagent reaction was carried out in 1.5 mL EP and detected in the colorimetric dish, the operation process of the detection system was relatively simple. The response time for each step was also relatively short. In this detection system, the linear equation was obtained for SA from 0.1 to 20 nM with a low detection limit of 0.068 nM (S/N = 3). In addition, this strategy has also achieved satisfactory results in the application of biological samples, which reveals the application prospect of this method in the future.

We have developed a new methodology for fluorescence turn-on detection of DNA methyltransferase (MTase) activity based on terminal deoxynucleotidyl transferase (TdT) using a thioflavin T probe. This method is highly selective and sensitive. The fluorescence intensity was direct proportion to Dam MTase concentration in the range from 0.1 to 8.0 U/mL with a detection limit of 0.1 U/mL. And because no labeling with a fluorophore-quencher pair was required, it is simple and low cost. We envision that our novel fluorescent detection method for Dam MTase activity could be applied as a useful tool in biomedical research.

We have developed a new methodology for the label-free detection of alkaline phosphatase (ALP) activity based on a DNA hairpin and thioflavin T (ThT) probes. In the presence of ALP, the 3′-phosphate of the primer DNA is dephosphorylated, triggering a primer extension reaction to open the hairpin probe and leading to release of the G-quartets. These then bind to ThT to form ThT/G-quadruplexes with obvious fluorescence generation. The method is highly sensitive, and the ALP detection limit was estimated to be 0.001 U mL−1, which is superior or comparable to those of reported assays. The assay avoids complicated modifications or chemical labeling, making it simple and cost-effective. Furthermore, the inhibition of ALP activity by the inhibitor theophylline is demonstrated, indicating the potential for the discovery and characterization of ALP-targeted drug candidates in pharmaceutical development.

Here, we have developed a novel fluorescence strategy for sensitive detection of uracil DNA glycosylase (UDG) activity based on G-quadruplex formation by using a label-free DNA hairpin probe. In the presence of UDG, it catalyzed the hydrolysis of the uracil bases in the hairpin DNA, resulting in the conformational transition of the hairpin structure. Then, the probe DNA can be recognized quickly by the thioflavin T (ThT) dye resulting in an increase in fluorescence. This strategy could detect UDG activity as low as 0.01 U mL−1. In addition, the strategy was also applied for the detection of UDG activity in HeLa cell lysate. It is simple and of low cost without the requirement of labeling with a fluorophore–quencher pair. Furthermore, UDG activity inhibition by uracil glycosylase inhibitor (UGI) is shown, demonstrating the potential for the discovery and characterization of UDG-targeted drug candidates in pharmaceutical development.

We have developed a new assay for the detection of T4 polynucleotide kinase/phosphatase activity (T4 PNKP) based on a terminal transferase-aided photoinduced electron transfer (PIET) strategy. The method is highly sensitive and the T4 PNKP detection limit was estimated to be 0.01 U mL−1, which is superior or comparable to previously reported assays. Furthermore, the proposed method was also applied to assay the inhibition of T4 PNKP activity. This approach may offer potential applications in drug screening, clinical diagnostics and some other related biomedical research.

In this study, we developed an aptamer-based fluorescent sensing platform for the detection of ochratoxin A (OTA) based on RecJf exonuclease-assisted signal amplification and interaction between graphene oxide (GO) and the OTA aptamer (OTA-apt). After optimizing the experimental conditions, the present aptamer-based sensing system can exhibit excellent fluorescent response in the OTA assay, with a limit of detection of 0.07 ng/mL. In addition to signal amplification, this strategy is also highly specific for other interfering toxins. Furthermore, this aptasensor can be reliably used for assessing red wine samples spiked with different OTA concentrations (2.4, 6 and 20 ng/mL). The proposed assay plays an important role in the field of food safety and can be transformed for detecting other toxins by replacing the sequence that recognizes the aptamer.

With the development of oncology and bioanalytical chemistry, liquid biopsy emerges as their fruit and manages to bring perspective chance of healthcare. To date, several typical biomarkers (i.e circulating tumor cells, extracellular vesicles, circulating nuclei acids, etc) have been well-established as promising targets of liquid biopsy, and numerous methods, of which optical and electrochemical methods occupy in majority, have been proposed for the idealized detection of these targets. However, the advancements in this field are massive from cancer biology and analytical technology. Therefore, it is necessary to review these advancements from both medical and chemical sides. Here in this paper, our group, of which academic background from medicine and chemistry, aim to provide some comprehensive and in-depth summarization on contribution of optical and electrochemical methods to liquid biopsy, considering both the view of oncologist and analytical chemists. Besides, we will discuss the remaining challenges and future trend of this field in the end. We hope this review can be inspirational to researcher in this field and give helpful introduction to people from various research area.

Herein, a turn-on fluorescence assay was introduced for alkaline phosphatase (ALP) detection based on ThT/G-quadruplex system. The basis of the method is that chelation of guanine bases at the binding sites by Ag+ blocks G-quadruplex formation and decreases the fluorescence intensity sharply. In the presence of ALP, ascorbic acid 2-phosphate (AAP) is hydrolyzed to form ascorbic acid (AA) which in turn reduces Ag+ to Ag0. As a result, the blockage ability of Ag+ is disrupted which augments the fluorescence intensity and relies on the concentration of ALP. Under the optimized parameters (500 nM DNA probe; 6 μM Ag+; 1 mM AAP; 30 min for Ag+ and DNA probe reaction time), fluorescence intensity correlates linear range between 1 and 100 U/L of ALP concentration with the detection limit of 0.503 U/L. In the inhibition assay, 50% of ALP inhibition is caused by Na3VO4 with a concentration of 0.254 mM. Furthermore, the assay was used to detect ALP activity in human serum samples in which the results were significant. Above all, the proposed strategy is potential, facile, and sensitive for analyzing ALP activity and screening ALP inhibitor.

Prostate specific antigen (PSA) is considered to be a well-established biomarker for prostate cancer, and the importance of PSA assay is increasing in order to improve the diagnosis and prognosis of prostate cancer. However, there are still some weaknesses with current methods of PSA detection including high-cost, heavy analyzer and complexed procedure, restricting the application of PSA assay in prevention of diseases. In this paper, we designed a novel fluorometric aptasensor for PSA assay. Inspired by the concept of enzyme-assisted target recycling (EATR), this novel aptasensor shows an excellent signal responsiveness by the exclusive recognition between aptamer and PSA, together with significant signal amplification through EATR. After systematic optimization, this method shows an excellent detective performance with a sensitive LOD at 0.043 pg mL−1(S/N = 3) and wide linear range of 0.05–150 pg mL−1. To verify the potential of practical application, a set of biomarkers that often co-exist in PSA samples and diluted human serum samples are used for selectivity and practical performances evaluation, and it turns out that this aptasensor exhibits selectivity and potential for clinical application. Specially, this method is easily performed under isothermal environment and needless of complexed preparation, making it more possible for wide application. In view of the superiority, a promising perspective can be provided by this method for practical PSA assay, further promoting the development of precise medicine and oncology.

Adenosine deaminase (ADA), able to catalyze the irreversible deamination of adenosine into inosine, can be found in almost all tissues and plays an important role in several diseases. In this work, we developed a label-free fluorescence method for the detection of adenosine deaminase activity and inhibition. In the presence of ADA, ATP has been shown to be hydrolyzed. The ATP aptamer was shown to form a G-quadruplex/thioflavin T (ThT) complex with ThT and exhibited an obvious fluorescence signal. However, the ATP aptamer could bind with ATP and exhibited a low fluorescence signal because of the absence of ADA. This assay showed high sensitivity to ADA with a detection limit of 1 U/L based on an SNR of 3 and got a good linear relationship within the range of 1&ndash, 100 U/L with R2 = 0.9909. The LOD is lower than ADA cutoff value (4 U/L) in the clinical requirement and more sensitive than most of the reported methods. This technique exhibited high selectivity for ADA against hoGG I, UDG, RNase H and &lambda, exo. Moreover, this strategy was successfully applied for assaying the inhibition of ADA using erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and, as such, demonstrated great potential for the future use in the diagnosis of ADA-relevant diseases, particularly in advanced drug development.

The authors describe a fluorometric assay for ochratoxin A (OTA) that is based on the use of graphene oxide and RNase H-aided amplification. On addition of OTA, cAPT is replaced from the APT/cAPT hybridization complex and then hybridizes with RNA labeled with a fluorophore at the 5'-end. Eventually, the fluorophore is released by RNase H cleavage. As the concentration of OTA increases, more cAPTs are displaced, this leading to fluorescence enhancement (best measured at excitation/emission wavelengths of 495/515 nm). This RNase H-assisted cycle response results in strong signal amplification. The limit of detection, calculated on the basis of a signal to noise ratio of 3, is 0.08 ng·mL-1. Response is linear in the 0.08-200 ng·mL-1 OTA concentration range. The method is highly selective for OTA over ochratoxin B and aflatoxin B1. It was applied to the determination of OTA in red wine samples spiked at levels of 1, 7, and 50 ng·mL-1, and the recoveries ranged from 90.9 to 112%. Graphical abstract Schematic of a novel fluorometric aptasensor for ochratoxin A based on the use of graphene oxide and RNase H-aided amplification.

Ochratoxin A (OTA) is one of the most common mycotoxins contaminating feed and foodstuffs. Therefore, a great deal of concern is associated with AFB1 toxicity. In this work, a fast and sensitive fluorescence aptamer biosensor has been proposed for the OTA assay. In the absence of OTA, the OTA aptamer can form a G-quadruplex structure with thioflavin T (ThT) dye, which results in increased fluorescence. After joining OTA, OTA aptamer combines with OTA and the G-quadruplex can be formed. Only faint fluorescence was finally observed when ThT weakly reacts with the quadruplex. Through this test method, the entire reaction and analysis process of OTA can be completed in 10 min. Under optimal experimental conditions (600 nM OTA-APT, 7 μM ThT, and 3 min incubation time), this proposed assay has a good limit of detection (LOD) of 0.4 ng/mL and shows a good linear relationship within the range of 1.2–200 ng/mL under the best experimental conditions. This method has a high specificity for OTA relative to Ochratoxin B (23%) and Aflatoxin B1 (13%). In addition, the quantitative determination of this method in real samples has been validated using a sample of red wine supplemented with a range of OTA concentrations (1.2 ng/mL, 12 ng/mL, and 40 ng/mL) with recoveries of 96.5% to 107%.

A real-time assay for DNA methyltransferase (MTase) activity has been developed. A hemimethylated smart probe is used as the substrate for DNA MTase. Cleavage of the methylated product leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. The method permits real-time monitoring of DNA methylation process and makes it easy to characterize the activity of DNA MTase. It also has the potential to screen suitable inhibitor drugs for DNA MTase.

Traditional methods of assaying polynucleotide kinase (PNK) activity are discontinuous, time-consuming, and laborious. Here we report a new quencher-free approach to real-time monitoring of PNK activity using a 2-aminopurine probe. When the 2-aminopurine probe was 5'-phosphorylated by PNK, it could be efficiently degraded by lambda exonuclease to release free 2-aminopurine molecules and generate a fluorescence signal. This method not only provides a universal approach to real-time monitoring of PNK activity, but also shows great potential for screening suitable inhibitor drugs for PNK.

In this work, we developed a facile fluorescence method for quantitative detection of human serum albumin (HSA) based on the inhibition of poly(thymine) (poly T)-templated copper nanoparticles (CuNPs) in the presence of HSA. Under normal circumstances, poly T-templated CuNPs can display strong fluorescence with excitation/emission peaks at 340/610 nm. However, in the presence of HSA, it will absorb cupric ion, which will prevent the formation of CuNPs. As a result, the fluorescence intensity will become obviously lower in the presence of HSA. The analyte HSA concentration had a proportional linear relationship with the fluorescence intensity of CuNPs. The detection limit for HSA was 8.2 × 10−8 mol·L−1. Furthermore, it was also successfully employed to determine HSA in biological samples. Thus, this method has potential applications in point-of-care medical diagnosis and biomedical research.