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MicroRNAs (miRNAs) are commonly used as biomarkers for the diagnosis of tumors. Since miRNA expression is strongly correlated to carcinogenesis, the detection of miRNA concentration in cells would be valuable for the diagnosis and evaluation of tumors. In this study, we proposed a system using two strands of DNA, one modified by a phosphate group at the 5' end, called Cap, and the other with a hairpin structure, called HP. The Cap chain could open the hairpin structure of HP and expose the sequences rich in G bases to form the G-quadruplex structure. Then, a strong fluorescence signal was emitted in the presence of N-methyl mesoporphyrin IX (NMM). However, with the addition of miRNA-21, Cap hybridized with it to form double chains, which were then cleaved by the digestion of lambda exonuclease, resulting in a weak fluorescent signal. The proposed method could detect miRNA-21 at a concentration of 1.4 pM with a broad dynamic linear range from 5 pM to 5 nM.

As a representative biochemical indicator, alkaline phosphatase (ALP) is of great importance in indicating and diagnosing clinical diseases. Herein, we developed a signal-on fluorescence sensing method for sensitive ALP activity detection based on the enzyme-assisted target recycling (EATR) technique. In this method, a two-step signal amplification process is designed. In the presence of ALP, the 3' phosphate group of an ss-DNA is removed explicitly by ALP, thus releasing free 3'-OH. Terminal deoxynucleotidyl transferase (TdT) can subsequently extend this substrate to generate poly(A) tails, converting the trace-level ALP information into multiple sequences and achieving the first-time amplification. A poly(T) Taqman probe labeled with FAM and BHQ1 provides the second one under the assistance of T7 exonuclease (T7 Exo) through alternate hybridization and degradation of ds-DNA regions. The previously quenched fluorescence is recovered due to the departure of FAM/BHQ1 during the cleavage of T7 Exo. Thus, taking advantage of template-free TdT-mediated polymerization and T7 Exo-based EATR, this strategy shows a sensitive LOD at 0.0074 U/L (S/N = 3) and a linear range of 0.01-8 U/L between ALP concentration and fluorescence intensity. To further verify the specificity and accuracy in practical application, we challenged it in a set of co-existing interference and biological environments and have gained satisfying results. The proposed method successfully quantified the ALP levels in clinical human serum samples, suggesting its applicability in practical application. Moreover, we have used this method to investigate the inhibition effects of Na

Base excision repair (BER) is vital for maintaining the integrity of the genome under oxidative damage. DNA glycosylase initiates the BER pathway recognizes and excises the mismatched substrate base leading to the apurinic/apyrimidinic site generation, and simultaneously breaks the single-strand DNA. As the aberrant activity of DNA glycosylase is associated with numerous diseases, including cancer, immunodeficiency, and atherosclerosis, the detection of DNA glycosylase is significant from bench to bedside. In this review, we summarized novel DNA strategies in the past five years for DNA glycosylase activity detection, which are classified into fluorescence, colorimetric, electrochemical strategies, etc. We also highlight the current limitations and look into the future of DNA glycosylase activity monitoring.

Alkaline phosphatase is one of the most important tool enzymes and diseases indicator, monitoring ALP activity with convenient, precise, efficient and sensitive methods plays a fundamental role in modern life and healthcare industries. In this study, we described a novel method for ALP analysis based on Pb

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.

Alkaline phosphatase (ALP) has become a representative enzyme of modern life industries. As a critical biomarker of numerous diseases and a major tool enzyme of experiments, classic methods for ALP assays fail to meet the emerging demand of sensitivity, precision, interference immunity and wide targeted types of sample. Therefore, numerous studies have been made to develop ALP detection strategies to solve these problems. In this view, we summarize and compare these studies with their working principals and major advantages or weakness. Also, the future prospective and trend are discussed on this topic, hoping to provide some inspirations for future work.

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.