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We develop a new strategy for single-molecule monitoring of telomerase based on proximity ligation-transcription circuit-powered exponential amplifications.

We develop a new fluorescent biosensor for flap endonuclease 1 assay based on CRISPR/Cas12-enhanced single-molecule counting.

We demonstrate that target-activated cascade transcription amplification lights up RNA aptamers for label-free detection of metalloproteinase-2 (MMP-2) activity with zero background. This assay exhibits good specificity and high sensitivity with a limit of detection (LOD) of 0.6 fM. Moreover, it can analyze enzyme kinetic parameters, screen inhibitors, and accurately quantify MMP-2 in cancer cells and clinical serums.

We have developed a simple and rapid mix-and-read assay for the sensitive detection of O6-methylguanine DNA methyltransferase activity.

We design mismatched fluorescent probes to directly monitor the long noncoding RNA (lncRNA) in living cells. The introduction of mismatched bases in the fluorescent probe greatly enhances the strand displacement reaction rate toward the target lncRNA. These mismatched probes can monitor the intracellular lncRNA expression level in various cell lines and discriminate cancer cells from normal cells, holding great potential in fundamental biomedical research and clinical disease diagnosis.

We develop for the first time a label-free fluorescent method for sensitive detection of fat mass and obesity-associated protein (FTO) activity using MazF-mediated primer generation rolling circle amplification. This method is very simple with ultrahigh sensitivity and good specificity, and it can detect FTO activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of FTO inhibitors.

We develop for the first time a label-free and self-circulated fluorescent biosensor for sensitive detection of ten-eleven translocation 1 (TET1) activity in cancer cells. This biosensor displays good specificity and high sensitivity without the need for any antibodies/exogenous primers and it can accurately quantify TET1 activity at the single-cell level. Moreover, it can be employed for the measurement of kinetic parameters and screening of inhibitors.

We demonstrate the construction of an aggregation-induced electrochemiluminescent (AIECL) sensor for glutathione (GSH) assay by integrating an aminal-linked covalent organic framework (A-COF) with manganese dioxide (MnO

We demonstrate the development of a new fluorescent biosensor for sensitive DNA methylation assay by integrating single-molecule detection with endo IV-assisted signal amplification. This biosensor possesses the characteristics of good selectivity and high sensitivity with a detection limit of 7.3 × 10-17 M. It can distinguish as low as 0.01% methylation level, and can analyze genomic DNA methylation even in a single cancer cell.

Single-molecule fluorescence imaging is among the most advanced analytical technologies and has been widely adopted for biosensing due to its distinct advantages of simplicity, rapidity, high sensitivity, low sample consumption, and visualization capability. Recently, a variety of nucleic acid amplification approaches have been developed to provide a straightforward and highly efficient way for amplifying low abundance target signals. The integration of single-molecule fluorescence imaging with nucleic acid amplification has greatly facilitated the construction of various fluorescent biosensors for in vitro and in vivo detection of DNAs, RNAs, enzymes, and live cells with high sensitivity and good selectivity. Herein, we review the advances in the development of fluorescent biosensors by integrating single-molecule fluorescence imaging with nucleic acid amplification based on enzyme (e.g., DNA polymerase, RNA polymerase, exonuclease, and endonuclease)-assisted and enzyme-free (e.g., catalytic hairpin assembly, entropy-driven DNA amplification, ligation chain reaction, and hybridization chain reaction) strategies, and summarize the principles, features, and in vitro and in vivo applications of the emerging biosensors. Moreover, we discuss the remaining challenges and future directions in this area. This review may inspire the development of new signal-amplified single-molecule biosensors and promote their practical applications in fundamental and clinical research.

We developed a single quantum dot-based fluorescence resonance energy transfer biosensor for antibody-free detection of ten-eleven translocation 1 (TET1). This biosensor can sensitively detect TET1 in a homogeneous manner without the involvement of any specific antibodies, and it can be used for accurate measurement of TET1 activity in human neuroblastoma cells and the screening of TET1 inhibitors.

We demonstrate the zirconium ion-mediated assembly of a single quantum dot (QD)-based nanosensor for accurate detection of protein kinases (PKA) and polynucleotide kinases (PNK). This nanosensor is very sensitive with a detection limit of 8.82 × 10-4 U mL-1 for PKA and 1.40 × 10-5 U mL-1 for PNK. Moreover, it can be used to analyze the enzyme kinetic parameters and screen the inhibitors of PKA and PNK, with potential applications in drug discovery and clinical diagnosis.

We developed a peptide-templated gold nanoparticle (AuNP) nanosensor for simultaneous detection of multiple posttranslational modification (PTM) enzymes with a detection limit of 28 pM for histone deacetylase (HDAC) and 0.8 pM for protein tyrosine phosphatase 1B (PTP1B), and it can be further applied for the screening of PTM enzyme inhibitors and the measurement of PTM enzymes in cancer cells.

We develop a label-free fluorescence method for ultrasensitive detection of apoptosis-associated caspase activity based on branched rolling circle amplification. This work demonstrates for the first time the use of a nucleic acid amplification strategy for the amplified detection of caspase activity, which allows for highly sensitive detection of endogenous caspase even at the single-cell level. This method can be further applied for kinetic analysis and inhibitor screening.

We demonstrate for the first time that host-guest recognition coupled with triple signal amplification endows an electrochemiluminescent (ECL) biosensor with enhanced sensitivity for uracil DNA glycosylase (UDG) assay. This biosensor exhibits good selectivity and extremely high sensitivity, and it can be used to screen UDG inhibitors and measure the cellular UDG activity as well.

We develop a new fluorescence method for the sensitive detection of protein tyrosine phosphatase 1B (PTP1B) based on dephosphorylation-directed tricyclic DNA amplification cascades. This method exhibits good specificity and high sensitivity with a detection limit of 0.24 pM. Moreover, it can be applied for kinetics analysis, inhibitor screening, and the accurate detection of PTP1B in a variety of cancer cells.

We report for the first time the integration of ultra-high-pressure liquid chromatography-tandem mass spectrometry with machine learning for identifying fatty acid metabolite biomarkers of ischemic stroke. In particular, we develop an optimal model to discriminate ischemic stroke patients from healthy persons with 100% sensitivity and 93.18% specificity. This research may facilitate understanding the roles of fatty acid metabolites in stroke occurrence, holding great potential in clinical stroke diagnosis.

We develop a new fluorescent method for sensitive detection of long noncoding RNAs (lncRNAs) on the basis of duplex-specific nuclease-actuated cyclic enzymatic repairing-mediated signal amplification. This method exhibits high sensitivity with a detection limit of 0.081 fM, and it can accurately detect the endogenous lncRNA HOTAIR in cancer cells, providing a new approach to study the physiological function of lncRNAs in human diseases.

We develop a label-free and high-throughput bioluminescence method for the sensitive detection of uracil DNA glycosylase (UDG) through enzyme-mediated tricyclic cascade signal amplification. This method exhibits high sensitivity with a detection limit as low as 0.00031 U mL-1, and it can be further applied for the measurement of enzyme kinetic parameters and the screening of UDG inhibitors as well as cancer cell analysis.

We develop a label-free fluorescence method for sensitive discrimination of 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) based on the specific oxidation of 5hmC to 5-formylcytosine (5fC) and the subsequent conversion to uracil (U) in combination with ligation-mediated rolling circle amplification (RCA). This method exhibits high sensitivity with a detection limit of as low as 34.8 fM. It can discriminate as low as the 0.01% 5hmC level from the mixture, and exhibits good performance in serum sample analysis as well.

We develop a new fluorescence method for the sensitive detection of alkaline phosphatase (ALP) based on dephosphorylation-initiated transcription reaction-mediated dual signal amplification. This research demonstrates for the first time a significant improvement in ALP assay performance by using a nucleic acid amplification strategy, which enables sensitive detection of ALP activity in vitro and in cell extracts. Importantly, this method can be further applied for kinetic analysis and ALP inhibition assay.

We develop a simple and label-free fluorescence method for the polynucleotide kinase (PNK) assay based on phosphorylation-triggered isothermal exponential amplification. This method exhibits ultrahigh sensitivity superior to most existing PNK assays. Importantly, this method enables sensitive detection of endogenous PNK activity at the single-cell level, holding great potential in clinical diagnosis and biomedical research.

We develop a simple and isothermal ligase-based cyclic amplification approach on the basis of a ligation-triggered duplex-specific nuclease (DSN)-mediated cleavage reaction without the involvement of any thermal cycling and separation procedures. As a proof of concept, we demonstrate the application of this method for the sensitive detection of polynucleotide kinase. Moreover, this method can be extended to the accurate quantification of DNA, proteins and ATP.

We develop a simple "mix-and-detection" method for the sensitive detection of telomerase from cancer cells under absolutely isothermal conditions. This method enables one-step and one-tube detection of telomerase with a detection limit of 3 cells, and it can be further applied for the screening of telomerase inhibitors and the discrimination of cancer cells from normal cells.

We combine single-molecule detection with magnetic separation for simultaneous measurement of human 8-oxoG DNA glycosylase 1 (hOGG1) and uracil DNA glycosylase (UDG) based on excision repair-initiated endonuclease IV (Endo IV)-assisted signal amplification. This method can sensitively detect multiple DNA glycosylases, and it can be further applied for the simultaneous measurement of enzyme kinetic parameters and screening of both hOGG1 and UDG inhibitors.

We developed a simple and rapid method for terminal deoxynucleotidyl transferase (TdT) assay on the basis of the polymerization-directed exonuclease-assisted construction of a single quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensor. This method is very sensitive with a detection limit as low as 1 × 10-6 U μL-1, and it can be used for the screening of TDT inhibitors and accurate quantification of TdT activity even in 5 cancer cells.

SUMOylation is a post-translational modification that plays critical roles in a multitude of cellular processes including transcription, cellular localization, DNA repair and cell cycle progression. Similar to ubiquitin, the small ubiquitin-like modifiers (SUMOs) are covalently attached to the epsilon amino group of lysine residues in the substrates. To understand the regulation and the dynamics of post-translational modifications (PTMs), the identification and quantification of SUMOylation is strictly needed. Although numerous proteomic approaches have been developed to identify hundreds of SUMO target proteins, the number of SUMOylation signatures identified from endogenous modified proteins is limited, and the identification of precise acceptor sites remains a challenge due to the low abundance of in vivo SUMO-modified proteins and the high activity of SUMO-specific proteases in cell lysates. In particular, very few sensitive strategies are available for accurate quantification of SUMO target proteins. Within the past decade, mass spectrometry-based strategies have been the most popular technologies for proteome-wide studies of SUMOylation. Recently, some new approaches such as single-molecule detection have been introduced. In this review, we summarize the strategies that have been exploited for enrichment, purification and identification of SUMOylation substrates and acceptor sites as well as ultrasensitive quantification of SUMOylation. We highlight the emerging trends in this field as well.

We develop a new fluorescence method for real-time monitoring of thymine DNA glycosylase (TDG) activity through cyclic enzymatic repairing-mediated dual-signal amplification. This method exhibits excellent sensitivity with a detection limit of 5.6 × 10−7 U μL−1, and it can be used to determine kinetic parameters and quantify TDG activity from even single cancer cells.

We develop a sensitive and selective method for the multiplex detection of histone-modifying enzymes (HMEs) through the integration of antibody-based fluorescence labeling with total internal reflection fluorescence (TIRF)-based single-molecule detection. This method exhibits excellent specificity and high sensitivity with a detection limit of 21 pM for histone acetyltransferase GcN5 and 12 pM for histone methyltransferase G9a, and it can be applied for the screening of HME inhibitors as well.

The repair ligation-mediated light-producing DNA machine can produce light through transforming the repetitive DNA cleavage/ligation motions into optical energy without the requirement of either external reporting reagents or excitation light, and it can be applied for sensitive and selective detection of DNA, thrombin, adenosine, potassium ions (K(+)) and endonuclease even in human serum.

We develop a new method for the sensitive detection of polynucleotide kinase (PNK) using rolling circle amplification-induced chemiluminescence. This method exhibits high sensitivity with a detection limit of 2.20 × 10(-4) U mL(-1), which is superior to most reported approaches. Moreover, this method can be used to screen both the inhibitors and the activators of PNK, and can be further applied for real sample analysis.

We develop a riboadenosine-substituted DNA probe-based bioluminescence method for real-time monitoring of exonuclease III (Exo III) activity and Exo III-assisted target recycling. This method employs the target-dependent ATP as the bioluminescence reporter without the involvement of any sophisticated luciferase manipulations, and the glow-type reaction kinetics facilitates the performance of imaging assay.

A telomere-triggered isothermal exponential amplification-based DNAzyme biosensor is developed for highly sensitive detection of telomerase in cancer cells even at the single-cell level. This biosensor can be further applied for the screening of telomerase inhibitors for anticancer drug development.

Multilayered ZnS shells are coated on a CdSe quantum dot (QD) core by a tri-n-octylphosphine-assisted successive ionic layer adsorption and reaction (TOP-SILAR) method. Notably, the quantum yield (QY) of obtained core-shell CdSe/ZnS QDs can reach up to 95% after coating with a 3-monolayered ZnS shell, and is maintained even after coating with a 6-monolayered ZnS shell, breaking through the bottleneck of low QY at high coverage of the shell.