A simple "mix-and-detection" method based on template-free amplification for sensitive measurement of human cellular FEN1.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that can specially identify and cleave 5' flap of branched duplex DNA, and it plays a critical role in DNA metabolic pathways and human diseases. Herein, we propose a simple "mix-and-detection" strategy for sensitive measurement of human cellular FEN1 on basis of template-free amplification. We design a dumbbell probe with 5' flap as a substrate of FEN1 and a NH2-labeled 3' termini to prevent nonspecific amplification. When FEN1 is present, the 5' flap is cleaved to release a free 3'-OH termini, initiating Ribonuclease HII (RNase HII)-assisted terminal deoxynucleotidyl transferase (TdT)-induced amplification for the production of a significant fluorescence signal. Due to the high exactitude of TdT-mediated extension reaction and RNase HII-induced single ribonucleotide excise, this assay shows excellent specificity and high sensitivity with a detection limit of 5.64 × 10-6 U/µL. Importantly, it can detect intracellular FEN1 activity with single-cell sensitivity under isothermal condition in a "mix-and-detection" manner, screen the FEN1 inhibitors, and even discriminate tumor cells from normal cells, offering a new platform for disease diagnosis and drug discovery.

Bidirectional Polymerization-Transcription Amplification-Encoded Dual-Color Fluorescent Biosensor for Label-Free and Primer-Free Detection of Multiple piRNAs.

PIWI-interacting RNAs (piRNAs) are a type of endogenous noncoding RNAs with a length of 24-31 nucleotides, and they can specifically bind with PIWI proteins to form the piRNA/PIWI complexes for regulating multiple physiological and pathological processes. Herein, we develop a bidirectional polymerization-transcription amplification-encoded dual-color fluorescent biosensor for label-free and primer-free measurements of multiple piRNAs. The designed hairpin probe contains a palindromic tail, and it can serve as the target recognition unit, polymerization primer, and transcription template. In the presence of target piRNAs, the hairpin probes are opened to expose a palindromic sequence that can trigger bidirectional polymerization and transcription reaction with the assistance of KF polymerase and T7 RNA polymerase for the production of numerous RNA aptamers. The aptamers subsequently bind with the corresponding fluorophores (DFHBI-1T/MG) to form the RNA aptamer-fluorophore complexes for the generation of enhanced fluorescence signals. This biosensor can sensitively detect piR-36026 with a limit of detection (LOD) of 82.08 aM and piR-36743 with a LOD of 44.44 aM. Moreover, it can quantify cellular piRNAs with single-cell sensitivity and distinguish cancer cells from normal cells. Furthermore, it has the capability of distinguishing the expression of piRNAs in the tissues of breast cancer patients and healthy individuals. By simply altering the target recognition site of the hairpin probe, this biosensor can be extended to detect various piRNAs, offering a powerful platform for piRNA-related clinical diagnostics and therapeutics.

Development of a DNAzyme-Driven Fluorescent Light-Up Aptasensor for Label-Free Detection of Multiple lncRNAs.

Long noncoding RNAs (lncRNAs) act as the dynamic regulatory molecules that control the expression of genes and affect numerous biological processes, and their dysregulation is associated with tumor progression. Herein, we develop a fluorescent light-up aptasensor to simultaneously measure multiple lncRNAs in living cells and breast tissue samples based on the DNAzyme-mediated cleavage reaction and transcription-driven synthesis of light-up aptamers. When target lncRNAs are present, they can be recognized by template probes to form the active DNAzyme structures, initiating the T4 PNK-catalyzed dephosphorylation-triggered extension reaction to generate double-strand DNAs with the T7 promoter sequences. The corresponding T7 promoters can initiate the transcription amplification catalyzed by the T7 RNA polymerase to generate abundant Broccoli aptamers and malachite green aptamers, which can bind DFHBI-1T and MG to generate strong fluorescence signals. Taking advantage of the good selectivity of DNAzyme-mediated cleavage of lncRNAs, high amplification efficiency of T7 transcription-driven amplification reaction, and bright fluorescence of the RNA aptamer-fluorophore complex, this method exhibits high sensitivity with a detection limit of 21.4 aM for lncRNA HOTAIR and 18.47 aM for lncRNA MALAT1, and it can accurately measure multiple lncRNAs in both tumor cell lines and breast tissue samples, providing a powerful paradigm for biomedical research and early clinic diagnostics.

Construction of a Spatial-Confined Self-Stacking Catalytic Circuit for Rapid and Sensitive Imaging of Piwi-Interacting RNA in Living Cells.

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs that repress transposable elements to maintain genome integrity. The canonical catalytic hairpin assembly (CHA) circuit relies on random collisions of free-diffused reactant probes, which substantially slow down reaction efficiency and kinetics. Herein, we demonstrate the construction of a spatial-confined self-stacking catalytic circuit for rapid and sensitive imaging of piRNA in living cells based on intramolecular and intermolecular hybridization-accelerated CHA. We rationally design a 3WJ probe that not only accelerates the reaction kinetics by increasing the local concentration of reactant probes but also eliminates background signal leakage caused by cross-entanglement of preassembled probes. This strategy achieves high sensitivity and good specificity with shortened assay time. It can quantify intracellular piRNA expression at a single-cell level, discriminate piRNA expression in tissues of breast cancer patients and healthy persons, and in situ image piRNA in living cells, offering a new approach for early diagnosis and postoperative monitoring.

Construction of a 3D Quantum Dot Nanoassembly with Two-Step FRET for One-Step Sensing of Human Telomerase RNA in Breast Cancer Cells and Tissues.

Telomerase is an important biomarker for early diagnosis of cancers, but current telomerase assays usually rely on measuring the extension products of telomerase substrates, which increases the assay complexity. More evidence indicates that human telomerase RNA (hTR), as a core component of telomerase, is positively correlated with the telomerase activity. Herein, we demonstrate the development of a duplex-specific nuclease (DSN)-propelled 3D quantum dot (QD) nanoassembly with two-step Föster resonance energy transfer (FRET) for the one-step sensing of hTR in breast cancer cells and tissues. This assay involves only one hairpin probe modified with a Cy5 at the sixth base from the 5'-biotin end and a BHQ2 at the 3'-terminus, which integrates three functions of target recognition, target recycling amplification, and signal readout. The anchoring of the hairpin probe on the 605QD surface results in the formation of a 3D 605QD-Cy5-probe-BHQ2 nanoassembly in which two-step FRET occurs among the 605QD, Cy5, and BHQ2 quencher. Notably, the formation of 605QD-Cy5-probe-BHQ2 nanoassembly facilitates the reduction of background signal and the increase of signal-to-background ratio due to its dense, highly oriented nucleic acid shell-induced steric hindrance effect. This assay can achieve one-step and rapid detection of hTR with a detection limit of 2.10 fM, which is the simplest and most rapid hTR assay reported so far. Moreover, this assay can efficiently distinguish single-base mismatched sequences, and it can discriminate the hTR level between breast cancer patients and healthy donors with a high accuracy of 100%, with great prospects for early diagnosis of cancers.

One-Dimensional Covalent Organic Framework with Improved Charge Transfer for Enhanced Electrochemiluminescence.

We present a dimensional regulating charge transfer strategy to achieve an enhanced electrochemiluminescence (ECL) by constructing a one-dimensional pyrene-based covalent organic framework (1D-COF). The dual-chain-like edge architecture in 1D-COF facilitates the stabilization of aromatic backbones, the enhancement of electronic conjugations, and the decrease of energy loss. The 1D-COF generates enhanced anodic (92.5-fold) and cathodic (3.2-fold) signals with tripropylamine (TPrA) and K2S2O8 as the anodic and cathodic coreactants, respectively, compared with 2D-COF. The anodic and cathodic ECL efficiencies of 1D-COF are 2.08- and 3.08-fold higher than those of 2D-COF, respectively. According to density functional theory (DFT), the rotational barrier energy (ΔE) of 1D-COF enhances sharply with the increase of dihedral angle, suggesting that the architecture in 1D-COF restrains the intramolecular spin of aromatic chains, which facilitates the decrease of nonradiative transitions and the enhancement of ECL. Furthermore, 1D-COF can be used to construct an ECL biosensor for sensitive detection of dopamine.

Autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of long noncoding RNA in human lung tissues.

Aberrant long noncoding RNA (lncRNA) expression is linked to varied pathological processes and malignant tumors, and lncRNA can serve as potential disease biomarkers. Herein, we demonstrate the autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of lncRNA in human lung tissues on the basis of multiple primer generation-mediated rolling circle amplification (mPG-RCA). This assay involves two padlock probes that act as both a detection probe for recognizing target lncRNA and a domain for producing complementary DNAzyme. Two padlock probes can hybridize with target lncRNA at different sites, followed by ligation to form a circular template with the aid of RNA ligase. The circular template can initiate mPG-RCA to generate abundant Mg2+-dependent DNAzymes that can specifically cleave signal probes to induce the recovery of Cy3 fluorescence. The inherent characteristics of ligase-based ligation reaction and DNAzymes endow this assay with excellent specificity, and the introduction of multiple padlock probes endows this assay with high sensitivity. This strategy can rapidly and sensitively measure lncRNA with a wide linear range of 1 fM - 1 nM and a detection limit of 678 aM within 1.5 h, and it shows distinct advantages of simplicity and immobilization-free without the need of precise temperature control and tedious procedures of nanomaterial preparation. Moreover, it enables accurate measurement of lncRNA level in normal cells and malignant tumor cells as well as differentiation of lncRNA expressions in tissues of non-small cell lung cancer (NSCLC) patients and normal individuals, with promising applications in biomedical studies and disease diagnosis.

Recent advances in nucleic acid signal amplification-based aptasensors for sensing mycotoxins.

Mycotoxin contamination in food products may cause serious health hazards and economic losses. The effective control and accurate detection of mycotoxins have become a global concern. Even though a variety of methods have been developed for mycotoxin detection, most conventional methods suffer from complicated operation procedures, low sensitivity, high cost, and long assay time. Therefore, the development of simple and sensitive methods for mycotoxin assay is highly needed. The introduction of nucleic acid signal amplification technology (NASAT) into aptasensors significantly improves the sensitivity and facilitates the detection of mycotoxins. Herein, we give a comprehensive review of the recent advances in NASAT-based aptasensors for assaying mycotoxins and summarize the principles, features, and applications of NASAT-based aptasensors. Moreover, we highlight the challenges and prospects in the field, including the simultaneous detection of multiple mycotoxins and the development of portable devices for field detection.

Elongation and Ligation-Mediated Differential Coding for Label-Free and Locus-Specific Analysis of 8-Oxo-7,8-dihydroguanine in DNA.

Oxidative DNA damage is closely associated with the occurrence of numerous human diseases and cancers. 8-Oxo-7,8-dihydroguanine (8-oxoG) is the most prevalent form of DNA damage, and it has become not only an oxidative stress biomarker but also a new epigenetic-like biomarker. However, few approaches are available for the locus-specific detection of 8-oxoG because of the low abundance of 8-oxoG damage in DNA and the limited sensitivity of existing assays. Herein, we demonstrate the elongation and ligation-mediated differential coding for label-free and locus-specific analysis of 8-oxoG in DNA. This assay is very simple without the involvement of any specific labeled probes, complicated steps, and large sample consumption. The utilization of Bsu DNA polymerase can specifically initiate a single-base extension reaction to incorporate dATP into the opposite position of 8-oxoG, endowing this assay with excellent selectivity. The introduction of cascade amplification reaction significantly enhances the sensitivity. The proposed method can monitor 8-oxoG with a limit of detection of 8.21 × 10-19 M (0.82 aM), and it can identify as low as 0.001% 8-oxoG damage from a complex mixture with excessive undamaged DNAs. This method can be further applied to measure 8-oxoG levels in the genomic DNA of human cells under diverse oxidative stress, holding prospect potential in the dynamic monitoring of critical 8-oxoG sites, early clinical diagnosis, and gene damage-related biomedical research.

Construction of an Enzymatically Controlled DNA Nanomachine for One-Step Imaging of Telomerase in Living Cells.

Telomerase is a basic reverse transcriptase that maintains the telomere length in cells, and accurate and specific sensing of telomerase in living cells is critical for medical diagnostics and disease therapeutics. Herein, we demonstrate for the first time the construction of an enzymatically controlled DNA nanomachine with endogenous apurinic/apyrimidinic endonuclease 1 (APE1) as a driving force for one-step imaging of telomerase in living cells. The DNA nanomachine is designed by rational engineering of substrate probes and reporter probes embedded with an enzyme-activatable site (i.e., AP site) and their subsequent assembly on a gold nanoparticle (AuNP). Upon recognition and cleavage of the AP site in the substrate probe by APE1, the loop of the substrate probe unfolds, exposing telomeric primer (TP) with the 3'-OH end. Subsequently, the TP is elongated by telomerase at the 3'-OH end to generate a long telomeric product. The resultant telomeric product acts as a swing arm that can hybridize with a reporter probe to initiate the APE1-powered walking reaction, ultimately generating a significantly enhanced fluorescence signal. Notably, endogenous APE1 is used as the driving force of the DNA nanomachine, avoiding the introduction of exogenous auxiliary cofactors into the cellular microenvironment. Owing to the high kinetics and high amplification efficiency of the APE1-powered DNA nanomachine, this strategy enables one-step sensitive sensing of telomerase in vitro and in vivo. It can successfully discriminate telomerase activity between cancer cells and normal cells, screen telomerase inhibitors, and monitor the variations of telomerase activity in living cells, offering a prospective platform for molecular diagnostics and drug discovery.

Integration of Demethylation-Activated DNAzyme with a Single Quantum Dot Nanosensor for Sensitive Detection of O6-Methylguanine DNA Methyltransferase in Breast Tissues.

O6-Methylguanine-DNA-methyltransferase (MGMT) is a demethylation protein that dynamically regulates the O6-methylguanine modification (O6 MeG), and dysregulated MGMT is implicated in various malignant tumors. Herein, we integrate demethylation-activated DNAzyme with a single quantum dot nanosensor to sensitively detect MGMT in breast tissues. The presence of MGMT induces the demethylation of the O6 MeG-caged DNAzyme and the restoration of catalytic activity. The activated DNAzyme then specifically cleaves the ribonucleic acid site of hairpin DNA to expose toehold sequences. The liberated toehold sequence may act as a primer to trigger a cyclic exponential amplification reaction for the generation of enormous signal strands that bind with the Cy5/biotin-labeled probes to form sandwich hybrids. The assembly of sandwich hybrids onto 605QD obtains 605QD-dsDNA-Cy5 nanostructures, inducing efficient FRET between the 605QD donor and Cy5 acceptor. Notably, the introduction of a mismatched base in hairpin DNA can greatly minimize the background and improve the signal-to-noise ratio. This nanosensor achieves a dynamic range of 1.0 × 10-8 to 0.1 ng/µL and a detection limit of 155.78 aM, and it can screen MGMT inhibitors and monitor cellular MGMT activity with single-cell sensitivity. Moreover, it can distinguish the MGMT level in tissues of breast cancer patients and healthy persons, holding great potential in clinical diagnostics and epigenetic research studies.

Construction of single-molecule counting-based biosensors for DNA-modifying enzymes: A review.

DNA-modifying enzymes act as critical regulators in a wide range of genetic functions (e.g., DNA damage & repair, DNA replication), and their aberrant expression may interfere with regular genetic functions and induce various malignant diseases including cancers. DNA-modifying enzymes have emerged as the potential biomarkers in early diagnosis of diseases and new therapeutic targets in genomic research. Consequently, the development of highly specific and sensitive biosensors for the detection of DNA-modifying enzymes is of great importance for basic biomedical research, disease diagnosis, and drug discovery. Single-molecule fluorescence detection has been widely implemented in the field of molecular diagnosis due to its simplicity, high sensitivity, visualization capability, and low sample consumption. In this paper, we summarize the recent advances in single-molecule counting-based biosensors for DNA-modifying enzyme (i.e, alkaline phosphatase, DNA methyltransferase, DNA glycosylase, flap endonuclease 1, and telomerase) assays in the past four years (2019 - 2023). We highlight the principles and applications of these biosensors, and give new insight into the future challenges and perspectives in the development of single-molecule counting-based biosensors.

Construction of a glycosylation-mediated fluorescent biosensor for label-free measurement of site-specific 5-hydroxymethylcytosine in cancer cells with zero background signal.

BACKGROUND: 5-hydroxymethylcytosine (5hmC) as an epigenetic modification can regulate gene expression, and its abnormal level is related with various tumor invasiveness and poor prognosis. Nevertheless, the current methods for 5hmC assay usually involve expensive instruments/antibodies, radioactive risk, high background, laborious bisulfite treatment procedures, and non-specific/long amplification time. RESULTS: We develop a glycosylation-mediated fluorescent biosensor based on helicase-dependent amplification (HDA) for label-free detection of site-specific 5hmC in cancer cells with zero background signal. The glycosylated 5hmC-DNA (5ghmC) catalyzed by ß-glucosyltransferase (ß-GT) can be cleaved by AbaSI restriction endonuclease to generate two dsDNA fragments with sticky ends. The resultant dsDNA fragments are complementary to the biotinylated probes and ligated by DNA ligases, followed by being captured by magnetic beads. After magnetic separation, the eluted ligation products act as the templates to initiate HDA reaction, generating abundant double-stranded DNA (dsDNA) products within 20 min. The dsDNA products are measured in a label-free manner with SYBR Green I as an indicator. This biosensor can measure 5hmC with a detection limit of 2.75 fM and a wide linear range from 1 × 10-14 to 1 × 10-8 M, and it can discriminate as low as 0.001% 5hmC level in complex mixture. Moreover, this biosensor can measure site-specific 5hmC in cancer cells, and distinguish tumor cells from normal cells. SIGNIFICANCE: This biosensor can achieve a zero-background signal without the need of either 5hmC specific antibody or bisulfite treatment, and it holds potential applications in biological research and disease diagnosis.

Construction of Gold/Rhodium Freestanding Superstructures as Antenna-Reactor Photocatalysts for Plasmon-Driven Nitrogen Fixation.

Precisely controlling the architecture and spatial arrangement of plasmonic heterostructures offers unique opportunities to tailor the catalytic property, whereas the lack of a wet-chemistry synthetic approach to fabricating nanostructures with high-index facets limits their practical applications. Herein, we describe a universal synthetic strategy to construct Au/Rh freestanding superstructures (SSs) through the selective growth of ordered Rh nanoarrays on high-index-faceted Au nanobipyramids (NBPs). This synthetic strategy works on various metal nanocrystal substrates and can yield diverse Au/Rh and Pd/Rh SSs. Especially, the obtained Au NBP/Rh SSs exhibit high photocatalytic activity toward N2 fixation as a result of the spatially separated architecture, local electric field enhancement, and the antenna-reactor mechanism. Both theoretical and experimental results reveal that the Au NBPs can function as nanoantennas for light-harvesting to generate hot charge carriers for driving N2 fixation, while the Rh nanoarrays can serve as the active sites for N2 adsorption and activation to synergistically promote the overall catalytic activity in the Au NBP/Rh SSs. This work offers new avenues to rationally designing and constructing spatially separated plasmonic photocatalysts for high-efficiency catalytic applications.

Controllable Assembly of a Quantum Dot-Based Aptasensor Guided by CRISPR/Cas12a for Direct Measurement of Circulating Tumor Cells in Human Blood.

Accurate and sensitive analysis of circulating tumor cells (CTCs) in human blood provides a non-invasive approach for the evaluation of cancer metastasis and early cancer diagnosis. Herein, we demonstrate the controllable assembly of a quantum dot (QD)-based aptasensor guided by CRISPR/Cas12a for direct measurement of CTCs in human blood. We introduce a magnetic bead@activator/recognizer duplex core-shell structure to construct a multifunctional platform for the capture and direct detection of CTCs in human blood, without the need for additional CTC release and re-identification steps. Notably, the introduction of magnetic separation ensures that only a target-induced free activator can initiate the downstream catalysis, efficiently avoiding the undesired catalysis triggered by inappropriate recognition of the activator/recognizer duplex structure by crRNAs. This aptasensor achieves high CTC-capture efficiency (82.72%) and sensitive detection of CTCs with a limit of detection of 2 cells mL-1 in human blood, holding great promise for the liquid biopsy of cancers.

Exogenous Co-Reactant-Free Electrochemiluminescent Biosensor for Ratiometric Measurement of α-Glucosidase Based on a ZIF-67-Regulated Hydrogen-Bonded Organic Framework.

The development of highly sensitive and selective analytical approaches for monitoring enzymatic activity is critical for disease diagnosis and biomedical research. Herein, we develop an exogenous co-reactant-free electrochemiluminescence (ECL) biosensor for the ratiometric measurement of α-glucosidase (α-Glu) based on a zeolitic imidazolate framework (ZIF-67)-regulated pyrene-based hydrogen-bonded organic framework (HOF-101). Target α-Glu can hydrolyze maltose to α-d-glucose, which can subsequently react with GOx to produce gluconic acid. The resultant gluconic acid can dissolve ZIF-67, leading to the recovery of the HOF-101 cathodic ECL signal and the decrease of the luminol anodic ECL signal. The long-range ordered structure of HOF-101 can speed up charge transfer, resulting in a stable and strong cathodic ECL signal. Moreover, ZIF-67 can not only efficiently quench the ECL signal of HOF-101 due to ECL resonance energy transfer between HOF-101 and ZIF-67 as well as the steric hindrance effect of ZIF-67 but also enhance the anodic ECL emission of luminol in dissolved O2 system because of its ordered and porous crystalline structure and the atomically dispersed Co2+. Notably, HOF-101 possesses a higher ECL efficiency (32.22%) compared with the Ru(bpy)32+ standard. Importantly, this ratiometric ECL biosensor shows high sensitivity (a detection limit of 0.19 U L-1) and a broad linear range (0.2-50 U L-1). This biosensor can efficiently eliminate systematic errors and enhance detection reliability without the involvement of exogenous co-reactants, and it displays good assay performance in human serum samples, holding great promise in biomedical research studies.

Synthesis of silica-encapsulated tetraphenylethylene with aggregation-induced electrochemiluminescence resonance energy transfer for sensitively sensing microcystin-LR.

The reported organic electrochemiluminescence (ECL) luminophors for the detection of various markers often suffer from intermolecular π-π stacking-induced luminophore quenching. Herein, we demonstrate one-pot synthesis of a new aggregation-induced electrochemiluminescence (AIECL) emitter (i.e., TPE@SiO2/rGO composite) for sensitive measurement of microcystin-leucine arginine (MC-LR). The TPE@SiO2/rGO composite is constructed by embedding the silica-encapsuled 1,1,2,2-tetra(4-carboxylphenyl)ethylene (TPE) in the reduced graphene oxide. In comparison with the monomer TPE, this composite exhibit high luminescence efficiency and strong ECL emission, because the AIECL phenomenon triggered by the spatial confinement effect in the SiO2 cage induces the restriction of the internal motion and vibration of molecules. Notably, this composite has distinct advantages of easy preparation, simple functionalization, and stable luminescence. Especially, the TPE@SiO2/rGO-based ECL-RET system exhibits a high quenching efficiency (ΦET) of 69.7%. When target MC-LR is present, it triggers DNA strand displacement reaction (SDR), inducing the quenching of the ECL signal of TPE@SiO2/rGO composite due to ECL resonance energy transfer between TPE@SiO2/rGO composite and methylene blue (MB). The proposed biosensor enables highly sensitive, low-cost, and robust measurement of MC-LR with a large dynamic range of 7 orders of magnitude and a detection limit of 3.78 fg/mL, and it displays excellent detection performance in complex biological matrices, holding potential applications in food safety and water monitoring.

Construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues.

Fat mass and obesity-associated protein (FTO) is a crucial eraser of RNA N6- methyladenosine (m6A) modification, and abnormal FTO expression level is implicated in pathogenesis of numerous cancers. Herein, we demonstrate the construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues. When FTO is present, it specifically erases the methyl group in m6A, inducing the cleavage of demethylated DNA by endonuclease DpnII and the generation of a single-stranded DNA (ssDNA) with a 3'-hydroxyl group. Subsequently, terminal deoxynucleotidyl transferase (TdT) promotes the incorporation of dTTPs into the ssDNA to obtain a long polythymidine (T) DNA sequence. The resultant long poly (T) DNA sequence can act as a template to trigger hyperbranched strand displacement amplification (HSDA), yielding numerous DNA fragments that may be stained by SYBR Gold to produce an enhanced fluorescence signal. This biosensor processes ultrahigh sensitivity with a detection limit of 1.65 × 10-10 mg/mL (2.6 fM), and it can detect the FTO activity in a single MCF-7 cell. Moreover, this biosensor can screen the FTO inhibitors, evaluate enzyme kinetic parameters, and discriminate the FTO expression levels in the tissues of breast cancer patients and healthy persons.

Ultrathin-FeOOH-coated MnO2 nanozyme with enhanced catalase-like and oxidase-like activities for photoelectrochemical and colorimetric detection of organophosphorus pesticides.

Herein, we develop a dual-mode biosensor for photoelectrochemical and colorimetric detection of organophosphate pesticides (OPPs) based on ultrathin-FeOOH-coated MnO2 (MO@FHO) nanozyme. In this biosensor, OPPs can inhibit the alkaline phosphatase (ALP) activity and hinder the dephosphorylation of l-ascorbic acid-2-phosphate, preventing the decomposition of MO@FHO nanozyme and inducing both a photoelectrochemical (PEC) signal and the colorimetric change. The MO@FHO nanozyme not only possesses an enhanced catalase-like activity to degrade H2O2 for the generation of an improved cathodic photocurrent, but also exhibits an excellent oxidase-like activity to oxidize 3,3,5,5-tetramethylbenzidine with high catalytic efficiency. This biosensor displays a detection limit of 50 pmol/L for the PEC mode and a detection limit of 0.8 nmol/L for the colorimetric mode. Moreover, this biosensor exhibits excellent performance in complex biological matrices, and the smartphone-based visual sensing platform facilitates rapid and sensitive detection of OPPs, holding promising applications in food safety monitoring, and on-site detection.

Ligase detection reaction amplification-activated CRISPR-Cas12a for single-molecule counting of FEN1 in breast cancer tissues.

We construct a simple fluorescent biosensor for single-molecule counting of flap endonuclease 1 (FEN1) based on ligase detection reaction (LDR) amplification-activated CRISPR-Cas12a. This biosensor exhibits excellent selectivity and high sensitivity with a detection limit (LOD) of 1.31 × 10-8 U. Moreover, it can be employed to screen the FEN1 inhibitors and quantitatively measure the FEN1 activity in human cells and breast cancer tissues, holding great promise in clinical diagnosis and drug discovery.