Heavy metal Cu2+ emitted in industry and residues of glyphosate pesticides are pervasive in ecosystems, accumulated in water bodies and organisms\' overtime, constituting hazard to human and ecological balance. The development of rapid, highly selective, reversibility and sensitive biosensor in vivo detection for Cu2+ and glyphosate was imminent. A novel dual-recognition fluorescence biosensor MPH was successfully synthesized based on triphenylamine, which demonstrated remarkable ratiometric fluorescence quenching toward Cu2+, while MPH-Cu2+ (1:1) ensemble exhibited ratiometric fluorescence restoration for glyphosate, both with observable color changes in daylight and UV lamp. The biosensor exhibited rapid, outstanding selectivity, anti-interference, and multiple cycles reversibility through \"turn-off-on\" fluorescence towards Cu2+ and glyphosate, respectively. Surprisingly, the clearly binding mechanisms of MPH to Cu2+ and MPH-Cu2+ ensemble to glyphosate were determined, respectively, based on the Job\'s plot, FT-IR, ESI-HRMS, 1H NMR titration and theoretical calculations of dynamics and thermodynamics. In addition, biosensor MPH demonstrated successful detection of Cu2+ and glyphosate across diverse environmental samples including tap water, extraction solutions of traditional Chinese medicine honeysuckle and soil samples. In the meantime, fluorescence imaging of Cu2+ and glyphosate at both micro and macro scales in various living organisms, such as rice roots, MCF-7 cells, zebrafish, and mice, were successfully achieved. Overall, this work was expected to become a promising and versatile fluorescence biosensor for rapid and reversible detection of Cu2+ and glyphosate both in vitro and vivo.

Sialyl-Lewisx (SLex) is a tetrasugar, which plays an important role in initial inflammation and cancer cell metastasis, and can be used as a marker for cancer diagnosis and prognosis or a therapeutic target. Detecting SLex from complex biological media remains a significant challenge. Herein, a single-stranded DNA aptamer of SLex was screened based on the double-stranded DNA library-modified magnetic bead (MB)-SELEX technology. After 14 rounds of screening, 12,639 sequences were obtained and divided into nine families. Three representative sequences were selected based on the number of sequence repeats and Gibbs binding free energy, and the aptamer SLex-Apt2 with 80 nt length (Kd = 23.01 nM) had the best affinity and relatively high specificity for targeting SLex. Then, a novel dual-recognition fluorescent biosensor for SLex-sensitive detection based on aptamer SLex-Apt2 bio-dots and 3-aminobenzoboric acid-modified MB was developed. This method can detect SLex as low as 32 μM and has a good linear response in the range 100 μM to 2 mM. It has the advantages of low preparation cost, good targeting, and avoiding the occurrence of false-positive and false-negative detection results, which makes the biosensor more valuable in biological detection and clinical diagnosis.

Detection of disease biomarkers constitutes a major challenge for the development of personalized and predictive diagnostics as well as companion assays. Protein kinases (PKs) involved in the coordination of cell cycle progression and proliferation that are hyperactivated in human cancers constitute attractive pharmacological targets and relevant biomarkers. Although it is relatively straightforward to assess the relative abundance of PKs in a biological sample, there is not always a direct correlation with enzymatic activity, which is regulated by several posttranslational mechanisms. Studies of relative abundance therefore convey limited information, and the lack of selective, sensitive, and standardized tools together with the inherent complexity of biological samples makes it difficult to quantify PK activities in physio-pathological tissues. To address this challenge, we have developed a toolbox of fluorescent biosensors that report on CDK activities in a sensitive, selective, dose-dependent, and quantitative fashion, which we have implemented to profile CDK activity signatures in cancer cell lines and biopsies from human tumors. In this study, we report on a standardized and calibrated biosensing approach to quantify CDK1,2,4, and 6 activities simultaneously through a combination of four different biosensors in a panel of 40 lung adenocarcinoma and 40 follicular lymphoma samples. CDK activity profiling highlighted two major patterns which were further correlated with age, sex of patients, tumor size, grade, and genetic and immunohistochemical features of the biopsies. Multiplex CDKACT biosensing technology provides new and complementary information relative to current genetic and immunohistochemical characterization of tumor biopsies, which will be useful for diagnostic purposes, potentially guiding therapeutic decision. These fluorescent peptide biosensors offer promise for personalized diagnostics based on kinase activity profiling.

BACKGROUND: Tetrodotoxin (TTX) is a potent neurovirulent marine biotoxin that is present in puffer fish and certain marine animals. It is capable of causing severe neurotoxic symptoms and even death when consumed through contaminated seafood. Due to its high toxicity, developing an effective assay for TTX determination in seafood has significant benefits for food safety and human health. Currently, it remains challenging to achieve on-site determination of TTX in seafood. To facilitate mass on-site assays, more affordable technologies utilizing accessible equipment that require no skilled personnel are needed.
RESULTS: A smartphone-based portable fluorescent biosensor is proposed for TTX determination by using metal-organic framework (MOF) biocomposites and cotton swabs. Oriented antibody (Ab)-decorated and fluorescent quantum dot (QD)-loaded MOF biocomposites (QD@MOF*Ab) are rapidly synthesized for binding targets and fluorescent responses by utilizing the tunability of zinc-based MOF. Moreover, facile Ab-immobilized household cotton swabs are utilized as TTX capture tools. TTX forms sandwich immune complexes with QD@MOF*Ab probes, achieving signal amplification. These probes are excited by a portable device to generate bright fluorescent signals, which can be detected by the naked eye, and TTX quantitative results are obtained using a smartphone. When observed with the naked eye, the limit of detection (LOD) is 0.4 ng/mL, while intelligent quantitation presents an LOD of 0.13 ng/mL at logarithmic concentrations of 0.2-400 ng/mL.
CONCLUSIONS: This biosensor is convenient to use, and an easy-to-operate analysis is completed within 15 min, thus demonstrating excellent performance in terms of detection speed and portability. Furthermore, it successfully determines TTX contents in puffer fish and clam samples, demonstrating its potential for monitoring seafood. Herein, this work provides a favorable rapid sensing platform that is easily portable.

To address the limitations of the CRISPR/Cas12f1 system in clinical diagnostics, which require the complex preparation of single-stranded DNA (ssDNA) or in vitro transcripts (RNA), we developed a fluorescent biosensor named PDTCTR (PAM-dependent dsDNA Target-activated Cas12f1 Trans Reporter). This innovative biosensor integrates Recombinase Polymerase Amplification (RPA) with the Cas12f_ge4.1 system, facilitating the direct detection of double-stranded DNA (dsDNA). PDTCTR represents a significant leap forward, exhibiting a detection sensitivity that is a hundredfold greater than the original Cas12f1 system. It demonstrates the capability to detect Mycoplasma pneumoniae (M. pneumoniae) and Hepatitis B virus (HBV) with excellent sensitivity of 10 copies per microliter (16.8 aM) and distinguishes single nucleotide variations (SNVs) with high precision, including the EGFR (L858R) mutations prevalent in non-small cell lung cancer (NSCLC). Clinical evaluations of PDTCTR have demonstrated its high sensitivity and specificity, with rates ranging from 93%-100% and 100%, respectively, highlighting its potential to revolutionize diagnostic approaches for infectious diseases and cancer-related SNVs.This research underscores the substantial advancements in CRISPR technology for clinical diagnostics and its promising future in early disease detection and personalized medicine.

Amyloid fibrils are formed from various pathological proteins. Monitoring their aggregation process is necessary for early detection and treatment. Among the available detection techniques, fluorescence is simple, intuitive, and convenient due to its sensitive and selective mode of detection. It has certain disadvantages like poor photothermal stability and detection state limitation. Research has focused on minimising the limitation by developing hybrid fluorescence techniques. This review focuses on the two ways fluorescence (intrinsic and extrinsic) has been used to monitor amyloid fibrils. In intrinsic/label free fluorescence: i) The fluorescence emission through aromatic amino acid residues like phenylalanine (F), tyrosine (Y) and tryptophan (W) is present in amyloidogenic peptides/protein sequence. And ii) The structural changes from alpha helix to cross-β-sheet structures during amyloid formation contribute to the fluorescence emission. The second method focuses on the use of extrinsic fluorophores to monitor amyloid fibrils i) organic dyes/small molecules, ii) fluorescent tagged proteins, iii) nanoparticles, iv) metal complexes and v) conjugated polymers. All these fluorophores havetheir own limitations. Developing them into hybrid fluorescence techniques and converting it into biosensors can contribute to early detection of disease.

Cancer detection is still a major challenge in public health. Identification of oncogene is the first step toward solving this problem. Studies have revealed that various cancers are associated with miRNA expression. Therefore, the sensitive detection of miRNA is substantially important to solve the cancer problem. In this study, let-7a, a representative substance of miRNA, was selected as the detection target. With the assistance of magnetic beads commonly used in biosensors and self-synthesized graphene oxide materials, specificity and sensitivity detection of the target gene let-7a were achieved via protease-free signal amplification. The limit of detection (LOD) was as low as 15.015pM. The fluorescence signal intensity showed a good linear relationship with the logarithm of let-7a concentration. The biosensor could also detect let-7a in complex human serum samples. Overall, this fluorescent biosensor is not only simple to operate, but also strongly specificity to detect let-7a. Therefore, it has substantial potential for application in the early diagnosis of clinical medicine and biological research.

The quantitative detection of luteinising hormone (LH) is critical for the study of the physiological mechanism of reproductive function and the assessment of infertility and the clinical treatment of reproductive disorders. However, conventional approaches for LH detection are mostly based on an antibody recognition module with the limitations of sensitivity, simplicity and cost. The development of robust LH sensing methods is therefore highly demanded for facilitating the diagnosis of LH-related diseases. We establish a convenient, amplified and sensitive fluorescent aptamer LH assay based on new target-triggered and cascaded autocatalytic hairpin assembly (C-aCHA) circuit amplification means via initiator sequence replication. Target LH molecules bind the aptamers in the aptamer/initiator duplexes to release the initiator sequences, which trigger CHA formation of DNA three-way junctions (TWJs) and the unfolding of fluorescently quenched signal hairpins to show amplified fluorescence. The TWJs further activate another CHA cycle for the yield of more initiator sequences to form the C-aCHA circuit amplification cycles, which lead to the unfolding of many signal hairpins to exhibit substantially magnified fluorescence recovery for detecting LH down to 8.56 pM in the range from 10 pM to 50 nM. In addition, the monitoring of trace LH in diluted serums by this sensing approach has been also verified. Our LH assay clearly outperforms current existing antibody-based methods and the C-aCHA signal amplification strategy can be easily extended as a robust means for sensitively monitoring various biomolecular markers with simple replacement of the corresponding aptamers for diverse applications.

To address the food safety issues caused by toxins, we established a fluorescent copper nanocluster biosensor based on magnetic aptamer for the visual and quantitative detection of ZEN. Specifically, we utilized the docking-aided rational tailoring (DART) strategy to analyze intermolecular force and interaction sites between zearalenone (ZEN) and the aptamer, and optimize the long-chain aptamer step by step to enhance the binding affinity by 3.4 times. The magnetic bead-modified aptamer underwent conformational changes when competing with complementary sequences to bind with ZEN. Then, the released complementary sequences will be amplified in template-free mode with the presence of the terminal deoxynucleotidyl transferase (TdT), and generating T-rich sequences as the core sequences for the luminescence of copper nanoclusters. The luminescence could be visualized and quantitatively detected through ultraviolet irradiation. The proposed label-free aptasensor exhibited high sensitivity and specificity, with a low limit of detection (LOD) of 0.1 ng/mL.

Target specificity, one of aptamer characteristics that determine recognition efficiency of biosensors, is generally considered to be an intrinsic property of aptamer. However, a high-affinity aptamer may have additional target binding specificity, little is known about the specificity of aptamer binding to multiple targets, which may result in false-positive results that hinder the accuracy of detection. Herein, an aptamer OBA3 with dual target ochratoxin A (OTA) and norfloxacin (NOR) was used as an example to explore the binding specificity mechanism and developed rapid fluorescent aptasensing methods. The nucleotide 15th T of aptamer OBA3 was demonstrated to be critical for specificity and affinity binding of target OTA via site-saturation mutagenesis. Substituting the 15th T base for C base could directly improve recognition specificity of aptamer for NOR and remove the binding affinity for OTA. The combination of π-π stacking interactions, salt bridges and hydrogen bonds between loop pocket of aptamer and quinolone skeleton, piperazinyl group may contributes to the fluoroquinolone antibiotics (NOR and difloxacin)-aptamer recognition interaction. Based on this understanding, a dual-aptamer fluorescent biosensor was fabricated for simultaneous detection of OTA and NOR, which has a linear detection range of 50-6000 nM with a detection limit of 31 nM for OTA and NOR. Combined with T15C biosensor for eliminating interference of OTA, the assay was applied to milk samples with satisfactory recovery (94.06-100.93%), which can achieve detection of OTA and NOR individually within 40 min.