Biosensors and Bioelectronics最新文献

Rapid, low-cost, and multiplexed nucleic acid testing is essential for human health but remains challenging. Although CRISPR-Cas systems offer high specificity, their integration into Multiplexed platforms suitable for near-patient testing has been limited. Here, we introduce a biosensing platform that combines CRISPR-initiated enzymatic amplification with quantum-dot encoded microbeads and deep-learning based image analysis for Multiplexed detection of human papillomavirus (HPV) DNA. The high specificity of CRISPR/Cas9 first triggers an exponential amplification. The products are then specifically captured on the microbead surface for localized fluorescent readout. A custom deep-learning algorithm automatically quantifies the bead fluorescence, enabling robust and automated analysis. The integrated approach achieves simultaneous detection of HPV16, HPV18, and HPV33 with detection limits as low as 0.2 pM. By using recognition-triggered amplification and a simple deep-learning assisted fluorescence readout, the workflow is significantly simplified. The platform establishes a universal and practical strategy for molecular diagnostics, demonstrating strong potential for near-patient testing.

Glycine is an essential metabolite and a valuable precursor for various industrially relevant compounds. However, the lack of efficient high-throughput detection methods has limited progress in metabolic engineering for glycine bioproduction. In this study, we developed a transcription factor-based glycine biosensor in Escherichia coli by reprogramming the native GcvA/GcvR regulatory complex. By fine-tuning the expression balance between GcvA and GcvR, the biosensor exhibited significantly reduced basal fluorescence while achieving strong glycine-dependent induction of up to 16.45-fold. The optimized system demonstrated rapid response kinetics, high sensitivity, and excellent specificity toward exogenously supplied glycine. The biosensor accurately reflected intracellular glycine concentrations in engineered glycine-producing strains, with fluorescence output correlating with production levels. Furthermore, its application enabled high-throughput screening of SerC (phosphoserine aminotransferase) mutants with enhanced glycine production. The best-performing SerC variant resulted in a 48.54-fold increase in fluorescence intensity and a 2.25-fold improvement in glycine accumulation, validating the biosensor as an effective tool for directed enzyme evolution. This work established a robust and tunable platform for glycine biosensing, offering broad potential for metabolic pathway optimization and enzyme engineering related to glycine metabolism.

Enhancing the electrochemiluminescence (ECL) of Au nanoclusters (AuNCs)-based system is a crucial approach for broadening their applications. Unlike traditional strategies that relied solely on coreaction accelerators to promote coreactant oxidation, this work innovatively adopted a "luminophore-coreaction accelerator" synergistic catalysis strategy to enhance the ECL of the AuNCs-Triethylamine (TEA) system. Specifically, Pd nanocubes (PdNCs), serving as a coreaction accelerator, formed a heterostructure with AuNCs on the working electrode surface. This structural design enhanced the ECL signal of the AuNCs-TEA system via two mechanisms. On one hand, PdNCs acted as efficient "electron traps" to capture electrons injected from TEA into the lowest unoccupied molecular orbital (LUMO) of AuNCs, thus significantly promoting the oxidation of TEA. On the other hand, the incorporation of PdNCs effectively improved the electrochemically active surface area of AuNCs, thus providing more active sites to catalyze the oxidation of TEA. Together, these two mechanisms accelerated the generation of TEA free radicals (TEA^•) and the subsequent excited state of AuNCs (AuNCs*), thereby enhancing the ECL of the system. As an application, the developed ECL aptasensor exhibited favorable analytical performance for As³⁺. This study has opened up a new way to improve the ECL properties of metal nanoclusters.

Urea is a key biomarker for diagnosing various kidney and liver disorders, which can be assessed through sensing platforms. However, existing urea sensors have performance issues in real samples. Herein, a novel fiber-organic electrochemical transistor (F-OECT) sensing platform was constructed by sequentially depositing cobalt-based metal-organic framework material (ZIF-67), carboxylated multi-walled carbon nanotubes, poly (3,4-ethylenedioxythiophene), and urease on cotton fibers for urea determination. The resulting F-OECT sensor exhibited excellent performance toward urea detection with a high sensitivity (82.91 μA/decade). This performance is attributed to the synergistic effect of the hierarchical composite: the significantly enlarged electrochemically active surface area of PEDOT/f-MWCNT/ZIF-67 facilitates high-density urease immobilization, while the conductive network ensures efficient signal transduction, coupled with the intrinsic signal amplification of the OECT architecture. Under optimized conditions, the F-OECT sensor illustrated a wide linear response range (1 pM-10 mM) and a low detection limit of 1 pM toward the detection of urea, coupled with an excellent selectivity, reproducibility, and stability. More importantly, the F-OECT sensor was able to detect urea in real sweat and urine samples. Based on these encouraging data, the F-OECT sensor was connected to a printed circuit board to transmit signals via Bluetooth, and the results showed a sensor capable of visually quantifying urea. Overall, the proposed F-OECT sensor looks promising for future non-invasive and sensitive liquid detection of urea, thereby deserving further research and development.

African swine fever (ASF), a highly fatal and contagious animal disease caused by ASF virus, leads to substantial economic losses. Early detection is critical, as the viral load is initially low, demanding highly sensitive detection of the viral p30 antigen. Although the existing digital ELISA enables sensitive detection of p30, its performance is constrained by inefficient microcavity utilization (resulting in Poisson noise-limited sensitivity) and low image recognition accuracy. This study presents an open-surface digital ELISA (OS-dELISA) platform that integrates an open-space magnetic bead (MB) array with artificial intelligence. The platform uses magnetic trapping to form the MB array, which acts as an open-space microcavity. Combined with reciprocating-flow microfluidics and in-situ tyramine signal amplification, this design eliminates the additional step of encapsulating immunocomplexes into enclosed microcavities, enhances microcavity utilization and antigen capture efficiency, and improves sensitivity. An improved mask region-based convolutional neural network enables intelligent and accurate image recognition for large-scale datasets. OS-dELISA achieves highly sensitive p30 detection within 17 min, with a detection limit of 21 fg/mL. Serum samples validation showed 100% sensitivity and 95% specificity. By combining microfluidics and artificial intelligence, OS-dELISA provides a powerful tool for intelligent animal disease diagnostics.

The integration of DNA hydrogel with electrochemiluminescence (ECL) technology represents a synergistic enhancement through molecular-level precision design and nanoscale coordination. This strategic integration confers biosensors novel functionalities including intelligent responsiveness and environmental adaptability. In this study, a cascaded hybridization chain reaction (HCR) and CRISPR/Cas12a-integrated DNA hydrogel paper chip was engineered for ultrasensitive microRNA 622 (miRNA 622) detection. Target miRNA 622 triggered HCR amplification via hairpin DNA assembly, while Cas12a recognized protospacer adjacent motif (PAM) sequences within the HCR-generated double-stranded products to activate its trans-cleavage ability. The DNA hydrogel was constructed through copolymerization of acrylamide-modified DNA single strands (SA and SB) with Ru (II) complex-functionalized linker DNA. Activated Cas12a cleaved single-stranded DNA within the DNA hydrogel network, thereby releasing Ru (II) complexes. AuPd nanoparticles (AuPd NPs) served as the co-reactant accelerator, amplifying the cathodic ECL signals of the liberated Ru (II) complexes. The developed platform demonstrated a dynamic detection range from 0.001 to 500 pM with a detection limit of 0.33 fM, establishing a groundbreaking approach for detecting miRNA 622 in clinical diagnostics.

Early screening is crucial for improving the survival rate of gastric cancer (GC). MiRNA-106a is abnormally overexpressed in GC tissues, making it an ideal biomarker for liquid biopsy. In this study, a floating-gate carbon nanotube field-effect transistor (FG CNTFET) biosensor, functionalized with tetrahedral DNA nanostructure (TDN) probes was developed to enable label-free, highly sensitive detection of miRNA 106a. The FG layer not only physically isolates the CNT channel from the complex biological environment-shielding it against moisture, ions, and impurities-but also amplifies surface potential changes via capacitive coupling, thereby enhancing both device stability and sensitivity. The rigid framework of TDN probes overcomes the issues of aggregation and entanglement issues associated with single-stranded DNA (ssDNA), reducing steric hindrance and improving target accessibility. In static mode, the biosensor exhibited a linear detection range of 1 fM-1 μM with a detection limit (LOD) as low as 7 aM. Under dynamic conditions, it enabled real-time tracking of miRNA-106a binding events with an LOD of 1 pM. Preliminary analysis of 12 pilot clinical serum samples demonstrated the biosensor's capability of distinguishing patients from healthy donors, despite the limited sample size. These findings validate the potential of TDN-functionalized FG CNTFET architecture for aM-level early GC screening, laying a technological foundation for the development of low-cost, portable point-of-care testing (POCT) devices.

Direct measurement of mitochondrial oxygen tension in vivo provides direct information on tissue metabolism and could facilitate new approaches in disease detection, function monitoring, and treatment efficacy assessment with cellular level data, with far superior sensitivity to oxygen changes than blood saturation measures. Here, a platform system was engineered to quantify fast sampling of oxygen partial pressure (pO₂) inside tissue by utilizing the inherent rolling shutter readout of a smartphone CMOS camera detector for measuring the oxygen-sensitive time-resolved delayed fluorescence (DF) signal from Protoporphyrin IX (PpIX) which naturally occur in mitochondria for most tissues. The CMOS rolling shutter readout produces a microsecond-level time difference in the pixels row-by-row detection of light, here utilized as a time-gated shutter to sample the time distributed DF intensity. This novel technique eliminated the necessity of high-speed intensified camera with excitation isolation facility as well as advanced precise time synchronization system as required in the conventional time-resolved fluorescence lifetime measurement platforms for quantifying time-dependent very low intensity DF distribution of PpIX conjugated with prompt fluorescence. Both steady state and dynamic performance of the instrument were validated in tissue phantoms at different PpIX concentrations (0.5-10 μM) for wide range of pO₂ detection (0-160 mmHg) with tunable fast response time (1-3.5 s) which is substantially faster than electrode-based systems that measure over 10's of seconds. Finally, it was tested in vivo to assess the impact of dynamic inhaled oxygen concentration variation on skin tissue pO₂ under the conditions of normoxia, hyperoxia and hypoxia.

miRNA, the non-coding RNA comprised of about 22 nucleotides, serves as the biomarker in various biomedical scenarios based on different expression level. The more miRNAs are characterized, the more accurate result will be obtained, highlighting the necessary of multiplex miRNA quantification. Although PCR-based methods have been widely applied, the limited specificity and sensitivity restrict their application in liquid biopsy, prenatal diagnosis and forensic identification due to the trace of target and degraded specimen. Despite the higher sensitivity and specificity of loop-mediated isothermal amplification (LAMP), the short fragment renders miRNA detection impossible, let alone multiplex quantification. In this study, a novel strategy for multiplex miRNA quantification is developed based on the response of difunctional molecular beacon to hairpin probe-triggered isothermal amplification. Two hairpin probes are designed to hybridize with target miRNA and fused by ligase, forming a dumbbell structure as the trigger to initiate LAMP. The difunctional molecular beacon, featured with 3'-overhang, serves as loop primer by annealing to the dumbbell structure and participates in amplification. The quencher-fluorophore pair located on molecular beacon is separated through strand displacement, generating fluorescence to monitor amplification of each target for multiplex miRNA quantification. Hairpin probe addresses the incompatibility between template length requirement of LAMP and short fragment of miRNA, even extended cDNA from reverse transcription. Besides, specificity is improved by hairpin probe fusion with single nucleotide distinguishability, which is also not possessed in reverse transcription. The paradigm contains universal hairpin probe and molecular beacon, providing a general platform for multiplex miRNA analysis.