Nucleic acid detection plays an important role in pathogen monitoring and disease diagnosis. CRISPR one-pot assays combined with isothermal amplification are emerging as promising point-of-care technologies that simplify workflows while increasing sensitivity and specificity. However, the incompatibility inherent in the one-pot reaction of isothermal amplification and CRISPR detection limits their practical application. This review comprehensively analyzes diverse advanced one-pot CRISPR-based isothermal amplification strategies developed to overcome this fundamental challenge. These strategies primarily encompass physical separation strategies (utilizing lid-bottom, internal ledge, nested tube, and membrane approaches), phase separation strategies (employing glycerol, sucrose, and gel matrices), reaction system optimization strategies (fine-tuning reaction parameters and incorporating specialized additives), non-PAM and suboptimal PAM strategies, improved Cas enzyme strategies (enhanced Cas12 and Cas13 variants), light-controlled approaches (PC-oligonucleotides, NPOM-dt modification, and acylation modification), and microfluidic chip integration strategies (centrifugal microfluidic chips, droplet microfluidic chips, and microarray chips). These methodological approaches have achieved important advances in simplifying operational processes, enhancing sensitivity, shortening detection cycles, and minimizing cross-contamination risks. The review further synthesizes critical insights regarding current opportunities, technical challenges, and future directions for one-pot CRISPR-based isothermal amplification technologies in nucleic acid detection, providing valuable guidance for researchers and practitioners in this evolving field.

Extracellular vesicles (EVs), which carry a variety of molecules such as proteins and nucleic acids, have great potential for broad application in liquid biopsy. However, achieving highly sensitive detection of biomarkers within EVs remains a significant challenge. The emergence of CRISPR/Cas systems─adaptive immune mechanisms found in bacteria and archaea that defend against foreign genetic elements─offers new opportunities to address this issue through powerful nucleic acid recognition and cleavage capabilities. Compared to other EV detection techniques, CRISPR/Cas-based biosensors exhibit superior sensitivity, specificity, and operational efficiency, making them a compelling platform for clinical translation. Thus, to promote the application of EVs in disease diagnosis, disease monitoring, and therapeutic evaluation, this review focuses on the state-of-the-art CRISPR/Cas systems (specifically CRISPR/Cas9, CRISPR/Cas12, CRISPR/Cas13, and CRISPR/Cas14) as well as the latest applications of CRISPR/Cas-based EV detection techniques.

Lateral flow assays (LFAs) are point-of-care devices that are known for their affordability, speed, and simplicity. However, LFA sensitivity is often limited by the need for fast associative rates between the assay components. This work presents a strategy toward reducing the demand for fast test line associative kinetics via a "capture-and-release" approach. Using HER2 protein as a model biomarker system, this methodology─termed the "AmpliFold" approach─involves the initial sequestration of analyte-bound complexes, which undergo triggered release and are rebound, using high-affinity hapten interactions, resulting in enhanced signal-to-noise detection. Using anti-HER2 Fab fragments modified with cleavable biotin linkers to achieve triggered release, the importance of linker length and bioconjugation strategy on the efficiency of analyte-bound complex release is described. Cleavable Fab fragment conjugates were combined with 'dual-affinity' gold nanoparticles (AuNPs) highly decorated with fluorescein-tagged anti-HER2 antibodies to facilitate signal amplification. The utility of the AmpliFold approach is demonstrated by titrating capture receptor density to modulate the signal distribution across test lines. Larger capture areas in the AmpliFold approach were shown to overcome poor capture kinetics associated with low receptor densities, achieving up to a 16-fold improvement in LFA sensitivity. The AmpliFold approach was further shown to address the poor diffusivity and surface binding kinetics of large nanoparticles in LFA systems. Using high capture receptor densities and a 150 nm AuNP example, a 12-fold sensitivity enhancement was achieved when using AmpliFold to detect the target analyte spiked into both buffer and human serum samples. Incorporated into a folding "two-strip" LFA design and performed via a multistep (capture, wash, and linker cleavage) workflow, the AmpliFold approach represents a proof-of-concept strategy that utilizes established protein modification chemistries to provide a rapid (<30 min), equipment-free, and tractable route toward enhancing LFA kinetics and sensitivity.

Exosomes that can cross the blood-brain barrier are promising biomarkers for glioma diagnosis, yet highly specific and sensitive detection of glioma-derived exosomes remains a challenge. Herein, a strategy of "proximity effect-mediated DNA self-assembly" has been proposed to achieve highly specific, sensitive, and flexible detection of glioma-derived exosomes. Exosomes are separated and enriched by CD63 aptamer-modified nanomagnetic beads; a pair of proximity probes simultaneously binds to PDPN and EGFR on the surface of exosomes, which will induce proximity effect-mediated DNA self-assembly with linker probes and the subsequent invertase-labeled signal amplification probes, thus converting one target exosome in the presence of multiple invertases. Benefiting from the dual signal amplification from nucleic acid self-assembly and enzymatic reaction, highly sensitive and flexible detection of glioma-derived exosomes can be achieved by using a portable blood glucose meter, with a limit of detection of 3 × 104 particles/mL. Of note, the combined detection of multiple exosomal surface markers (CD63/PDPN/EGFR) based on proximity hybridization significantly improves the specificity of glioma-derived exosome detection, enabling efficient discrimination of glioma cells from normal microglia and various other tumor cells. Furthermore, the level of CD63/PDPN/EGFR-positive exosomes in glioma patients was significantly higher than that of healthy subjects (P < 0.0001); compared with the CD63/PDPN- and CD63/EGFR-positive exosomes (AUCs of 0.852 and 0.895), the detection of CD63/PDPN/EGFR-based exosomes provides a remarkably accurate diagnosis of glioma (AUC of 0.98). Additionally, this strategy can be easily extended to the detection of other disease-derived exosomes just by replacing the corresponding recognition units.

To understand the gas-sensing mechanism of COFs and explore an effective modulation way to regulate their sensing properties, the adsorption and sensing behaviors of NO2, NO, SO2, O2, H2O, CO2, H2S, CO, N2, and NH3 gas molecules on the surface of pristine and n-doped (Na-adsorption) Tr-Th COFs are explored theoretically with first-principles calculations in this work. Attributed to the lowest unoccupied molecular orbital (LUMO) energy level of NO2, the adsorption of NO2 on Tr-Th leads to a larger increase in carrier concentration increment (n = 1.79 × 1012 to 6.26 × 1010 cm-2) and a greater work function shift (ΔΦ = 0.178 eV) compared to other gases, which suggest that Tr-Th is a highly promising material for NO2 sensing applications. n-Type doping elevates the Fermi level of COFs, resulting in a greater carrier concentration increment (n = 1.83 × 1012 cm-2 ∼ 2.52 × 1012 cm-2) and a larger work function shift (ΔΦ = 0.08 eV ∼ 0.30 eV) upon exposure to NO2, NO, SO2, or O2 compared to other gases. It means that apart from NO2, NO, SO2, and O2 gases will also trap electrons in n-doped Tr-Th COFs, increase the electrical resistance dramatically, and then quench the source leakage current of the COFs-FET gas sensor. Our study provides more detailed information about the gas-sensing mechanism of COFs and highlights the key role that surface doping strategy plays in regulating the gas adsorption and selection behaviors for its practical gas sensor applications.

In situ monitoring of biomarkers in wound fluids using wearable potentiometric sensors is especially significant for early wound infection prediction and effective wound management. However, the antifouling abilities of such sensors in wound fluids have rarely been investigated. Herein, we propose a simple and versatile strategy based on a self-adhesive hydrogel coating to impart antifouling capabilities to a wearable potentiometric sensing chip for on-body wound monitoring. A catechol-functionalized zwitterionic hydrogel is synthesized via a dopamine-triggered gelation strategy, which exhibits a low swellability, good biocompatibility, and high stretchability. Notably, owing to its self-adhesive properties, a thin hydrogel layer could be readily immobilized on various surfaces of the sensing chip without elaborate modification protocols. Using a polymeric membrane-based H+-sensing chip as a model, the hydrogel-coated chip exhibits significantly enhanced antifouling performance and decreased cytotoxicity compared to the pristine one. These improvements are evidenced by the markedly suppressed adsorption of bacteria, proteins, and cells, and an ∼35% increase in cell viability. In situ bacterial infection monitoring in a series of on-body experiments indicates the capability of the proposed antifouling sensing chip to measure both spatial and temporal pH changes in wound microenvironments.

Optical coherence tomography (OCT) is a powerful tool for in vivo corneal imaging; however, its diagnostic precision is hampered by a lack of molecular specificity. To overcome this limitation, we designed and synthesized CD44 aptamer-conjugated gold nanoparticles (AP-GNPs) as a molecularly targeted OCT contrast agent for the quantitative assessment of corneal injury. AP-GNPs specifically bind to CD44, a biomarker overexpressed in injured corneal tissue, leading to enhanced signal intensity and prolonged retention at the lesion site compared to nontargeted nanoparticles. In the alkali burn mouse model, AP-GNP-enhanced OCT enabled noninvasive and highly sensitive detection of mild and severe injuries, demonstrating a significant increase in the signal-to-noise ratio from 12 to 24 h postinsult, a feat not achievable by conventional diagnostics such as fluorescein staining. This was quantified by a significant amplification of the signal-to-noise ratio at 12 h by 23.6% for mild injuries and 54.5% for severe injuries, relative to nonenhanced controls. Comprehensive safety profiling confirmed the excellent biocompatibility of the nanoprobes after topical administration. This study establishes an aptamer-based contrast enhancement strategy for OCT, providing the first demonstration of quantitative in vivo improvement in detection sensitivity for corneal pathology and offering a promising platform for both preclinical research and clinical diagnostics.

Sensitive detection of low-abundance biomarkers in blood is essential for the early diagnosis of Alzheimer's disease (AD). Although the single molecule array (Simoa) platform offers femtomolar-level sensitivity and surpasses conventional assays such as enzyme-linked immunosorbent assay, its broader clinical utility is constrained by issues like nonspecific binding and a limited dynamic range. In this study, we present a surface-engineered microfluidic platform incorporating engineered beads to reduce nonspecific binding, electrostatic bead-microwell pairing for improved capture efficiency, and an algorithmic calibration model to extend the dynamic range. These innovations collectively improve the analytical sensitivity and quantification accuracy for plasma Aβ1-42 and pTau181. Statistical analyses assessed correlation between our microfluidic platform and Quanterix Simoa, and evaluated our diagnostic ability using the Quanterix Simoa measurements as the reference standard in individuals with AD (N = 107) and cognitively normal controls (N = 100). We further validated diagnostic cutoff values in an independent cohort. The microfluidic platform demonstrates a superior diagnostic performance and enables reliable longitudinal monitoring of plasma biomarkers. Our results highlight the potential of the surface-engineered microfluidic platform for clinical translation in neurodegenerative disease diagnostics.

The preclinical window for acute brain injury is characterized by its considerable narrowness, thus necessitating the development of clinical evaluation methods that are both highly efficient and sensitive. Herein, we constructed a highly miniaturized photothermal immunoassay platform integrated onto a pipet tip for on-site detection of the central nervous system-specific protein S100β. Signal amplification was performed using a liposome-embedded CuS nano-octahedral framework (CuS NOFs). The template-synthesized CuS NOFs demonstrated a high photothermal conversion efficiency (30.2%). Chitosan gel served as the sensitization and immobilization matrix, functionalizing the inner wall of pipet-compatible tips with capture probes for signal transduction in the presence of targets. The developed photothermal assay protocol based on pipet tips satisfied the specificity requirements for S100β in blood samples and enables rapid and low-cost (1.0 $ per test) testing across an ultrawide range of 0.05-500 ng·mL-1. Using 3D printing technology, all components were fabricated and integrated, including a pipet, a support platform, a smartphone, a near-infrared laser, and a dedicated sample chamber. This work provides novel insights for developing rapid point-of-care photothermal immunoassays for acute brain injury.

N-acetyl-β-d-glucosaminidase (NAG) is an important biomarker that indicates early renal tubular damage. Its activity level in urine has been widely used for early detection and monitoring of chronic kidney disease (CKD), particularly diabetic nephropathy. However, current detection methods rely on complex instrumentation, which hinders their suitability for point-of-care testing (POCT) that requires portability and user-friendliness. To address this challenge, we developed a sequential dual-reagent paper-based analytical device (SeDR-PAD) that utilizes structural partitioning and a delayed-trigger mechanism to achieve time-programmed control of the NAG enzymatic reaction and color development process. The system uses VRA-GlcNAc as the substrate to generate a chromogen under the catalysis of NAG. A portable device then releases an alkaline catalyst after a programmed delay to initiate color development, followed by optical quantitative analysis. This method provides a linear detection range of 0-200 U/L, with a detection limit of 0.524 U/L and high reproducibility (CV < 6%, n = 5). In a methodological comparison involving 70 clinical urine samples, the SeDR-PAD platform demonstrated strong concordance with the standard clinical method, with a correlation coefficient R2 of 0.9833. These findings highlight the strong potential of SeDR-PAD for clinical POCT of uNAG in individuals at high risk for kidney disease.