Digital bioassays are emerging as a pivotal technology in disease prevention and diagnostics due to their single-molecule level detection capability. However, elaborate microchamber fabrication, high-end equipment, and skilled manipulation are required to enable the end - point digital signal readout. Herein, we propose a novel concept of artificial intelligence (AI)-facilitated real-time digital micromotor tracking-enabled immunoassay (AI-dMIA), which leverages a self-developed multi-microparticle tracking system to monitor micromotor motion trajectories in a real-time manner for digital protein analysis. In this design, even a single molecule-bridged immunobinding event on the fully-open microparticle's surface can induce obvious motion behaviors and trajectories, forming a positive micromotor that can be accurately tracked and discriminated from the negative ones in real time by a bright-field microscope integrated with the AI algorithm. The AI-dMIA gets rid of the complex process of microchamber fabrication and fluorescence signal yielding/amplification to generate digital counting events. It also exhibits powerful processing capacity to simultaneously track up to thousands of motor trajectories on a large scale, no longer requiring the high-end equipment that is essential to traditional end-point digital bioassays. The AI-dMIA not only demonstrates a robust immunosensing approach but also provides a new alternative for developing next-generation digital micromotor-based platforms.

Digital assays have become a state-of-the-art technology for the sensing of nucleic acid molecules. Nevertheless, most of the prevalent digital nucleic acid assays heavily rely on sealed microchambers and sophisticated nucleic acid amplification strategies to achieve an end-point digital signal readout. Herein, we proposed an artificial intelligence (AI)-facilitated, digital fluorescence nanomotor tracking-powered assay (AI-dFNA) for one-step, amplification-free, and highly sensitive detection of microRNAs (miRNAs). In this design, even a single target miRNA-templated amplification-free ligation can introduce a sufficient driving force on the surface of a fully open fluorescence nanoparticle to cause remarkable motion behaviors. With the assistance of a self-developed AI algorithm, clear discrimination of positive and negative fluorescent nanomotors can be achieved, enabling real-time digital miRNA sensing. The AI-dFNA avoids the use of enclosed microchambers and intricate signal amplification techniques, which could even identify varying fluorescence colors of nanomotors to allow for the analysis of different kinds of miRNA molecules, pioneering a new direction toward next-generation digital nucleic acid sensing.

Glioma is the most common brain neoplasm that features aggressive behavior with a dismal prognosis. Isocitrate dehydrogenase (IDH) gene mutation in glioma is an early genetic event in gliomagenesis that occurs in virtually every tumor cell and can cause profound metabolic changes. In this manuscript, we report for the first time the analysis of Raman optical signatures of IDH genotypes for human glioma using visible resonance Raman (VRR) spectroscopy. We demonstrated that VRR is a rapid, label-free, and objective method as an alternative to the existing methods for the rapid intraoperative determination of IDH mutation status with high accuracy. This study shows AI-assisted VRR has the potential to provide a new optical molecular biomarker and perform early diagnosis of glioma, which is of great importance for current guiding surgical strategies and even for targeting in situ therapies in the future.

Digital bioassays have emerged as cutting-edge techniques for highly sensitive biomarker analysis. However, the requirement of sophisticated operation and equipment, fully sealed microchambers, and well-trained professionals confines its application to central laboratories. Herein, we present dMSink, a new conceptual mix-and-read d igital biosensing platform based on the single microbead ( M B) floating/ sink ing mechanism that is compatible in general biolabs and even in-field testing. Specifically, MBs will float on the surface of a liquid with a finely tuned critical density to ensure that even a single target molecule binding event-introduced extra tiny force (e.g., magnetic force) on the MB is sufficient to induce its sinking. Therefore, only by digitally counting the number of sunken MBs with a conventional microscope can ultrasensitive quantification of diverse biomarkers be facilely achieved. The dMSink empowers the digital bioassay for both speed and sensitivity, significantly simplifying its implementation to in-field multiplexed diagnosis.

Digital immunoassay is a state-of-the-art technique for quantifying low-abundance protein biomarkers, but the prevalent digital platforms are generally limited by the precise microfabrication of enclosed reaction chambers and reliance on fluorescence-based readouts. Herein, we propose a sealed chamber-free, persistent-chemiluminescence (CL)-powered digital bead-counting immunoassay (dCCIA) for ultrasensitive protein sensing, well eliminating the need for physical target compartmentalization and the light-scattering/photobleaching interference associated with fluorescence readout. This dCCIA platform rationally integrates tyramide signal amplification with phenothiazine derivative-enhanced HRP-H2O2-luminol CL (E-CL) system, enabling highly enhanced and stable CL emission spatially confined around the target-loaded microbeads. This spatially confined, long-lasting CL signaling enables single target molecule-loaded microbead to function as an independently identifiable digital counting unit, allowing clear binary classification into "CL-positive" or "CL-negative" events and ensuring highly reliable digital readout. The dCCIA achieves attomolar-level sensitivity for prostate-specific antigen, which exhibits over 100-fold improvement in sensitivity compared to both conventional bulk CL measurement mode and fluorescence-based signal readout under identical experimental conditions, establishing a new digital paradigm for ultrasensitive protein detection.

How to precisely discriminate homologous microRNA sequences from each other has always been a big challenge. Herein, we proposed an artificial mismatch-assisted Cas12a crRNA splicing mechanism that enables completely interference-free discrimination of homologous microRNAs with single-nucleotide resolution. Specifically, a truncated crRNA (tcrRNA) can recover its ability to activate Cas12a trans-cleavage activity when spliced with a miRNA. Since the miRNA splicing region itself is relatively short, rationally tuning the tcrRNA splicing length can render the proposed system with high sensitivity for base-variation discrimination. More importantly, we innovatively introduced an extra artificial mismatch in the miRNA splicing region, which enables the system to distinguish single-nucleotide variants in an interference-free manner. In this way, only the perfectly matched target miRNA can trigger the complete assembly of the spliced crRNA to active Cas12a, while non-target RNAs even with a single-base mismatch cannot. This mechanism ensures that the target miRNA can be accurately hooked even in the presence of a large excess of non-target homologous RNAs (100-fold). The discrimination capability of the proposed method has been proven by achieving the interference-free discrimination of the members within the let-7 family, which can hardly be achieved by conventional hybridization-based amplification methods. This work fully taps into the potential of the Cas12a system in interference-free homologous miRNA discrimination, offering a new tool for precise RNA analysis in molecular diagnostics. (c) 2026 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.

The accurate and on-site measurement of trace water content in industrial processes is essential for product quality and production safety. However, most existing methods remain restricted to laboratory environments and are not readily applicable under complex field conditions. Herein, a universal visual sensing platform for rapid and accurate trace water content monitoring is proposed, based on H2O-actuated lattice regulation of the lead halide perovskite nanocrystals (HPNCs). Water specifically triggers the redox reaction between iodine and sulfur dioxide, and the resulting iodide ions subsequently undergo a halogen exchange with bromide ions in the HPNCs, thereby modulating their band gap and inducing distinct changes in emission wavelength and fluorescence color, thus enabling visual detection of trace water content. Despite the simple operation and instrument-free visual detection characteristics, its performance is comparable to the standard Karl Fischer volumetric titration method. Owing to the highly specific and rapid kinetics of H2O-triggered modulation of the perovskite structure, this strategy enables selective detection of water content within minutes. Integrated with a standard colorimetric card or a common smartphone, it enables semi-quantitative or quantitative on-site determination of water content in various real samples, pioneering a completely new platform for portable trace H2O sensing.

A rapid one-step flow cytometry immunoassay was developed by counting the number of target protein-induced nanoaggregates. Without any separation or washing steps, it quantifies Tau protein rapidly with a lowest detectable concentration of 5 pg mL-1 in a mix-and-read manner.

Digital biosensing has emerged as an irreplaceable, ultrasensitive biosensing method across research and clinical diagnosis. The frontier leap achieved by partitioning target molecules into individual units, reaching single-molecule-level sensitivity, pioneers a new biosensing direction. This review summarizes the fundamental principles, the biosensing performance, the methodological limitations, and the future opportunities of emerging digital platforms integrated with different signal output modes, particularly including fluorescence, Raman scattering, electrochemistry, and particle motion.

The overuse of antibiotics has emerged as a critical global public health challenge. On-site antibiotic detection is of great significance for promoting rational antibiotic use, ensuring food safety, and safeguarding public health. In this work, a visual sensing strategy based on dynamic bandgap modulation of perovskite nanocrystals (PNCs) is developed for the on-site detection of penicillin. The hydrolysis products of penicillin react with iodine to generate iodide ions, which induce lattice structural regulation in CsPbBr3 PNCs through ion exchange, leading to a pronounced redshift in fluorescence emission and an observable fluorescence color change. The concentration of penicillin exhibits a strong correlation with the maximum emission wavelength shift of PNCs, enabling direct visual detection through naked-eye observation of fluorescence color changes without reliance on complex or costly instrumentation. This method demonstrates high specificity, enabling effective discrimination between penicillin and cephalosporin. What is more, no complex pretreatment is required, allowing for on-site detection of penicillin within 60 min. This method has been successfully applied to detecting penicillin in pharmaceuticals, foods, and environmental samples, offering a robust strategy for efficient and rapid screening of penicillin residues in complex matrices.

A versatile TtAgo cleavage-aided isothermal amplification strategy (TAC-IAS) is devised for precise detection of DNA methylation with high flexibility and universality compared to methylation-restriction endonuclease, reliably identifying methylation with an abundance of 0.1% in a large pool of unmethylated fragments.

Microbeads (MBs) aggregation-based immunoassay, which is independent of multistep bead washing, signal labeling, and even reporter eluting procedures, has emerged as a promising label-free route for protein biomarker analysis. However, their wide application is not only subjected to challenges from target-actuated low aggregation efficiency derived from the steric hindrance and high weight of micrometer-sized beads but also suffers from the lack of precise method to exactly measure the uncontrollable aggregation process/state. Herein, a new mechanism of metastable DNA hybridization-accelerated programmable immuno-aggregation of MBs is proposed, which enables the facile mix-and-read and flow cytometric detection of protein biomarkers. The introduction of auxiliary metastable DNA hybridization and magnetic facilitation can remarkably boost the immunoreaction efficiency between two kinds of MBs, achieving an ∼200-fold increase of detection sensitivity. What is more, benefiting from the powerful ability of flow cytometry to precisely interrogate the light scattering and fluorescence information on individual events one-by-one, the distinct discrimination and precise quantification of MB aggregates from MB monomers can be easily achieved. Additionally, fluorescent color and intensity coencoded MBs can be easily acquired by simply adjusting the amounts of fluorescent probes to achieve the multiplexed analysis of protein targets. With these advantages, the proposed method demonstrated a successful application for mix-and-read protein detection, showing great potential in diverse biomedical applications.

We present a robust 'splice-at-will' CRISPR RNA (crRNA) engineering mechanism that overcomes the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in directly detecting ultrashort RNAs. In this strategy, an intact Cas12a crRNA can be split from almost any site of the spacer region to obtain a truncated crRNA (tcrRNA) that cannot activate Cas12a even after binding an auxiliary DNA activator. While splicing tcrRNAs with a moiety of ultrashort RNA, the formed combination can work together to activate Cas12a efficiently, enabling 'splice-at-will' crRNA engineering. Importantly, the 'splice-at-will' crRNA exhibits almost the same trans-cleavage activation efficiency as that of a conventional intact crRNA. Therefore, by rationally designing a DNA auxiliary activator with a conserved tcrRNA-complementary sequence and an arbitrary short RNA-of-interest recognition domain, a general sensing system is established that directly utilizes traditional DNA-activated Cas12a to detect ultrashort RNAs. This 'splice-at-will' crRNA engineering strategy could faithfully detect ultrashort RNA sequences as short as 6-8 nt, which cannot be achieved by conventional Cas12a and Cas13a systems. Additionally, through flexible splicing site design, our method can precisely distinguish single-base differences in microRNA and other short RNA sequences. This work has significantly expanded the Cas12a-based diagnostic toolbox and opened new avenues for ultrashort RNA detection.

High-sensitive detection of circulating biomarkers is in high demand because many of them are found at low concentrations in bioliquids. Herein, we report an immuno-transcription-amplified single microbead (MB) assay (IT-SMA) based on the specific S9.6 antibody-DNA/RNA hybrid recognition strategy for the sensitive and universal quantification of protein biomarkers. This design rationally converts the immunoreaction events into amplified nucleic acid transcription to produce numerous RNA molecules, which can efficiently enrich fluorescent signals onto a single MB through a specific S9.6 antibody-DNA/RNA hybrid recognition mechanism, enabling sensitive protein analysis. This method exhibits excellent specificity and high sensitivity for protein analysis with a low detection limit at the fg/mL level. Furthermore, the S9.6 antibody-aided IT-SMA allows for universal detection of various proteins and even exosomes, testing target proteins in serum samples, and differentiating cancer patients from healthy individuals by directly analyzing the exosomes in human blood samples. These features make the IT-SMA strategy a promising tool for the quantitative detection of a variety of biomarkers toward precision diagnostics.

In contrast to conventional CRISPR/Cas12a systems, which rely on complex functional nucleic acids, protein switches, or allosteric transcription factor (aTF)-based signal conversion for non-nucleic acid analysis, this work achieves more facile quantification of non-nucleic acid biomarkers through a novel heparin-mediated Cas12a inhibition mechanism.

Digital biosensing is the state-of-the-art technique for precisely quantifying low-abundance biomarkers but heavily limited to sophisticated fabrication of sealed microchambers and fluorescence signal readout. Herein, a novel persistent, enhanced-chemiluminescence (E-CL)-enabled microchamber-free digital counting strategy is proposed for miRNA analysis by using fully open microbeads (MBs) as independent microreactors and signaling units. The employment of a phenothiazine derivative enhancer efficiently transfers the flash-type CL of horseradish peroxidase (HRP)-H2O2-luminol into persistent and stable E-CL with more than 103-fold signal enhancement. More importantly, by leveraging single miRNA molecule-activated deposition of HRP, the driving power of E-CL, on the miRNA-loaded MBs, the long-lasting E-CL can be finely sustained on the MBs' surface, achieving CL-based binary MB counting for digital miRNA quantification at the aM level. This persistent E-CL-powered microchamber-free digital design may well complement prevalent fluorescence-based digital bioassays by effectively addressing their inherent drawbacks (photobleaching/quenching and scattering light background), thus expanding the digital biosensing toolbox.

We propose a novel single microbead “chemiluminescence (CL) super enriching and imaging” strategy that efficiently concentrates highly intense, long-lasting, and ultrastable CL shining on only a single microbead (SMB), allowing for the ultrasensitive quantification of various biomarkers at the attomolar level. Aided by a phenothiazine derivative enhancer, the traditional flash-type CL of horseradish peroxidase (HRP)-H2O2-luminol can be efficiently converted into a significantly enhanced (103-fold), persistent, and ultrastable CL emission (E-CL) that can be exactly confined and accurately imaged on the microbead interface. Then, by employing only one minuscule-sized SMB as the sole reaction and signaling unit, the target as well as the aroused E-CL signal concentrated on the SMB can further achieve 104 “super-enriching” efficiency. Therefore, by “super enriching” target-responsive E-CL signal on an SMB, the SMB E-CL imaging strategy provides a powerful tool for the attomolar-level detection of various biomarkers including proteins, microRNAs, and virus DNA.

Despite the unique advantage of the isothermal exponential amplification reaction (EXPAR) for the rapid detection of short nucleic acids, it severely suffers from the drawback of sequence-dependent amplification bias, mainly arising from the secondary structures of the EXPAR template under the commonly used reaction temperature (55 °C). As such, the limits of detection (LOD) for different target sequences may vary considerably from aM to nM. Here we report a sequence-generic exponential amplification reaction (SG-EXPAR) that eliminates sequence-dependent amplification bias and achieves similar amplification performance for different targets with generally sub-fM LODs. The assay innovatively employs a thermophilic nicking enzyme that allows SG-EXPAR to work efficiently at higher temperatures (60-70 °C) while eliminating the secondary structures of the templates, which is the basis for eliminating the amplification bias. Furthermore, we increased the probability of trigger/template binding through rational modification of the locked nucleic acids and template optimization, further ensuring the high amplification efficiency for various targets. According to these critical principles, we have developed an automated design platform that allows nonspecialists to obtain the optimal SG-EXPAR template for any desired sequence. The robust performance of the proposed methodology was demonstrated by quantifying microRNA, SARS-CoV-2, monkeypox virus, and HPV B19 at the 1 fM level without sequence screening. SG-EXPAR significantly expands the potential applications of EXPAR and facilitates the development of reliable point-of-care nucleic acid assays.

Surface modification is the prerequisite for yielding reliable bionanomaterials for a variety of applications including molecular diagnosis. Among a library of nanomaterials, upconversion nanoparticles (UCNPs) have emerged as promising candidates for biomolecule sensing toward disease diagnosis. As is well acknowledged, surface modification of UCNPs is required because the as-synthesized UCNPs are generally covered with organic ligands. However, current surface functionalized UCNPs cannot support in-situ signal amplification to boost the sensing performance. In this work, ultra-stable surface modification has been achieved to render UCNPs endurable to high temperatures, allowing for target microRNA (miRNA)-mediated in-situ thermal cycling click ligation on the UCNPs' surface. By integrating with facile magnetic separation, the content of the target miRNA can be faithfully reflected by the upconversion fluorescence intensity in the reaction system, enabling a simple way for nucleic acid quantification. This design exhibits a lowest detectable concentration that is one order of magnitude lower than that of the one-step conjugation without amplification, and is proven to work well in biological medium. We have also demonstrated its potential in multiplexed miRNA sensing by using UCNPs of different emitting colors. This work achieves thermal cycling-mediated nucleic acid signal amplification on the surface of UCNPs, providing a new avenue for providing UCNP-based biosensors.

Quantification of low-abundance proteins in biofluids using affordable and portable biosensors stands as the top priority for point-of-care testing and in-field disease screening/monitoring. Herein, we propose a cellphone-based portable immunoassay platform coupled with an intelligent single microbead (SMB) bubbling visualization mechanism, which allows for facile, low-cost, and highly sensitive protein analysis. The use of solely an SMB as the immunocarrier can remarkably boost the sensitivity by superenriching the target-mediated, platinum nanoparticle (PtNP)-involved immunocomplexes on its surface. As such, even trace amounts of target proteins can bring highly gathered PtNPs on the SMB, which rapidly catalyze the production of noticeable and highly centralized oxygen (O2) bubbles firmly adhered to the SMB in just 50 s. The SMB bubbles can be facilely captured and imaged by a cellphone-attached lens, and the area of bubbles is employed as a novel signal readout mode for the ultrasensitive analysis of proteins down to the aM levels. Assisted by a self-written image analysis algorithm, the SMB-bubble imaging, recognition, area calculation, and target qualification are all automatically streamlined into an all-in-one cellphone application (App), achieving simple yet sensitive protein detection. This proposed intelligent platform provides a highly sensitive and cost-effective substitute for critical biomarker testing and paves a new way for cellphone-enabled portable biosensing.