A major challenge for ultrasensitive analysis is the high-efficiency determination of different target single molecules in parallel with high accuracy. Herein, we developed a quantitative fluoro-electrochemical imaging approach for direct multiplexed single-molecule counting with a SiC-nanofilm-modified indium tin oxide transparent electrode. The nanofilm could control local pH through proton-coupled electron transfer in a lower potential range and further induce direct electrochemical oxidation of the dye molecules with a higher applied potential. The fluoro-electrochemical responses of immobilized single molecules with different pH values and redox behaviors could thus be distinguished within the same fluorescence channels. This method yields nonamplified direct counting of single molecules, as indicated by excellent linear responses in the picomolar range. The successful distinction of seven different randomly mixed dyes underscores the versatility and efficacy of the proposed method in the highly accurate determination of single dye molecules, paving the way for highly parallel single-molecule detection for diverse applications.

With significant advancements in understanding gene functions and therapy, the potential misuse of gene technologies, particularly in the context of sports through gene doping (GD), has come to the forefront. This raises concerns regarding the need for point-of-care testing of various GD candidates to counter illicit practices in sports. However, current GD detection techniques, such as PCR, lack the portability required for on-site multiplexed detection. In this study, we introduce an integrated microfluidics-based chip for multiplexed gene doping detection, termed MGD-Chip. Through the strategic design of hydrophilic and hydrophobic channels, MGD-Chip enables the RPA and CRISPR-Cas12a assays to be sequentially performed on the device, ensuring minimal interference and cross-contamination. Six potential GD candidates were selected and successfully tested simultaneously on the platform within 1 h. Demonstrating exceptional specificity, the platform achieved a detection sensitivity of 0.1 nM for unamplified target plasmids and 1 aM for amplified ones. Validation using mouse models established by injecting IGFI and EPO transgenes confirmed the platform\'s efficacy in detecting gene doping in real samples. This technology, capable of detecting multiple targets using portable elements, holds promise for real-time GD detection at sports events, offering a rapid, highly sensitive, and user-friendly solution to uphold the integrity of sports competitions.

Traumatic brain injury (TBI) is widely recognized as a global public health crisis, affecting millions of people each year, leading to permanent neurologic, emotional, and occupational disability, and highlighting the urgent need for rapid, sensitive, and early assessment. Here, we design a novel and simple lithography-free method for preparing dual-channel graphene-based field-effect transistors (G-FETs) and integrating them with microfluidic channels for simultaneously multiplexed detection of key blood TBI biomarkers: neurofilament light chain (NFL) and glial fibrillary acidic protein (GFAP). The G-FET utilizes an ingenious dual-channel electrode array design, where the source is shared between channels and the drains are independent of each other, which is the key to achieving simultaneous output of dual detection signals. At the same time, the microfluidic chip realizes microscale fluidic control and fast sample response time. This integrated detection system shows excellent sensitivity in biological fluids for the TBI biomarkers with detection limits as low as 55.63 fg/mL for NFL and 144.45 fg/mL for GFAP in phosphate-buffered saline (PBS) buffer, respectively. Finally, the clinical sample analysis shows promising performance for TBI detection, with an area under the curve (AUC) of 0.98 for the two biomarkers. And the combined dual-protein assay is also a good predictor of intracranial injury findings on computed tomography (CT) scans (AUC = 0.907). The integrated microfluidic G-FET device with a dual-signal output strategy has important potential for application in clinical practice, providing more comprehensive information for brain injury assessment.

A growing number of applications require simultaneous detection of multiplexed nucleic acid targets in a single reaction, which enables higher information density in combination with reduced assay time and cost. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-Cas system have broad applications for the detection of nucleic acids due to their strong specificity, high sensitivity, and excellent programmability. However, realizing multiplexed detection is still challenging for the CRISPR-Cas system due to the nonspecific collateral cleavage activity, limited signal reporting strategies, and possible cross-reactions. In this review, we summarize the principles, strategies, and features of multiplexed detection based on the CRISPR-Cas system and further discuss the challenges and perspective.

Pandemics caused by respiratory viruses, such as the SARS-CoV-1/2, influenza virus, and respiratory syncytial virus, have resulted in serious consequences to humans and a large number of deaths. The detection of such respiratory viruses in the early stages of infection can help control diseases by preventing the spread of viruses. However, the diversity of respiratory virus species and subtypes, their rapid antigenic mutations, and the limited viral release during the early stages of infection pose challenges to their detection. This work reports a multiplexed microfluidic immunoassay chip for simultaneous detection of eight respiratory viruses with noticeable infection population, namely, influenza A virus, influenza B virus, respiratory syncytial virus, SARS-CoV-2, human bocavirus, human metapneumovirus, adenovirus, and human parainfluenza viruses. The nanomaterial of the nanozyme (Au@Pt nanoparticles) was optimized to improve labeling efficiency and enhance the detection sensitivity significantly. Nanozyme-binding antibodies were used to detect viral proteins with a limit of detection of 0.1 pg/mL with the naked eye and a microplate reader within 40 min. Furthermore, specific antibodies were screened against the conserved proteins of each virus in the immunoassay, and the clinical sample detection showed high specificity without cross reactivity among the eight pathogens. In addition, the microfluidic chip immunoassay showed high accuracy, as compared with the RT-PCR assay for clinical sample detection, with 97.2%/94.3% positive/negative coincidence rates. This proposed approach thus provides a convenient, rapid, and sensitive method for simultaneous detection of eight respiratory viruses, which is meaningful for the early diagnosis of viral infections. Significantly, it can be widely used to detect pathogens and biomarkers by replacing only the antigen-specific antibodies.

Fluorescence dye-based loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detection method, but is limited to single-plex detection and may yield non-specific signals. In this study, we propose a bifunctional probe-based real-time LAMP amplification method for single-plexed or multiplexed detection. The bifunctional probe is derived by modifying the 5\' end of the fluorophore and an internal quencher on one of the LAMP primers; therefore, it can simultaneously be involved in the LAMP process and signal amplification. The fluorescence intensity undergoes a cumulative exponential increase during the incorporation of the bifunctional probe into double-stranded DNA amplicons. The bifunctional probe-based LAMP method is simplified and cost-effective, as the primer design and experimental operations align entirely with the ordinary LAMP. Different from other current probe-based methods, this method does not require additional enzymes, sequences, or special probe structures. Also, it is 10 min faster than several other probe-based LAMP methods. The bifunctional probe-based LAMP method allows the simultaneous detection of the target Vibrio parahaemolyticus DNA and the internal amplification control in a one-pot reaction, demonstrating its potential for multiplexed detection.

Hepatocellular carcinoma (HCC), as one of the most lethal cancers, significantly impacts human health. Attempts in this area tends to develop novel technologies with sensitive and multiplexed detection properties for early diagnosis. Here, we present novel hydrogel photonic crystal (PhC) barcodes with tyramine deposition amplified enzyme-linked immunosorbent assay (ELISA) for highly sensitive and multiplexed HCC biomarker screening. Because of the abundant amino groups of acrylic acid (AA) component, the constructed hydrogel PhC barcodes with inverse opal structure could facilitate the loading of antibody probes for subsequent detection of tumor markers. By integrating tyramine deposition amplified ELISA on the barcode, the detection signal of tumor markers has been enhanced. Based on these features, it is demonstrated that the hydrogel PhC barcodes with tyramine deposition amplified ELISA could realize highly sensitive and multiplexed detection of HCC-related biomarkers. It was found that this method is flexible, sensitive and accurate, suitable for multivariate analysis of low abundance tumor markers and future cancer diagnosis. These features make the newly developed PhC barcodes an innovation platform, which possesses tremendous potential for practical application of low abundance targets.

The multiplexed detection of metabolites in parallel within a single biosensor plate is sufficiently valuable but also challenging. Herein, we combine the inherent light addressability of silicon with the high selectivity of enzymes, for the construction of multiplexed photoelectrochemical enzymatic biosensors. To conduct a stable electrochemistry and reagentless biosensing on silicon, a new strategy involving the immobilization of both redox mediators and enzymes using an amide bond-based hydrogel membrane was proposed. The membrane characterization results demonstrated a covalent coupling of ferrocene mediator to hydrogel, in which the mediator acted as not only a signal generator but also a renewable sacrifice agent. By adding corresponding enzymes on different spots of hydrogel membrane modified silicon and recording local photocurrents with a moveable light pointer, this biosensor setup was used successfully to detect multiple metabolites, such as lactate, glucose, and sarcosine, with good analytical performances. The limits of detection of glucose, sarcosine and lactate were found to be 179 μM, 16 μM, and 780 μM with the linear ranges of 0.5-2.5 mM, 0.3-1.5 mM, and 1.0-3.0 mM, respectively. We believe this proof-of-concept study provides a simple and rapid one-step immobilization approach for the fabrication of reagentless enzymatic assays with silicon-based light-addressable electrochemistry.

Respiratory infections and food contaminants pose severe challenges to global health and the economy. A rapid on-site platform for the simultaneous detection of multiple pathogens is crucial for accurate diagnosis, appropriate treatment, and a reduced healthcare burden. Herein, we present a spheres-on-sphere (SOS) platform for multiplexed detection using a portable Coulter counter, which employs millimeter- and micron-sized spheres coupled with antibodies as multitarget probes. The assay allows for quantitative detection of multiple analytes within 20 min by simple mixing, enabling on-site detection. The platform shows high accuracy in identifying three respiratory viruses (SARS-CoV-2, influenza A virus, and parainfluenza virus) from throat swab samples, with LOD of 50.7, 32.4, and 49.1 pg/mL. It also demonstrates excellent performance in quantifying three mycotoxins (aflatoxin B1, deoxynivalenol, and ochratoxin A) from food samples. The SOS platform offers a rapid on-site approach with high sensitivity and specificity for applications in resource-limited settings.

Biocoding technology constructed by readable tags with distinct signatures is a brand-new bioanalysis method to realize multiplexed identification and bio-information decoding. In this study, a novel fluorescence intensity coding technology termed Tetra-FICT was reported based on tetrahedron DNA nanostructure (TDN) carrier and Főrster Resonance Energy Transfer (FRET) effect. By modulating numbers and distances of Cy3 and Cy5 at four vertexes of TDN, different fluorescence intensities of twenty-six samples were produced at ∼565.0 nm (FICy3) and ∼665.0 nm (FICy5) by detecting fluorescence spectra. By developing an error correction mechanism, eleven codes were established based on divided intensity ranges of the final FICy3 together with FICy5 (Final-FICy3&FICy5). These resulting codes were used to construct barcode probes, with three miRNA biomarkers (miRNA-210, miRNA-199a and miRNA-21) as cases for multiplexed bio-assay. The high specificity and sensitivity were also demonstrated for the detection of miRNA-210. Overall, the proposed Tetra-FICT enriched the toolbox of fluorescence coding, which could be applied to multiplexing biomarkers detection.