CRISPR/Cas Multiplexed Biosensing: Advances, Challenges, and Perspectives.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems are renowned for their high sensitivity and specificity, enabling them as a powerful diagnostic toolbox. Multiplexed detection of panels of targets, as opposed to single targets, is imperative for reliable and conclusive disease diagnostics. However, multiplex application of the CRISPR/Cas system has long been hindered by indistinguishable signals from specific targets due to nonspecific chaotic trans-cleavage.

Multiplexed Pathogenic Bacteria Detection via a Two-Dimensional Encoded Fluorescent Microsphere System.

We developed an advanced microscopy imaging platform enabling amplification-free, multiplex detection of pathogenic bacteria in food and clinical samples, eliminating the need for DNA extraction. This platform leverages two-dimensional encoded polystyrene (PS) microspheres and an Argonaute-based decoding system to create multiplexed signal libraries. Each PS microsphere probe, encoded with spectrally distinct fluorophores and differing particle sizes, achieves high fluorescence through a tetrahedral DNA-enhanced hybridization chain reaction (TDNA-HCR), significantly enhancing signal intensity and reducing reaction time by 67%.

Enhanced CRISPR/Cas12a Fluorimetry via a DNAzyme-Embedded Framework Nucleic Acid Substrate.

CRISPR/Cas12a fluorimetry has been extensively developed in the biosensing arena, on account of its high selectivity, simplicity, and rapidness. However, typical CRISPR/Cas12a fluorimetry suffers from low sensitivity due to the limited trans-cleavage efficiency of Cas12a, necessitating the integration of other preamplification techniques. Herein, we develop an enhanced CRISPR/Cas12a fluorimetry via a DNAzyme-embedded framework nucleic acid (FNAzyme) substrate, which was designed by embedding four CLICK-17 DNAzymes into a rigid tetrahedral scaffold.

Guide DNA dephosphorylation-modulated Pyrococcus furiosus Argonaute fluorescence biosensor for the detection of alkaline phosphatase and aflatoxins B.

Foodborne hazardous factors pose a significant risk to public health, emphasizing the need for the development of sensitive and user-friendly detection strategies to effectively manage and control these risks in the food supply chain. Pyrococcus furiosus argonaute (PfAgo)-based biosensing approaches have been extensively explored due to its built-in signal amplification. However, the property that PfAgo is a DNA-guided DNA endonuclease has enabled almost all the existing PfAgo-based reports to be used for the detection of nucleic acids.

Detection of foodborne methicillin-resistant Staphylococcus aureus via fluorescence-encoded microsphere and Argonaute-mediated decoding.

Molecular diagnosis of foodborne methicillin-resistant Staphylococcus aureus (MRSA) is crucial for controlling its dissemination and ensuring food safety. However, existing genetic methods are limited by susceptibility to aerosol contamination and restricted to single-gene detection. Herein, a fluorescent biosensor employing fluorescence-encoded microspheres and Argonaute-mediated decoding is developed, enabling ultrasensitive, accurate, and duplex detection of MRSA genes.

Artificial Intelligence-Based Imaging Transcoding System for Multiplex Screening of Viable Foodborne Pathogens.

Multiplex detection of viable foodborne pathogens is critical for food safety and public health, yet current assays suffer trade-offs between cost, assay complexity, sensitivities, and the specificity between live and dead bacteria. We herein developed a sensing method using artificial intelligence transcoding (SMART) for rapid, sensitive, and multiplex profiling of foodborne pathogens. The assay utilizes the programmable polystyrene (PS) microspheres to encode different pathogens, inducing subsequent visible signals under conventional microscopy that can be analyzed using a customized, artificial intelligence-computer vision, which was trained to decode the intrinsic properties of PS microspheres to reveal the numbers and types of pathogens.

CRISPR/Cas12a-Assisted Chemiluminescence Sensor for Aflatoxin B Detection in Cereal Based on Functional Nucleic Acid and In-Pipet Rolling Circle Amplification.

Herein, we report a CRISPR/Cas12a-assisted chemiluminescence sensor for aflatoxin B (AFB) detection based on functional nucleic-acid-mediated target recognition and in-pipet rolling circle amplification-mediated signal amplification. In this sensor, we performed rolling circle amplification on the inside of the pipet to enrich horseradish peroxidase (pipet-poly-HRP). When AFB is present, it interacts with functional nucleic acids and results in the release of the activator.

Rapid and quantitative determination of deoxynivalenol in cereal through the combination of magnetic solid-phase extraction and optical fiber-based homogeneous chemiluminescence immunosensor.

An integrated strategy for the rapid and sensitive detection of deoxynivalenol in cereals was developed by combining FeO magnetic nanoparticle-modified metal organic framework-5-based magnetic solid-phase extraction and the optical fiber-based homogeneous chemiluminescence immunosensor. The hybrid magnetic material was prepared and characterized, exhibiting good enrichment capacity up to 1.68 mg/g.

CRISPR/Cas12a-based magnetic relaxation switching biosensor for nucleic acid amplification-free and ultrasensitive detection of methicillin-resistant Staphylococcus aureus.

Herein, we develop a CRISPR/Cas12a-based magnetic relaxation switching (C-MRS) biosensor for ultrasensitive and nucleic acid amplification-free detection of methicillin-resistant Staphylococcus aureus (MRSA) in food. In this biosensor, mecA gene in MRSA was recognized by CRISPR-RNA, which will activate the trans-cleavage activity of Cas12a and release the fastened alkaline phosphatase (ALP) on the particle. The freed ALP can then use to hydrolyze substrate to produce ascorbic acid that trigger the click reaction between magnetic probe.

Optical Biosensor for Ochratoxin A Detection in Grains Using an Enzyme-Mediated Click Reaction and Polystyrene Nanoparticles.

Herein, we develop an optical biosensor for highly sensitive and facile detection of ochratoxin A (OTA) using an enzyme-mediated click reaction for signal amplification and polystyrene nanoparticles (PNPs) for signal readout. Alkaline phosphatase was employed to hydrolyze the ascorbic acid-phosphate to generate ascorbic acid, which reduces Cu(II) to Cu(I). Cu(I) can catalyze the click reaction between alkyne-functionalized magnetic beads and azide-functionalized PNPs to form complexes, while unbound PNPs acted as the signal probe.

Aptamer-based colorimetric detection of methicillin-resistant Staphylococcus aureus by using a CRISPR/Cas12a system and recombinase polymerase amplification.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) with superior accuracy, timeliness, and simplicity is highly valuable in clinical diagnosis and food safety. In this study, an aptamer-based colorimetric biosensor was developed to detect MRSA by using a CRISPR/Cas12a system and recombinase polymerase amplification (RPA). The aptamer of silver ion (Ag) pre-coupled to magnetic nanoparticles was employed not only as the substrate of trans-cleavage in the CRISPR/Cas12a system, but also as the modulator of Ag-3,3',5,5'-tetramethylbenzidine (TMB) chromogenic reaction, innovatively integrating the powerful CRISPR/Cas12a system with convenient colorimetry.

Integrating magnetic metal-organic frameworks-based sample preparation with microchannel resistance biosensor for rapid and quantitative detection of aflatoxin B.

Aflatoxin B, a secondary metabolite produced by fungi, is one of the most toxic mycotoxins that poses a major food security and public health threat worldwide. Effective sample pretreatment and high sensitivity detection techniques are urgently needed due to its trace amount in complex samples. Herein, an integrated detection strategy was developed by combining Mg/Zn-metal organic framework-74 modified FeO magnetic nanoparticles (Mg/Zn-MOF-74 @FeO MNPs)-based sample preparation and microchannel resistance biosensor for rapid and highly sensitive detection of aflatoxin B in food samples.

Enzyme-modulated photothermal immunoassay of chloramphenicol residues in milk and egg using a self-calibrated thermal imager.

Highly sensitive and accurate detection of chloramphenicol is of paramount importance for food safety. Herein, an enzyme-modulated photothermal immunosensor that uses a self-calibrated thermal imaging system (SCTIS) as signal read-out was developed for detecting chloramphenicol. In this immunosensor, alkaline phosphatase was used as a modulator of the photothermal conversion.

Click Chemistry-Mediated Particle Counting Sensing via Cu(II)-Polyglutamic Acid Coordination Chemistry and Enzymatic Reaction.

An electrical resistance-based particle counter (ERPC) with simple operation and high resolution has proved to be a promising biosensing toolkit, whereas amplification-free ERPC biosensors are incapable of analyzing trace small molecules due to their relatively low sensitivity. In this work, click chemistry-mediated particle counting sensing of small-molecule hazards in food samples with high sensitivity was developed. In this strategy, unbound alkyne-functionalized polystyrene microspheres were collected by magnetic separation from the copper-ion-mediated click reaction between alkyne-functionalized polystyrene microspheres and azido-functionalized magnetic beads, which could be used as signal probes for the readout.

Direct Transverse Relaxation Time Biosensing Strategy for Detecting Foodborne Pathogens through Enzyme-Mediated Sol-Gel Transition of Hydrogels.

In this work, we develop a direct transverse relaxation time () biosensing strategy and employ it for assaying foodborne pathogens relying on the alkaline phosphatase (ALP)-mediated sol-gel transition of hydrogels. ALP can catalyze the reaction to generate an acidic environment to transform the sol-state alginate solution to hydrogel, and this hydrogelation process can directly regulate the diffusion rate of water protons that results in a change of water molecules. By means of enzyme-modulated sol-gel transition and antigen-antibody interactions, this biosensor displays high sensitivity for detecting 50 CFU/mL within 2 h.