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. Our approach enabled the rapid and simultaneous detection of multiple bacteria from egg samples of <102 CFU/mL without DNA amplification and showed strong consistency with the standard microbiologic and genotypic methods. We adopted our assay through phage-guided targeting to enable the discrimination between live and dead bacteria.

CRISPR/Cas12a-Assisted Chemiluminescence Sensor for Aflatoxin B1 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 B1 (AFB1) 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 AFB1 is present, it interacts with functional nucleic acids and results in the release of the activator. The activator is designed to activate the CRISPR/Cas12a system, which cleaves the pipet-poly-HRP to liberate HRP. The freed HRP can then be measured by chemiluminescence to quantify AFB1. This CRISPR/Cas12a-assisted chemiluminescence sensor enables facile, highly sensitive, and specific detection of AFB1, with a linear range from 50 pg/mL to 100 ng/mL and a detection limit of 5.2 pg/mL. Furthermore, it exhibits satisfactory recovery and has successfully challenged AFB1 detection in cereal samples. The proposed sensor offers a novel rapid screening approach that holds great promise for food security monitoring.

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 Fe3O4 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. The competitive immunoassay-based homogeneous chemiluminescence immunosensor enabled washing-free and high-sensitivity detection of deoxynivalenol. Under optimized conditions, this immunosensor could detect deoxynivalenol as low as 46.7 pg/mL with a quantitatively linear range of 0.1 to 1000 ng/mL. The recoveries of this integrated detection strategy in rice, corn, and wheat ranged from 80.0 % to 118.2 %, 91.1 % to 116.7 %, and 80.0 % to 91.5 %, respectively, with a relative standard deviation that did not exceed 9.11 %. More importantly, it shows great consistency with the high-performance liquid chromatography-mass spectrometry in blind sample analysis. This integrated detection strategy provides a convenient approach for mycotoxins screening in cereals.

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. The transverse relaxation time of the unbound magnetic probe can be measured for signal readout. By incorporating collateral activity of CRISPR/Cas12a, on-particle rolling circle amplification, and ALP-triggered click chemistry into background-free MRS, as low as 16 CFU/mL MRSA can be detected without any nucleic acid pre-amplification, which avoids carryover contamination, but without compromising sensitivity. Moreover, this C-MRS biosensor could distinguish 0.01% target DNA from the single-base mutant. Recovery in eggs, milk and pork ranged from 75% to 112%, 82%-104%, and 81%-91%, respectively, revealing its satisfactory accuracy and applicability in the complex food matrix. The developed C-MRS biosensor fleshes out the CRISPR toolbox for food safety and provides a new approach for the sensitive and accurate detection of foodborne drug-resistant bacteria.

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. This strategy utilized the high efficiency of click chemistry and the inherent optical absorption properties of PNPs, which effectively improved the sensitivity of conventional immunoassays and simplified the procedures using magnetic separation technology. This optical biosensor enabled OTA detection in a linear range of 0.1 to 50 ng/mL with a detection limit of 54 pg/mL. Moreover, it has been successfully challenged with OTA detection in maize samples, revealing its potential as a promising tool for mycotoxin screening.

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. The utilized aptamer containing consecutive and interrupted cytosine: cytosine mismatched base pairs also served as a signal amplifier because of the one-to-multiple binding of the aptamer to Ag+. Using triple amplification of RPA, multiple-turnover nuclease activity of Cas12a, and cytosine-Ag+-cytosine coordination chemistry, MRSA was detected as low as 8 CFU mL-1. Moreover, its satisfactory accuracy in the analysis of real samples, together with visualization and simplicity, revealed the great potential of the proposed biosensor as a robust antibiotic-resistant bacteria detection platform.

Integrating magnetic metal-organic frameworks-based sample preparation with microchannel resistance biosensor for rapid and quantitative detection of aflatoxin B1.

Aflatoxin B1, 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 Fe3O4 magnetic nanoparticles (Mg/Zn-MOF-74 @Fe3O4 MNPs)-based sample preparation and microchannel resistance biosensor for rapid and highly sensitive detection of aflatoxin B1 in food samples. The synthesis and characterization of Mg/Zn-MOF-74 @Fe3O4 MNPs was reported, which exhibited efficient separation and enrichment capacity when exposed to complex grain samples. The competitive immunoassay-based microchannel resistance biosensor enabled specific and high-sensitive analysis of aflatoxin B1 by using current as a readout, which caused by the blocking effect between the functionalized-polystyrene microspheres and microchannel. Under optimized conditions, this biosensor was capable to quantitatively analysis aflatoxin B1 from 10 pg/mL to 20 ng/mL, and with a limit of detection of 4.75 pg/mL. This integrated detection strategy has been tested for the quantitative detection of aflatoxin B1 in grain samples that is a potential protocol for food safety control and environmental monitoring.

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. It could hydrolyze the substrate into ascorbic acid, thereby reducing oxidized 3,3',5,5'-tetramethylbenzidine, which exhibited a near-infrared laser-driven photothermal effect. For precise temperature measurement, the SCTIS was designed by using the temperature compensation of a ceramic chip to enable real-time self-calibration of the temperature. This SCTIS-based immunosensor could detect chloramphenicol with a LOD of 9 pg/mL in 2 h, and relative standard derivations from 3.95% to 13.58%. The average recoveries in milk and egg samples ranged from 76% to 114%. This versatile sensing strategy can detect various targets by altering recognition elements, thus has wide applicability in food safety testing and monitoring.

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. This click chemistry-mediated ERPC biosensor converts the detection of targets to the quantification of copper ions or ascorbic acid by performing competitive immunoassay-based coordination chemistry and enzymatic reaction, respectively. The sensitivity of the ERPC biosensor has been improved by an order of magnitude due to the signal amplification effects of click chemistry, coordination adsorption, and enzyme catalysis. Furthermore, because of the efficient separation and enrichment of immunomagnetic beads and the robustness of click chemistry, the interference from food matrixes and immunoassay is effectively reduced, and thus, our strategy is exceedingly suitable for detecting trace targets in complex samples.

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 (T2) 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 T2 change of water molecules. By means of enzyme-modulated sol-gel transition and antigen-antibody interactions, this T2 biosensor displays high sensitivity for detecting 50 CFU/mL S. enteritidis within 2 h. This biosensing strategy directly modulates the water molecules rather than magnetic probes in traditional methods, offering a straightforward, novel, and sensitive platform for pathogen detection.