It is of great significance to develop consensus, easy-to-use and conceptually new methods for guaranteeing food safety. CRISPR/Cas system is an emerging technology with excellent capability of DNA/RNA targeting, which has been repurposed as a powerful toolkit for nucleic acids detection. A number of nanomaterials have been created and manufactured via advanced nanotechnology and used in biosensors to revolutionize food safety detection by improving feasibility, compatibility and applicability. CRISPR/Cas-based detection is greatly benefited from the combination with nanomaterials to upgrade performance. This review paid attention to CRISPR/Cas-assisted biosensors with the help of nanomaterials and their enormous potential in food safety. First, the issues in food safety and principles of the CRISPR/Cas system were summarized. Then the different types of nanomaterials used in novel CRISPR/Cas-assisted biosensors were objectively introduced. The applications of nanomaterials-assisted CRISPR/Cas detection in food safety were elaborated and discussed. Lastly, discussions were conducted on challenges and future prospects. 

Nanomaterials-assisted CRISPR/Cas detection for food safety: Advances, challenges and future prospects

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-related proteins (CRISPR/Cas) are regarded as the next generation of molecular diagnostic systems. Due to its good biocompatibility, unique cutting activity, ease of operation and design, the CRISPR/Cas system plays an important role in biosensing applications, especially the trans-cutting activity of several Cas effectors. In recent years, CRISPR/Cas, as an emerging molecular recognition component and signal transduction module, has been combined with modern analytical techniques for detecting various analytes. This paper briefly introduces the classification and working principle of CRISPR sensor-related enzymes, summarizes the latest research on the detection of various Cas effectors on single (multiple) targets, and analyzes their application examples in practical and portable sensing from the perspective of multiple detection platforms. Finally, the technical bottleneck and application prospects were summarized. 

CRISPR molecular detection techniques: Advances from single to multiple detection methods

Food and waterborne illnesses are still a major concern in health and food safety areas. Every year, almost 0.42 million and 2.2 million deaths related to food and waterborne illness are reported worldwide, respectively. In foodborne pathogens, bacteria such as Salmonella, Shiga-toxin producer Escherichia coli, Campylobacter, and Listeria monocytogenes are considered to be high-concern pathogens. High-concern waterborne pathogens are Vibrio cholerae, leptospirosis, Schistosoma mansoni, and Schistosima japonicum, among others. Despite the major efforts of food and water quality control to monitor the presence of these pathogens of concern in these kinds of sources, foodborne and waterborne illness occurrence is still high globally. For these reasons, the development of novel and faster pathogen-detection methods applicable to real-time surveillance strategies are required. Methods based on biosensor devices have emerged as novel tools for faster detection of food and water pathogens, in contrast to traditional methods that are usually time-consuming and are unsuitable for large-scale monitoring. Biosensor devices can be summarized as devices that use biochemical reactions with a biorecognition section (isolated enzymes, antibodies, tissues, genetic materials, or aptamers) to detect pathogens. In most cases, biosensors are based on the correlation of electrical, thermal, or optical signals in the presence of pathogen biomarkers. The application of nano and molecular technologies allows the identification of pathogens in a faster and high-sensibility manner, at extremely low-pathogen concentrations. In fact, the integration of gold, silver, iron, and magnetic nanoparticles (NP) in biosensors has demonstrated an improvement in their detection functionality. The present review summarizes the principal application of nanomaterials and biosensor-based devices for the detection of pathogens in food and water samples. Additionally, it highlights the improvement of biosensor devices through nanomaterials. Nanomaterials offer unique advantages for pathogen detection. The nanoscale and high specific surface area allows for more effective interaction with pathogenic agents, enhancing the sensitivity and selectivity of the biosensors. Finally, biosensors’ capability to functionalize with specific molecules such as antibodies or nucleic acids facilitates the specific detection of the target pathogens.

Emerging Applications of Nanobiosensors in Pathogen Detection in Water and Food

Aflatoxin B1 (AFB1) is the food contaminant metabolite excreted by Aspergillus fungus. Many detection strategies have been developed due to AFB1 widespread distribution and intrinsic carcinogenic and mutagenic effects on living organism. Nanobiosensors are bioanalytical devices that employ the materials in nanoscale dimensions to improve their diagnosis performance. They present multiple advantages such as providing higher surface area/volume ratio, immobilization of biomolecules, signal generation, signal amplification and also signal transduction. The present review offers a description of multi classification of AFB1 nanobiosensors based on bioreceptors, signal transduction and applied nanoparticles (NPs). The comprehensive and updated of recent advances for detection of AFB1 toxin through nanobiosensors are described with emphasis of applied strategies and components in their working principles. Conventional and novel developed NPs are also defined and discussed to determine their practical roles in improving efficacy of AFB1 detection. In addition, the trends and future perspectives are included as the outlook. 

Nanobiosensors for aflatoxin B1 detection, current research trends and future outlooks

Construction of molecular logic gates using heavy metal ions as inputs based on catalytic hairpin assembly and CRISPR-Cas12a.

Contamination-free V-shaped ultrafast reaction cascade transferase signal amplification driven CRISPR/Cas12a magnetic relaxation switching biosensor for bacteria detection.

Chlorpyrifos (CPF) is the most frequently found organophosphate pesticide residue in solid food samples and can cause increasing public concerns about potential risks to human health. Traditional detection signals of such small molecules are mostly generated by target-mediated indirect conversion, which tends to be detrimental to sensitivity and accuracy. Herein, a novel magnetic relaxation switching detection platform was developed for target-mediated direct and sensitive detection of CPF with a controllable aggregation strategy based on a bioorthogonal ligation reaction between tetrazine (Tz) and trans-cyclooctene (TCO) ligands. Under optimal conditions, this sensor can achieve a detection limit of 37 pg/mL with a broad linear range of 0.1-500 ng/mL in 45 min, which is approximately 51-fold lower than that of the gas chromatography analysis and 13-fold lower than that of the enzyme-linked immunosorbent assay. The proposed click chemistry-mediated controllable aggregation strategy is direct, rapid, and sensitive, indicating great potential for residue screening in food matrices.

Magnetic Relaxation Switching Immunosensors via a Click Chemistry-Mediated Controllable Aggregation Strategy for Direct Detection of Chlorpyrifos.

Tyramine Signal Amplification on Polystyrene Microspheres for Highly Sensitive Quantification of Aflatoxin B1 in Peanut Samples

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Contamination-free V-shaped ultrafast reaction cascade transferase signal amplification driven CRISPR/Cas12a magnetic relaxation switching biosensor for bacteria detection.

Magnetic Relaxation Switching Immunosensors via a Click Chemistry-Mediated Controllable Aggregation Strategy for Direct Detection of Chlorpyrifos.

Tyramine Signal Amplification on Polystyrene Microspheres for Highly Sensitive Quantification of Aflatoxin B1 in Peanut Samples