An Innovative and Efficient Fluorescent Detection Technique for Salmonella in Animal-Derived Foods Using the CRISPR/Cas12a-HCR System Combined with PCR/RAA.

Here, we present a method for Salmonella detection using clustered regularly interspaced short palindromic repeats associated with the CRISPR-associated protein 12a-hybridization chain reaction (CRISPR/Cas12a-HCR) system combined with polymerase chain reaction/recombinase-assisted amplification (PCR/RAA) technology. The approach relies on the Salmonella invA gene as a biorecognition element and its amplification through PCR and RAA. In the presence of the target gene, Cas12a, guided by crRNA, recognizes and cleaves the amplification product, initiating the HCR. Fluorescently labeled single-stranded DNA (ssDNA) H1 and H2 were introduced, and the Salmonella concentration was determined based on the fluorescence intensity from the triggered HCR. Both assays demonstrate high specificity, sensitivity, simplicity, and rapidity. The detection range was 2 × 101-2 × 109 CFU/mL, with an LOD of 20 CFU/mL, and the entire process enabled specific and rapid Salmonella detection within 85-105 min. Field-incurred spiked recovery tests were conducted in mutton and beef samples using both assays, demonstrating satisfactory recovery and accuracy in animal-derived foods. By combining CRISPR/Cas12a with hybridization chain reaction technology, this study presents a rapid and sensitive Salmonella detection method that is crucial for identifying pathogenic bacteria and monitoring food safety.

Facile fabricate sandwich-structured molecularly imprinted dopamine polymer for simultaneously specific capture of Low-density lipoprotein and eliminate "bad cholesterol".

A simplified approach for preparation of sandwich type molecularly imprinted polymers (PPDA-MIPs) is proposed for simultaneously identify Low-density lipoprotein (LDL) and dispose "bad cholesterol". Porous polydopamine nanosphere (PPDA) is applied as a matrix for immobilization of LDL, and the imprinted layer is formed by dopamine acting as a functional monomer. Since imprinted cavities exhibit shape memory effects in terms of recognizing selectivity, the PPDA-MIPs exhibit excellent selectivity toward LDL and a substantial binding capacity of 550.3 µg mg-1. Meanwhile, six adsorption/desorption cycles later, the adsorption efficiency of 83.09 % is still achieved, indicating the adequate stability and reusability of PPDA-MIPs. Additionally, over 80 % of cholesterol is recovered, indicating the completeness of "bad cholesterol" removal in LDL. Lastly, as demonstrated by gel electrophoresis, PPDA-MIPs performed satisfactory behavior for the removal of LDL from the goat serum sample.

A novel phagomagnetic separation-ATP bioluminescence (PhMS-BL) for rapid and sensitive detection of viable Vibrio parahaemolyticus in aquatic product.

Detection of viable Vibrio parahaemolyticus (V. parahaemolyticus) is a major challenge due to its significant risk to food safety and human health. Herein, we developed a phagomagnetic separation-ATP bioluminescence (PhMS-BL) assay based on phage VPHZ6 for rapid and sensitive detection of viable V. parahaemolyticus. Phage as a recognition element was coupled to magnetic beads to capture and enrich V. parahaemolyticus, shortening detection time and improving method sensitivity. The intracellular ATP released by chemical lysis using CTAB was quantified using firefly fluorescein-adenosine triphosphate bioluminescence system to detect viable bacteria. So, PhMS-BL method was able to detect V. parahaemolyticus in a linear range of 2.3 × 102 to 1.3 × 107 CFU mL-1, with a detection limit of 78 CFU mL-1 within 15 min. It is successfully applied to detect V. parahaemolyticus in spiked lake water, lobster tail meat, and clam meat. The developed detection strategy can rapidly and sensitively detect viable V. parahaemolyticus in food matrixes.

Unveiling heterocyclic aromatic amines (HAAs) in thermally processed meat products: Formation, toxicity, and strategies for reduction - A comprehensive review.

This comprehensive review focuses on heterocyclic aromatic amines (HAAs), a class of chemicals that commonly form during the cooking or processing of protein-rich foods. The International Agency for Research on Cancer (IARC) has categorized certain HAAs as probable human carcinogens, highlighting the significance of studying their formation and control in food safety research. The main objective of this review is to address the knowledge gaps regarding HAAs formation and propose approaches to reduce their potential toxicity during thermal processing. By summarizing the mechanisms involved in HAAs formation and inhibition, the review encompasses both conventional and recent detection methods. Furthermore, it explores the distribution of HAAs in thermally processed meats prepared through various cooking techniques and examines their relative toxicity. Additionally, considering that the Maillard reaction, responsible for HAAs formation, also contributes to the unique flavors and aromas of cooked meat products, this review investigates the potential effects of inhibiting HAAs formation on flavor substances. A thorough understanding of these complex interactions provides a foundation for developing targeted interventions to minimize the formation of HAAs and other harmful compounds during food processing.

A visual colorimetric assay based on phage T156 and gold nanoparticles for the sensitive detection of Salmonella in lettuce.

In this study, a new technique was developed for visual and precise identification of Salmonella using phage T156-mediated aggregation of gold nanoparticles. The phage binds to gold nanoparticles in a dispersed and stable state under high NaCl concentrations. When Salmonella is introduced, the phage specifically recognizes and adsorbs the targeted bacteria, causing the AuNPs to undergo a discoloration reaction resulting in aggregation, which enables Salmonella visualization. The method has a detection range of 3.8 × 101-3.8 × 109 CFU/mL and a limit of detection of 38 CFU/mL and can produce results in approximately 80 min. The technique was also tested on field samples, including spiked lettuce, and was found to be accurate with a recovery rate of 81.0-119.2% and relative standard deviations ranging from 3.3% to 14.7%. Notably, this technique utilizes phages as recognition elements in colorimetric methods, offering simplicity, speed, and the ability to effectively distinguish between live and dead Salmonella. It demonstrates remarkable sensitivity, specificity, and accuracy. Furthermore, it presents a novel avenue for the rapid detection of other pathogenic bacteria.

Intermittent Fasting and Physical Exercise for Preventing Metabolic Disorders through Interaction with Gut Microbiota: A Review.

Metabolic disorders entail both health risks and economic burdens to our society. A considerable part of the cause of metabolic disorders is mediated by the gut microbiota. The gut microbial structure and function are susceptible to dietary patterns and host physiological activities. A sedentary lifestyle accompanied by unhealthy eating habits propels the release of harmful metabolites, which impair the intestinal barrier, thereby triggering a constant change in the immune system and biochemical signals. Noteworthy, healthy dietary interventions, such as intermittent fasting, coupled with regular physical exercise can improve several metabolic and inflammatory parameters, resulting in stronger beneficial actions for metabolic health. In this review, the current progress on how gut microbiota may link to the mechanistic basis of common metabolic disorders was discussed. We also highlight the independent and synergistic effects of fasting and exercise interventions on metabolic health and provide perspectives for preventing metabolic disorders.

Personal glucose meters coupled with signal amplification technologies for quantitative detection of non-glucose targets: Recent progress and challenges in food safety hazards analysis.

Ensuring food safety is paramount worldwide. Developing effective detection methods to ensure food safety can be challenging owing to trace hazards, long detection time, and resource-poor sites, in addition to the matrix effects of food. Personal glucose meter (PGM), a classic point-of-care testing device, possesses unique application advantages, demonstrating promise in food safety. Currently, many studies have used PGM-based biosensors and signal amplification technologies to achieve sensitive and specific detection of food hazards. Signal amplification technologies have the potential to greatly improve the analytical performance and integration of PGMs with biosensors, which is crucial for solving the challenges associated with the use of PGMs for food safety analysis. This review introduces the basic detection principle of a PGM-based sensing strategy, which consists of three key factors: target recognition, signal transduction, and signal output. Representative studies of existing PGM-based sensing strategies combined with various signal amplification technologies (nanomaterial-loaded multienzyme labeling, nucleic acid reaction, DNAzyme catalysis, responsive nanomaterial encapsulation, and others) in the field of food safety detection are reviewed. Future perspectives and potential opportunities and challenges associated with PGMs in the field of food safety are discussed. Despite the need for complex sample preparation and the lack of standardization in the field, using PGMs in combination with signal amplification technology shows promise as a rapid and cost-effective method for food safety hazard analysis.

Glucometer-based biosensor for the determination of ractopamine in animal-derived foods using rolling circle amplification.

Screening for persistent organic pollutants (POPs) in food is a complex and challenging process, as POPs can be present in very low levels and can be difficult to detect. Herein, we developed an ultrasensitive biosensor based on a rolling circle amplification (RCA) platform using a glucometer to determine POP. The biosensor was constructed using gold nanoparticle probes modified with antibodies and dozens of primers, magnetic microparticle probes conjugated with haptens, and targets. After competition, RCA reactions are triggered, numerous RCA products hybridize with the ssDNA-invertase, and the target is successfully transformed into glucose. Using ractopamine as a model analyte, this strategy obtained a linear detection range of 0.038-5.00 ng mL-1 and a detection limit of 0.0158 ng mL-1, which was preliminarily verified by screening in real samples. Compared with conventional immunoassays, this biosensor utilizes the high efficiency of RCA and the portable properties of a glucometer, which effectively improves the sensitivity and simplifies the procedures using magnetic separation technology. Moreover, it has been successfully applied to ractopamine determination in animal-derived foods, revealing its potential as a promising tool for POP screening.

Competitive fluorescent immunosensor based on catalytic hairpin self-assembly for multiresidue detection of organophosphate pesticides in agricultural products.

Simple and rapid multiresidue trace detection of organophosphate pesticides (OPs) is extremely important for various reasons, including food safety, environmental monitoring, and national health. Here, a catalytic hairpin self-assembly (CHA)-based competitive fluorescent immunosensor was developed to detect OPs in agricultural products, involving enabled dual signal amplification followed by a CHA reaction. The developed method could detect 0.01-50 ng/mL triazophos, parathion, and chlorpyrifos, with limits of detection (LODs) of 0.012, 0.0057, and 0.0074 ng/mL, respectively. The spiked recoveries of samples measured using this assay ranged from 82.8 % to 110.6 %, with CV values ranging between 5.5 % and 18.5 %. This finding suggests that the CHA-based competitive fluorescent immunosensor is a reliable and accurate method for detecting OPs in agricultural products. The results correlated well with those obtained from the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, indicating that the CHA-based biosensor is able to accurately detect OPs and can be used as a reliable alternative to the LC-MS/MS method. Additionally, the CHA-based biosensor is simpler and faster than LC-MS/MS, which makes it a more practical and cost-effective option for the detection of OPs. In summary, the CHA-based competitive fluorescent immunosensor can be considered a promising approach for trace analysis and multiresidue determination of pesticides, which can open up new horizons in the fields of food safety, environmental monitoring, and national health.

Properties of a Novel Salmonella Phage L66 and Its Application Based on Electrochemical Sensor-Combined AuNPs to Detect Salmonella.

Salmonella is widespread in nature and poses a significant threat to human health and safety. Phage is considered as a new tool for the control of food-borne pathogens. In this study, Salmonella phage L66 (phage L66) was isolated from sewage by using Salmonella Typhimurium ATCC 14028 as the host bacterium, and its basic properties were obtained by biological and bioinformatics analysis. Phage L66 had a broad host spectrum, with an optimal infection complex of 0.1 and an optimal adsorption rate of 90.06%. It also exhibited thermal stability between 30 °C~60 °C and pH stability pH from 3 to 12, and the average lysis amount was 46 PFU/cell. The genome sequence analysis showed that the genome length of phage L66 was 157,675 bp and the average GC content was 46.13%. It was predicted to contain 209 genes, 97 of which were annotated with known functions based on the evolutionary analysis, and phage L66 was attributed to the Kuttervirus genus. Subsequently, an electrochemical sensor using phage L66 as a recognition factor was developed and the working electrode GDE-AuNPs-MPA-Phage L66 was prepared by layer-by-layer assembly for the detection of Salmonella. The slope of the impedance was 0.9985 within the scope from 20 to 2 × 107 CFU/mL of bacterial concentration. The minimum detection limit of the method was 13 CFU/mL, and the average spiked recovery rate was 102.3% with a relative standard deviation of 5.16%. The specificity and stability of this sensor were excellent, and it can be applied for the rapid detection of Salmonella in various foods. It provides a phage-based electrochemical biosensor for the detection of pathogenic bacteria.

A highly sensitive bio-barcode immunoassay for multi-residue detection of organophosphate pesticides based on fluorescence anti-quenching.

Balancing the risks and benefits of organophosphate pesticides (OPs) on human and environmental health relies partly on their accurate measurement. A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs (triazophos, parathion, and chlorpyrifos) in apples, turnips, cabbages, and rice. Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs. DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification. The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore. The resulting fluorescence signal enables multiplexed quantification of triazophos, parathion, and chlorpyrifos residues over the concentration range of 0.01-25, 0.01-50, and 0.1-50 ng/mL with limits of detection of 0.014, 0.011, and 0.126 ng/mL, respectively. The mean recovery ranged between 80.3% and 110.8% with relative standard deviations of 7.3%-17.6%, which correlate well with results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proposed bio-barcode immunoassay is stable, reproducible and reliable, and is able to detect low residual levels of multi-residue OPs in agricultural products.

EIS biosensor based on a novel Myoviridae bacteriophage SEP37 for rapid and specific detection of Salmonella in food matrixes.

Recently, using bacteriophages as new molecular probes in reliable platforms for the detection of bacterial pathogens has attracted more and more increasing attentions. In this paper, a novel isolated Myoviridae bacteriophage SEP37 was covalently immobilized onto gold nanoparticles (AuNPs) modified gold disk electrode (GDE) surfaces using cysteamine (Cys) as a crosslinker. Substrates of GDE-AuNPs-Cys-Phage SEP37 and specific capture of Salmonella cells had been characterized using scanning electron microscopy (SEM) separately. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to study the electrochemical response of the biosensor interface manufacturing and bacterial capture process. Under the optimal experimental conditions, this phage-based EIS biosensor was able to detect Salmonella with a wide linear range from 2 × 101 to 2 × 106 colony forming unit (CFU)/mL within 30 min in spiked lake water and lettuce samples, with a limit of detection (LOD) of 17 CFU/mL. The detection linear range of spiked chicken samples was 2 × 102 to 2 × 105 CFU/mL, with a LOD of 1.3 × 102 CFU/mL. In combination with a pre-enrichment process for 3.5 h, this assay could reach a LOD of 1 CFU/mL in chicken breast meat samples. Besides, this phage-based EIS biosensor provided good reproducibility and stability. This phage-based EIS biosensor opens a new opportunity for the detection of pathogenic bacteria using the inherent selectivity of bacteriophage recognition.

A competitive immunoassay for detecting triazophos based on fluorescent catalytic hairpin self-assembly.

A rapid detection method is introduced for residual trace levels of triazophos in water and agricultural products using an immunoassay based on catalytic hairpin self-assembly (CHA). The gold nanoparticle (AuNPs) surface was modified with triazophos antibody and sulfhydryl bio-barcode, and an immune competition reaction system was established between triazophos and its ovalbumin-hapten (OVA-hapten). The bio-barcode served as a catalyst to continuously induce the CHA reaction to achieve the dual signal amplification. The method does not rely on the participation of enzymes, and the addition of fluorescent materials in the last step avoids interfering factors, such as a fluorescence burst. The emitted fluorescence was detected at 489/521 nm excitation/emission wavelengths. The detection range of the developed method was 0.01-50 ng/mL for triazophos, and the limit of detection (LOD) was 0.0048 ng/mL. The developed method correlates well with the results obtained by LC-MS/MS, with satisfactory recovery and sensitivity. In sum, the designed method is reliable and provides a new approach to detect pesticide residues rapidly and quantitatively.

A highly sensitive bio-barcode immunoassay for multi-residue detection of organophosphate pesticides based on fluorescence anti-quenching / 药物分析学报

Balancing the risks and benefits of organophosphate pesticides(OPs)on human and environmental health relies partly on their accurate measurement.A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs(triazophos,parathion,and chlorpyrifos)in apples,turnips,cabbages,and rice.Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs.DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification.The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore.The resulting fluorescence signal en-ables multiplexed quantification of triazophos,parathion,and chlorpyrifos residues over the concen-tration range of 0.01-25,0.01-50,and 0.1-50 ng/mL with limits of detection of 0.014,0.011,and 0.126 ng/mL,respectively.The mean recovery ranged between 80.3％and 110.8％with relative standard deviations of 7.3％-17.6％,which correlate well with results obtained by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The proposed bio-barcode immunoassay is stable,reproducible and reliable,and is able to detect low residual levels of multi-residue OPs in agricultural products.

Enhanced Bio-Barcode Immunoassay Using Droplet Digital PCR for Multiplex Detection of Organophosphate Pesticides.

A bio-barcode immunoassay based on droplet digital polymerase chain reaction (ddPCR) was developed to simultaneously quantify triazophos, parathion, and chlorpyrifos in apple, cucumber, cabbage, and pear. Three gold nanoparticle (AuNP) probes and magnetic nanoparticle (MNP) probes were prepared, binding through their antibodies with the three pesticides in the same tube. Three groups of primers, probes, templates, and three antibodies were designed to ensure the specificity of the method. Under the optimal conditions, the detection limits (expressed as IC10) of triazophos, parathion, and chlorpyrifos were 0.22, 0.45, and 4.49 ng mL-1, respectively. The linear ranges were 0.01-20, 0.1-100, and 0.1-500 ng mL-1, and the correlation coefficients (R2) were 0.9661, 0.9834, and 0.9612, respectively. The recoveries and relative standard deviations (RSDs) were in the ranges of 75.5-98.9 and 8.3-16.7%. This study provides the first insights into the ddPCR for the determination of organophosphate pesticides. It also laid the foundation for high-throughput detection of other small molecules.

A sensitive bio-barcode immunoassay based on bimetallic Au@Pt nanozyme for detection of organophosphate pesticides in various agro-products.

Organophosphate pesticides (OPs) are often used as insecticides and acaricides in agriculture, thus improving yields. OP residues may pose a serious threat, duetoinhibitionof the enzymeacetylcholinesterase(AChE). Therefore, a competitive bio-barcode immunoassay was designed for simultaneous quantification of organophosphate pesticide residues using AuNP signal amplification technology and Au@Pt catalysis. The AuNP probes were labelled with antibodies and corresponding bio-barcodes (ssDNAs), MNP probes coated with ovalbumin pesticide haptens and Au@Pt probes functionalized with the complementary ssDNAs were then prepared. Subsequently, pesticides competed with MNP probes to bind the AuNP probes. The recoveries of the developed assay were ranged from 71.26 to 117.47% with RSDs from 2.52 to 14.52%. The LODs were 9.88, 3.91, and 1.47 ng·kg-1, for parathion, triazophos, and chlorpyrifos, respectively. The assay was closely correlated with the data obtained from LC-MS/MS. Therefore, the developed method has the potential to be used as an alternative approach for detection of multiple pesticides.

A visual bio-barcode immunoassay for sensitive detection of triazophos based on biochip silver staining signal amplification.

Herein, a novel visual method for detecting triazophos based on competitive bio-barcode immunoassay was described. The competitive immunoassay was established by gold nanoparticles (AuNPs), magnetic microparticle (MMPs) and triazophos, combined with biochip hybridization system to detect the residual of triazophos in water and apple. Because AuNPs carried many bio-barcodes, which hybridized with labeled DNA on the biochip, catalyzed signal amplification using silver staining was detected by grayscale values as well as the naked eye. Notably, the grayscale values decreases with increasing the concentrations of triazophos, and the color change weakened gradually. The detection range was in between 0.05 and 10 ng/mL and the minimum detection limit was set at 0.04 ng/mL. Percent recovery calculated from spiked water and apple samples ranged between 55.4 and 107.8% with relative standard deviations (RSDs) of 12.4-24.9%. It has therefore been shown that this protocol provides a new insight for rapid detection of small molecule pesticides in various matrices.

A sensitive fluorometric bio-barcodes immunoassay for detection of triazophos residue in agricultural products and water samples by iterative cycles of DNA-RNA hybridization and dissociation of fluorophores by Ribonuclease H.

Although the toxicity of triazophos is high and it has been pulled from the market in many countries; it is still widely used and frequently detected in agricultural products. While conventional analyses have been routinely used for the quantification and monitoring of triazophos residues, those for detecting low residual levels are deemed necessary. Therefore, we developed a novel and sensitive fluorometric signal amplification immunoassay employing bio-barcodes for the quantitative analysis of triazophos residues in foodstuffs and surface water. Herein, monoclonal antibodies (mAbs) attached to gold nanoparticles (AuNPs) were coated with DNA oligonucleotides (used as a signal generator), and a complementary fluorogenic RNA was used for signal amplification. The system generated detection signals through DNA-RNA hybridization and subsequent dissociation of fluorophores by Ribonuclease H (RNase H). It has to be noted that RNase H can only disintegrate the RNA in DNA-RNA duplex, but not cleave single or double-stranded DNA. Hence, with iterative cycles of DNA-RNA hybridization, sufficient strong signal was obtained for reliable detection of residues. Furthermore, this method enables quantitative detection of triazophos residues through fluorescence intensity measurements. The competitive immunoassay shows a wide linear range of 0.01-100 ng/mL with a limit of detection (LOD) of 0.0032 ng/mL. The assay substantially meets the demand for the low residue detection of triazophos residues in agricultural products and water samples. Accuracy (expressed as spiked recovery %) and coefficient of variation (CV) were ranged from 73.4% to 116% and 7.04% to 17.4%, respectively. The proposed bio-barcodes immunoassay has the advantages of being stable, reproducible, and reliable for residue detection. In sum, the present study provides a novel approach for detection of small molecules in various sample matrices.

Competitive Bio-Barcode Immunoassay for Highly Sensitive Detection of Parathion Based on Bimetallic Nanozyme Catalysis.

A competitive sensitive bio-barcode immunoassay based on bimetallic nanozyme (Au@Pt: gold@platinum) catalysis has been designed for the detection of the pesticide parathion. Gold nanoparticles (AuNPs) were modified with single-stranded thiol oligonucleotides (ssDNAs) and monoclonal antibodies (mAbs) to form AuNP probes; magnetic nanoparticles (MNPs) were coated with ovalbumin (OVA)-parathion haptens as MNP probes, and bimetallic nanozyme (Au@Pt) nanoparticles functionalized with the complementary thiolated ssDNA were used as Au@Pt probes. The Au@Pt probes reacted with the AuNP probes through complementary base pairing. Further, parathion competed with MNP probes to bind the mAbs on the AuNP probes. Finally, the complex system was separated by a magnetic field. The released Au@Pt probes catalyzed a chromogenic system consisting of teramethylbenzidine (TMB). The bimetallic nanozyme-based bio-barcode immunoassay was performed on rice, pear, apple, and cabbage samples to verify the feasibility of the method. The immunoassay exhibited a linear response from 0.01 to 40 µg·kg-1, and the limit of detection (LOD) was 2.13 × 10-3 µg·kg-1. The recoveries and relative standard deviations (RSDs) ranged from 73.12 to 116.29% and 5.59 to 10.87%, respectively. The method was found to correlate well with data obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In conclusion, this method exhibits potential as a sensitive alternative method for the detection of a variety of pesticides, ensuring the safety of fruits and vegetables in agriculture.