A machine vision-assisted Argonaute-mediated fluorescence biosensor for the detection of viable Salmonella in food without convoluted DNA extraction and amplification procedures.

The precise identification viable pathogens hold paramount significance in the prevention of foodborne diseases outbreaks. In this study, we integrated machine vision and learning with single microsphere to develop a phage and Clostridium butyricum Argonaute (CbAgo)-mediated fluorescence biosensor for detecting viable Salmonella typhimurium (S. typhimurium) without convoluted DNA extraction and amplification procedures. Phage and lysis buffer was utilized to capture and lyse viable S. typhimurium, respectively. Subsequently, CbAgo can cleave the bacterial DNA to obtain target DNA that guides a newly targeted cleavage of fluorescent probes. After that, the resulting fluorescent signal accumulates on the streptavidin-modified single microsphere. The overall detection process is then analyzed and interpreted by machine vision and learning algorithms, achieving highly sensitive detection of S. typhimurium with a limit of detection at 40.5 CFU/mL and a linear range of 50-107 CFU/mL. Furthermore, the proposed biosensor demonstrates standard recovery rates and coefficients of variation at 93.22% - 106.02% and 1.47% - 12.75%, respectively. This biosensor exhibits exceptional sensitivity and selectivity, presenting a promising method for the rapid and effective detection of foodborne pathogens. ENVIRONMENTAL IMPLICATION: Bacterial pathogens exist widely in the environment and seriously threaten the safety of human life. In this study, we developed a phage and Clostridium butyricum Argonaute-mediated fluorescence biosensor for the detection of viable Salmonella typhimurium in environmental water and food samples. Compared with other Salmonella detection methods, this method does not need complex DNA extraction and amplification steps, which reduces the use of chemical reagents and experimental consumables in classic DNA extraction kit methods.

Dual cascade nucleic acid recycling-amplified assembly of hyperbranched DNA nanostructures to construct a novel plasmonic colorimetric biosensing method.

The plasmonic colorimetric biosensors are very favorable for the on-site testing and naked-eye screening of analytes from real samples, but how to realize their highly sensitive assays with simple manipulations is still a great challenge. Herein we designed a target-triggered dual cascade nucleic acid recycling strategy to amplify the assembly of a hyperbranched DNA nanostructure and thus developed a novel kanamycin colorimetric biosensing method. The first cycle arising from the aptamer recognition-triggered strand displacement reaction and its cascade cycle constructed on the catalytic reaction of two nucleases could release an output DNA to trigger the assembly of the DNA nanostructure. Based on the high capture of alkaline phosphatase at this DNA nanostructure to induce the localized surface plasmon resonance change of gold nanobipyramids (Au NBPs), an ultrasensitive colorimetric signal transduction strategy was constructed. Through the measurement of the shift of the characteristic absorption wavelength of Au NBPs, a very wide linear range from 10 fg mL-1 to 1 ng mL-1 and a very low detection limit of 1.4 fg mL-1 were obtained. Meanwhile, the obvious multicolor change of Au NBPs could be used for the visual semi-quantitative analysis of Kana residues. The whole homogeneous assay process well simplified the manipulation and also ensured the excellent repeatability. These excellent performances determine the great potential of the method for future applications.

Magnetic relaxation switching immunoassay based on "limited-magnitude" particles for sensitive quantification of aflatoxin B1.

Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic chemical substance that endangers food safety and human health. Magnetic relaxation switching (MRS) immunosensors are utilized in a variety of applications in food analysis due to its resistance to matrix interferences, but they often suffer from magnetic separation-based multi-washing steps and low sensitivity. Herein, we propose novel MRS strategy for the sensitive detection of AFB1 using "Limited-Magnitude" size particles: a single millimeter sized polystyrene spheres (PSmm) and 150 nm superparamagnetic nanoparticles (MNP150). Only a single PSmm is used as the microreactor to enhance all of the magnetic signal on its surface in high concentration by an immune competitive response, successfully preventing signal dilution, which can be transferred by pipette, simplifying the process of separation and washing. The established single polystyrene sphere magnetic relaxation switch biosensor (SMRS) was able to quantify AFB1 from 0.02 to 200 ng/mL with a detection limit of 14.3 pg/mL. SMRS biosensor has been successfully used for the detection of AFB1 in wheat and maize samples, and the results in agreement with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS). Benefiting from high sensitivity and convenient operation, the simple and enzyme-free method is promising in trace small molecules applications.

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.

The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli.

Escherichia coli has been developed as the most common host for recombinant protein expression. Unfortunately, there are still some proteins that are resistant to high levels of heterologous soluble expression in E. coli. Protein and peptide fusion tags are one of the most important methods for increasing target protein expression and seem to influence the expression efficiency and solubility as well. In this study, we identify a short 15-residue enhancing solubility peptide, the PCDS (protocatechuate 3,4-dioxygenase solubility) tag, which enhances heterologous protein expression in E. coli. This PCDS tag is a 45-bp long sequence encoding a peptide tag involved in the soluble expression of protocatechuate 3,4-dioxygenase, encoded by the pcaHG98 genes of Pseudomonas putida NCIMB 9866. The 45-bp sequence was also beneficial for pcaHG98 gene amplification. This tag was shown to be necessary for the heterologous soluble expression of PcaHG98 in E. coli. Purified His6-PcaHG98e04-PCDS exhibited an activity of 205.63±14.23U/mg against protocatechuate as a substrate, and this activity was not affected by a PCDS tag. This PCDS tag has been fused to the mammalian yellow fluorescent protein (YFP) to construct YFP-PCDS without its termination codons and YFPt-PCDS with. The total protein expressions of YFP-PCDS and YFPt-PCDS were significantly amplified up to 1.6-fold and 2-fold, respectively, compared to YFP alone. Accordingly, His6-YFP-PCDS and His6-YFPt-PCDS had 1.6-fold and 3-fold higher soluble protein yields, respectively, than His6-YFP expressed under the same conditions. His6-YFP, His6-YFP-PCDS, and His6-YFPt-PCDS also showed consistent fluorescence emission spectra, with a peak at 530nm over a scanning range from 400 to 700nm. These results indicated that the use of the PCDS tag is an effective way to improve heterologous protein expression in E. coli.

Molecular cloning, the characterization of metallothionein and catalase, and the evaluation of testicular toxicity of Cd in the Chinese fire-bellied newt (Cynops orientalis).

Cadmium (Cd) is an environmental toxicant and a nonessential metal. Cd can attack a wide range of organs, such as the liver, kidney, lung, ovary, testis, brain, and muscle in vertebrates. Among these organs, the testis might be the most sensitive organ to Cd toxicity. Metallothionein (MT) is a cysteine-rich protein with a low molecular weight, that can bind with Cd and eliminate reactive oxygen species (ROSs). Hydrogen peroxide, which as a crucial type of ROS that is induced by Cd, can be eliminated by catalase (CAT) in the self-protection of cells and to realize Cd toxicity resistance. To investigate the functions of MT and CAT in the testis of Cynops orientalis, we cloned the full-length MT and CAT genes of C. orientalis for the first time. Immunofluorescence results demonstrated that MT and CAT were expressed in Sertoli cells and all spermatogenic cells in the testis of C. orientalis. The results of the ultrastructural damage assay demonstrated that there were various impairments, which included organelle vacuolization, abnormal chromatin distribution, and apoptotic bodies, in somatic cells that were exposed to Cd. However, the anomalies of spermatozoa were located mainly in the mid-piece and head, many of which showed severely impaired structures. The results demonstrated that MT and CAT expression had distinct patterns in response to various Cd concentrations: an increase in MT mRNA levels with elevated Cd levels and a persistent increase in CAT mRNA levels with elevated Cd levels. These results suggested that MT and CAT play roles in Cd toxicity resistance in the testis and that the expression of CAT may be a better biomarker than the expression of MT for assessing Cd pollution.