Three-dimensional DNA nanomachine biosensor coupled with CRISPR Cas12a cascade amplification for ultrasensitive detection of carcinoembryonic antigen.

The detection of carcinoembryonic antigen (CEA) holds significant importance in the early diagnosis of cancer. However, current methods are hindered by limited accessibility and specificity. This study proposes a rapid and convenient Cas12a-based assay for the direct detection of CEA in clinical serum samples, aiming to address these limitations. The protocol involves a rolling machine operation, followed by a 5-min Cas12a-mediated cleavage process. The assay demonstrates the capability to detect human serum with high anti-interference performance and a detection limit as low as 0.2 ng/mL. The entire testing procedure can be accomplished in 75 min without centrifugation steps, and successfully reduced the limit of detection of traditional DNA walking machine by 50 folds. Overall, the testing procedure can be easily implemented in clinical settings.

Colorimetry/fluorescence dual-mode detection of Salmonella typhimurium based on a "three-in-one" nanohybrid with high oxidase-like activity for AIEgen.

A colorimetry/fluorescence dual-mode assay based on the aptamer-functionalized magnetic covalent organic framework-supported CuO and Au NPs (MCOF-CuO/Au@apt) was developed for Salmonella typhimurium (S. typhimurium) biosensing. The nanohybrid combined three functions in one: good magnetic separation characteristic, excellent oxidase-mimic activity for tetrap-aminophenylethylene (TPE-4A), and target recognition capability. The attachment of MCOF-CuO/Au@apt onto the surface of S. typhimurium resulted in a significant reduction in the oxidase-mimicking activity of the nanohybrid, which could generate dual-signal of colorimetry and fluorescence through the catalytic oxidation of TPE-4A. Based on this, S. typhimurium could be specifically detected in the linear ranges of 102- 106 CFU·mL-1 and 101- 106 CFU·mL-1, with LODs of 7.6 and 2.1 CFU·mL-1, respectively in colorimetry/fluorescence modes. Moreover, the smartphone and linear discrimination analysis-based system could be used for on-site and portable testing. In addition, this platform showed applicability in detecting S. typhimurium in milk, egg liquid and chicken samples.

Colorimetry/fluorescence dual-mode detection of Salmonella typhimurium based on self-assembly of MCOF with Au NPs nanozyme coupled AIEgen.

Sensitive, accurate, simple and quick monitoring of Salmonella typhimurium (S. typhimurium) in food is significant for preventing food poisoning, but still remains a challenge. Herein, a colorimetry/fluorescence dual-mode sensing strategy was fabricated to detect S. typhimurium by integrating the self-assembly of magnetic covalent organic framework (MCOF) with gold nanoparticles (Au NPs) as the peroxidase-mimicking nanozyme and aggregation-induced emission luminogen (AIEgen). S. typhimurium could competitive bind to aptamer conjugated Au NPs (Au NPs@apt), inhibit the self-assembly of MCOF with Au NPs, and shield the catalytic activity of AuNPs. After adding H2O2 and TPE-4A, the dark green solution changed to light with increasing S. typhimurium concentration, on the contrary, the fluorescent signals were generated. As a result, in colorimetry/fluorescence modes, S. typhimurium could be detected in the linear ranges of 103-108 CFU mL-1 and 101-107 CFU mL-1, with LODs of 1000 and 10 CFU mL-1, respectively. Importantly, different colors consistent with various S. typhimurium concentrations can be accurately classified by smartphone app and linear discriminant analysis (LDA). The smartphone-assisted data interpretation can generate complementary colorimetry and fluorescence signals without any sophisticated equipment and achieve on-site detection. Moreover, the proposed strategy could be explored for S. typhimurium monitoring in milk with satisfactory recoveries (97.6-100.4 %) in colorimetry and fluorescence mode and good classification and prediction performance in smartphone/LDA system, suggesting the feasibility and potential applications of the sensing platform.

A CRISPR/Cas12a-assisted bacteria quantification platform combined with magnetic covalent organic frameworks and hybridization chain reaction.

The total bacterial count is an important indicator of food contamination in food safety supervision and management. Recently, the CRISPR/Cas12a system integrated with nucleic acid amplification has increasingly shown tremendous potential in microorganism detection. However, a general quantification strategy for total bacteria count based on the CRISPR/Cas12a system has not yet been developed. Herein, we established a sensitive bacterial quantification strategy based on the CRISPR/Cas12a system combined with magnetic covalent organic frameworks (MCOFs) and hybridization chain reaction (HCR). MCOFs acted as a carrier, adsorbing the ssDNA as HCR trigger sequence through π-π stacking. Then, the HCR circuit produces DNA duplexes containing the PAM sequences that activate the trans-cleavage activity of Cas12a for further signal amplification. Under the optimal conditions, the proposed method can quantify total bacteria in 50 min with a minimum detection concentration of 10 CFU/mL. The successful applications in food samples confirmed the feasibility and broad application prospects.

Smartphone-assisted colorimetric detection of Salmonella typhimurium based on the catalytic reduction of 4-nitrophenol by ß-cyclodextrin-capped gold nanoparticles.

Salmonella typhimurium (S. typhimurium) is one of the most common pathogens in the environment, such as in drinking water and soil. Herein, an on-site detection method was developed by combining silver-coated magnetic nanoparticles (Fe3O4@Ag NPs) with the ß-cyclodextrin-capped gold nanoparticles (ß-CD-Au NPs) to achieve sensitive detection of S. typhimurium. After they formed a sandwich structure in the presence of S. typhimurium, the 4-nitrophenol was reduced to 4-aminophenol based on the nitro-reductase activity of ß-CD-Au NPs. The naked eyes were able to observe the color change from yellow to colorless. Under optimal conditions, the detection range of S. typhimurium was 10-107 CFU mL-1, and the limit of detection (LOD) was 10 CFU mL-1. The total detection time was 90 min, showing satisfactory performance in real samples. We combined a smartphone app with the colorimetric method, making it possible to semi-quantitatively detect S. typhimurium by analyzing the grey value. In conclusion, this assay detects S. typhimurium in environmental samples, offering an accurate and sensitive detection method without sophisticated equipment.

Genetic diversity, antibiotic resistance, and virulence profiles of Listeria monocytogenes from retail meat and meat processing.

Human listeriosis outbreaks are often associated with consumption of contaminated food, especially meat products. To better understand meat contamination of L. monocytogenes, whole genome sequencing(WGS) was performed on all detected isolates to investigate genetic relationships between retail markets and slaughterhouses. 110 and 13 isolates were isolated from 1914 food samples and 67 food and environmental samples, respectively. IIa (51/123,41.5%) and IIc (7/123,5.7%) were detected as the dominant serogroups of 123 L. monocytogenes isolates.Most isolates were penicillin-resistant (22/123,17.9%) in the phenotypic test, and all isolates were also found to be susceptible to ampicillin, meropenem, and vancomycin. All of them harbored virulence-associated genes and premature stop codons (PMSCs) in inlA genes were occurred in 35 strains. 22 multilocus sequence types and 19 clonal complexes were identified with ST9 being most common. This study also showed the prevalence and uniqueness of strains from Jilin, China compared with worldwide epidemic international strains. The findings of this study will contribute to the epidemiological understanding of transmission of L. monocytogenes from production and circulation in the region of northern China.

Prevalence, antibiotic resistance, and enterotoxin genes of Staphylococcus aureus isolated from milk and dairy products worldwide: A systematic review and meta-analysis.

Staphylococcal food poisoning is one of the common causes of food diseases, and the risk factor is staphylococcal enterotoxin. Milk and dairy products are often contaminated by antibiotic resistance and enterotoxins Staphylococcus aureus (S. aureus), which has become a critically important global public health concern. This study reviewed research studies on S. aureus in milk and dairy products worldwide published before October 2021 in PubMed, EMBASE, Scopus, and Web of Science to estimate the prevalence, antimicrobial resistance, and enterotoxin genes using a meta-analysis method. In addition, subgroup analysis, sensitivity analysis and regression analysis were conducted to explore the sources of the heterogeneity. The results showed that 140 eligible studies were published between 1992 and 2021. In raw milk, the prevalence (33.36%, 95% CI: 27.18-39.84%) was higher than that in dairy products and pasteurized milk, while it decreased over the publication period (P = 0.02). Subgroup analysis showed that the prevalence of S. aureus isolated from dairy plants was higher than that isolated from farms and retail markets. Among the 12 antibiotics, the resistance rates of penicillin (73.85%, 95% CI: 67.05-80.17%) and ampicillin (59.63%, 95% CI: 47.31-71.41%) were the highest, and the antibiotic resistance of ampicillin, gentamicin and chloramphenicol increased over time (P < 0.05). The pooled rate of classical staphylococcus enterotoxins was 39.31% (95% CI: 25.99-53.44%), and the highest rates were found for sec and sea genes. In conclusion, the hygiene and safety of raw milk can be guaranteed by improving the health of milking animals, elevating milking hygiene and using pasteurization. Developing ß-lactamase inhibitors and strengthening antibiotic resistance surveillance systems may alleviate antibiotic resistance issues. Transportation and storage according to regulation and standards may reduce the contamination of staphylococcus enterotoxins in raw milk.

On-site colorimetric detection of Salmonella typhimurium.

Rapid qualitative and quantitative detection of Salmonella typhimurium (S. typhimurium) takes an important role in ensuring food safety. Herein, a colorimetric assay aptasensor for S. typhimurium utilizing intrinsic peroxidase-like activity of gold nanoparticles embedded spherical covalent organic framework and the affinity and specificity of S. typhimurium-aptamer has been explored. This aptasensor can capture the S. typhimurium via the selective binding effect of aptamer, and the catalytically active sites were shielded. As a result, the colorimetric signals of the 3,3',5,5'-tetramethylbenzidine-H2O2 system were turned off. Under optimum conditions, the aptasensor gave a linear response over the range of 10 to 107 CFU/mL for S. typhimurium. The detection limit of 7 CFU/mL was obtained within 45 min and was effectively applied to detect S. typhimurium in milk and lake water samples with recoveries in the range from 96.4 to 101.0%. More importantly, combined with a self-developed smartphone-based image analysis system, the proposed aptasensor can be used for point-of-care testing applications.

Diagnostic accuracy of CRISPR technology for detecting SARS-CoV-2: a systematic review and meta-analysis.

OBJECTIVE: To evaluate the diagnostic accuracy of CRISPR-Cas technology for SARS-CoV-2. METHODS: RT-qPCR is defined as the reference standard. Data was collected and assessed by Quality Assessment of Diagnostic Accuracy Studies (QUADAS)-2 tool. A bivariate model for pooling was employed and subgroups analysis was used to explore heterogeneity. RESULTS: 2264 samples from 28 articles were extracted for evaluating the accuracy of CRISPR technology for diagnosing SARS-CoV-2. The pooled sensitivity and specificity of CRISPR technology were 0.98 (95% CI: 0.95-0.99) and 1.0 (95% CI: 0.98-1.00), respectively. High risks in patient selection bias and unclear risk of index test bias may affect accuracy. Subgroup analysis showed that CRISPR-Cas12 is applicable for molecular diagnostics for its active editing characteristics. RT-LAMP and RT-RPA are usually used for pre-amplification and fluorescence detection to output results quantitatively. Nasopharyngeal swabs and dual-genes perform greatly in our study. CONCLUSION: The results concluded from all studies showed that CRISPR technology is a promising molecular method for detecting SARS-CoV-2. Standard methods including comparable sample material, patient selection, operating procedure and operators should be established.

Genomic analysis, antibiotic resistance, and virulence of Staphylococcus aureus from food and food outbreaks: A potential public concern.

Transmission and outbreaks of Staphylococcus aureus among retail food highlights the need to comprehensive analysis the molecular characteristic of Staphylococcus aureus in foods. However, the information about Staphylococcus aureus in north China is limited. In this study, 97 and 28 S. aureus strains were isolated for analysis from 4262 samples of retail food and 61 samples food outbreaks with prevalence rate 2.28 % and 45.9 %, respectively in Jilin, China from 2014 to 2018. This study aimed to investigate the prevalence of S. aureus isolates and characterize by antimicrobial resistance testing, virulence profiles, spa typing, and multilocus sequence typing (MLST) analysis. 60 % (75/125) of the isolates contained at least enterotoxin genes including classic and new SEs genes as following: sea (40/125,32 %), see (36/125,28.8 %), sec (29/125,23.2 %), sell (29/125,23.2 %), seb (25/125,20 %), seh (22/125,17.6 %), sed (6/125,4.8 %), selq (6/125,4.8 %), and selk (6/125,4.8 %). In antimicrobial susceptibility tests, 59.2 % of the isolates (74/125) were considered as multi-drug-resistant isolates and four MRSA strains were all found with high multi-drug-resistance. Phenotype resistance to penicillin (94.4 %), erythromycin (84.2 %), clindamycin (63.9 %), and tetracycline (47.2 %) was observed which was corresponding with genotype resistance. The strains were classified to twenty-two sequence types (STs), fourteen clonal complexes (CCs), and forty-seven spa types. The predominant ST and spa types were ST1(22/125,17.6 %), ST25(20/125,16.00 %), ST398 (14/125,11.2 %) and t127 (20/125,16 %), t078 (14/125,11.2 %), t803 (7/125,5.6 %). The wgSNP analysis of these isolates in food represents showed close relatedness with food outbreaks which pose a potential health risk for consumers and warrants further attention.

Whole Genome Sequencing, Antibiotic Resistance, and Epidemiology Features of Nontyphoidal Salmonella Isolated From Diarrheic Children: Evidence From North China.

Nontyphoidal Salmonella (NTS) in children remains a growing burden on public health and often causes children to be hospitalized with diarrheic symptoms. In this work, 260 strains of human Salmonella isolated from Jilin, China were characterized by serotypes and antimicrobial resistance using whole genome sequencing (WGS). The most prevalent serotype was Salmonella enteritidis (47.3%), followed by S. I 4,[5],12:i:- (33.1%), and Salmonella Typhimurium (7.3%). Furthermore, the consistency between resistance phenotype and genotype was confirmed. Similarly, strains harbored bla TEM-1B and tetA genes were detected, which verified the level of resistant phenotype in ß-lactams and tetracyclines. The presence of a single mutation in parC, gyrA, and qnrS1 genes corresponding to quinolones was also observed. In our work, multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) were found to have a high resolution to molecular traceability, and the combination of both was conducive to practical application in an actual situation. Taking all of this into account, we suggested that the comprehensive surveillance of Salmonella infection in children should be carried out to monitor antimicrobial-resistant trends from various sources and to alert on outbreaks of foodborne diseases to protect public health.

Preparation of IgY Oriented Conjugated Fe3O4 MNPs as Immunomagnetic Nanoprobe for Increasing Enrichment Efficiency of Staphylococcus aureus Based on Adjusting the pH of the Solution System.

Immunomagnetic separation based on Fe3O4 magnetic nanoparticles (MNPs) has been widely performed in sample pretreatment. The oriented conjugation strategy can achieve a better capture effect than the N-(3-dimethylamlnopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) /N-hydroxysuccinimide (NHS) method. However, immunoglobulin yolk (IgY) cannot be oriented through an SPA strategy like immunoglobulin G (IgG). In this article, an oriented conjugation nanoprobe was prepared for the enrichment of bacteria based on pH adjusting. The main factors affecting the enrichment efficiency were studied, such as the pH of the buffer system, the concentration of IgY, the concentration of nanoprobe, and the enrichment time. Under the optimal conditions, the enrichment efficiency toward target bacteria could reach 92.8%. Combined with PCR, the limit of detection (LOD) was found to be 103 CFU/ml, which was lower than the PCR only. In conclusion, we provided a new protocol for the oriented conjugation of IgY and high sensitivity detection with simple pretreatment.

Colorimetric detection of Salmonella typhimurium based on hexadecyl trimethyl ammonium bromide-induced supramolecular assembly of ß-cyclodextrin-capped gold nanoparticles.

We developed an effective and specific colorimetric strategy to detect Salmonella typhimurium (S. typhimurium) based on hexadecyl trimethyl ammonium bromide (CTAB)-induced supramolecular assembly of ß-cyclodextrin-capped gold nanoparticles (ß-CD-AuNPs). In this study, ssDNA aptamer of S. typhimurium could combine with CTAB to form the supramolecular ssDNA-CTAB composite, so the ssDNA aptamer was applied to control the concentration of CTAB. In the presence of S. typhimurium, ssDNA aptamers selectively bound to S. typhimurium but not to CTAB, leading to the host-guest chemistry reaction of CTAB and ß-CD resulting in ß-CD-AuNP supramolecular assembly aggregation with an obvious color change. The ratio of absorption at 650 and 520 nm (A650nm/A520nm) has a linear correlation to the log scale of the concentration of the bacteria (1 × 102-1 × 107 CFU/mL) with a low limit of detection (LOD) of 13 CFU/mL. In addition, this optical sensor has good selectivity and practicability. In milk samples, the recovery was 93.55-111.32%, which suggested its potential application in real samples.

Promising application of polyoxometalates in the treatment of cancer, infectious diseases and Alzheimer's disease.

As shown in studies conducted in recent decades, polyoxometalates (POMs), as inorganic metal oxides, have promising biological activities, including antitumor, anti-infectious and anti-Alzheimer's activities, due to their special structures and properties. However, some side effects impede their clinical applications to a certain extent. Compared with unmodified POMs, POM-based inorganic-organic hybrids and POM-based nanocomposite structures show significantly enhanced bioactivity and reduced side effects. In this review, we introduce the biological activities of POMs and their derivatives and highlight the side effects of POMs on normal cells and organisms and their possible mechanisms of action. We then propose a development direction for overcoming their side effects. POMs are expected to constitute a new generation of inorganic metal drugs for the treatment of cancer, infectious diseases, and Alzheimer's disease.Graphical abstract.

A colorimetric sensor for Staphylococcus aureus detection based on controlled click chemical-induced aggregation of gold nanoparticles and immunomagnetic separation.

Staphylococcus aureus (S. aureus) is a pathogen closely associated with foodborne diseases. We prepared a reliable colorimetric sensor to detect S. aureus using click chemical reaction and immunomagnetic separation. Aptamer-functionalized and ALP-labeled Fe3O4 NPs act as separation and signal transduction elements. Under the optimized conditions, the Cu+ generated by signal transduction triggers a click chemistry reaction, which causes the aggregation of azides and alkyne-AuNPs and a color change. The net extinction ratio of Δ(A530/A760) was linearly correlated with the S. aureus concentration from 10 to 106 cfu mL-1, and the limit detection was 2.4 cfu mL-1. The recoveries were 91.15 ~ 106.36% for the analysis of spiked food and water samples without pre-enrichment. Therefore, we believe that the detection platform can be easily and accurately used for S. aureus detection, providing a broad prospect for on-site visual detection.

Colorimetric determination of Listeria monocytogenes using aptamer and urease dual-labeled magnetic nanoparticles and cucurbit[7]uril-mediated supramolecular assembly of gold nanoparticle.

A host-guest colorimetric strategy is described for the detection of Listeria monocytogenes (L. monocytogenes). The optical probes were self-assembled based on the supramolecular interactions between the carbonyl groups of cucurbit[7]uril portals and gold nanoparticles (CB[7]-AuNPs). Aptamer and urease modified magnetic nanoparticles were used to specifically recognize and binding to L. monocytogenes, simultaneously hydrolyzing urea to produce ammonium ion (NH4+) that can reverse CB[7] induced AuNPs aggregation. In the presence of L. monocytogenes, the above-mentioned magnetic conjugates preferentially bind to the bacterial surface, which results in blocking the catalytic active sites, thus inhibiting the production of ammonium ions. The normalized absorbance ratio of A700 nm/A525 nm was proportional to the L. monocytogenes concentration ranging from 10 to 106 cfu·mL-1, and the visual determination can be done down to 10 cfu·mL-1. For spiked food samples analyzed without pre-enrichment, recoveries of 98.4% to 99.3% were achieved could be verified and RSD were less than 10%. This work may offer a broad prospect for sensitive and specific determination  of pathogens.

Multi-functional magnetic molecular imprinting probe for visual detection of IgY antibodies.

Egg yolk antibodies (IgY) are an alternative to mammalian antibodies, which have been successfully applied in treatment of disease, as immunochemical reagents and as food additives. A fast, accurate, and easy-to-operate detection method for IgY antibodies is needed. Herein, we developed a rapid and cost-effective colorimetric assay for the ultrasensitive detection of anti-S. aureus IgY antibodies using multi-functional magnetic molecularly imprinted polymers (MMIPs). The prepared MMIPs can recognize, adsorb, and separate the analyte from the matrix efficiently, and oxidize TMB to generate a colorimetric signal within ~ 60 min. As low as 0.013 mg·mL-1 was able to be detected by using this method. The visual detection limit reached 0.02 mg·mL-1. By testing the IgY in milk samples, the feasibility was verified.

[The effect of nasal continuous positive airway pressure on endothelial function in obstructive sleep apnea-hypopnea syndrome with coronary heart disease].

OBJECTIVE: To study the association between the indices of endothelial function in obstruction sleep apnea-hypopnea syndrome (OSAHS) and coronary heart disease (CHD) and the effect of nasal continuous positive airway pressure (nCPAP) on the combination of OSAHS and CHD. METHODS: A total of 80 subjects were prospectively recruited into four groups including control, OSAHS, CHD, OSAHS with CHD groups, with 20 subjects each. The indices of sleep apnea, serum nitric oxide (NO), and plasma endothelial-1 (ET-1) were measured. The changes of concentration of ET-1 and NO were compared before and after nCPAP therapy. The associations between ET-1 and NO and MSpO2, CT90 were analyzed. RESULTS: (1) Multi-variable logistic analysis showed that OSAHS was one of the risk factors for CHD (OR = 0.511). (2) Compared with the control subjects and CHD group, there were significantly higher values of CT90, concentrations of ET-1 and lower values of MSpO2, concentrations of NO in both OSAHS and OSAHS with CHD groups (P < 0.01). There were no significant difference in sleep apnea indices between OSAHS and OSAHS with CHD groups (P > 0.05). However, in the group of OSAHS with CHD, the plasma ET-1 was significantly higher, whereas the serum NO was significantly lower than that in the group of OSAHS alone (P < 0.01). (3) The concentration of serum NO in the group of OSAHS was positively correlated with MSpO2 (r = 0.519, P < 0.05) and inversely correlated with CT90 (r = -0.529, P < 0.05). In addition, the concentration of plasma ET-1 was inversely correlated with MSpO2 (r = -0.457, P < 0.05) and positively correlated with CT90 (r = 0.476, P < 0.05). (4) In the groups of OSAHS and OSAHS with CHD, MSpO2, NO and NO/ET-1 after nCPAP therapy were higher than those before therapy, while CT90 and ET-1 were lower than those before therapy (P < 0.01). CONCLUSIONS: OSAHS is one of the risk factors for CHD. Endothelial function was significantly impaired in OSAHS patients, and more severe in patients with OSAHS with CHD. The impairment of endothelial function may be one of the main mechanisms for the development or deterioration of CHD in OSAHS patients. The vascular endothelial dysfunction can be ameliorated by nCPAP treatment, which is correlated with improvement of nocturnal hypoxemia.