Functionalized magnetic nanobeads for SERS-based detection of Staphylococcus aureus.

In this study, we introduced a Raman detection technique based on a combination of functionalized magnetic beads and surface-enhanced Raman scattering (SERS) tags to develop a rapid and sensitive strategy for the detection of Staphylococcus aureus (S. aureus), a typical foodborne pathogen. Polyethylene glycol (PEG) and bovine serum albumin (BSA) dual-mediated teicoplanin functionalized magnetic beads (TEI-BPBs) were prepared for separation of target bacteria. SERS tags were used to immobilize antibodies on gold surfaces with bifunctional linker proteins to ensure specific recognition of S. aureus. Under optimal conditions, the combination of TEI-BPBs and SERS tags showed reliable performance, exhibiting good capture efficiency even in the presence of 106 CFU mL-1 of non-target bacteria. The SERS tag provided an effective hot spot for subsequent Raman detection, presenting good linearity in the range of 102-107 CFU mL-1. Good performance has also been shown in detecting target bacteria in milk samples, where it has a recovery of 95.5-101.3%. Thus, the highly sensitive Raman detection technique combined with TEI-BPBs capture probes and SERS tags is a promising method for the detection of foodborne pathogens in food or clinical samples.

Amplification-free detection of HBV DNA mediated by CRISPR-Cas12a using surface-enhanced Raman spectroscopy.

Nucleic acid markers have been widely used in the detection of various virus-related diseases, including hepatitis B virus (HBV), which is spreading worldwide. The trans-activated CRISPR-Cas system has shown excellent sensitivity and specificity in nucleic acid detection. However, nucleic acid testing usually requires amplification of the target nucleic acid for more accurate and specific detection; furthermore, current nucleic acid assays are time-consuming, costly, and are limited by non-specific cross-reactivity. We developed an amplification-free viral DNA biosensor-based diagnostic method that uses a clustered regularly interspaced short palindromic repeats-associated system (CRISPR/Cas)-based approach with surface enhanced Raman spectroscopy. This method can specifically identify the target site by changing the crRNA sequence. In addition, the incubation period and development of the disease can be determined by quantitative detection of viral DNA. This system could achieve rapid and highly sensitive detection of HBV DNA within 50 min and vast detection range from 0.1 pM to 1 nM. Therefore, a combined CRISPR/Cas12a-SERS-based assay would improve the sensitivity of detection in assays using multiple biomarkers. In conclusion, our CRISPR/Cas12a-based biosensor would enable rapid, simple, and sensitive detection of HBV nucleic acids.

AuNP/Cu-TCPP(Fe) metal-organic framework nanofilm: a paper-based electrochemical sensor for non-invasive detection of lactate in sweat.

Effective and real-time detection of lactate (LA) content in human sweat has attracted considerable attention from researchers. In this work, a novel electrochemical paper-based analysis device (ePAD) was developed for the non-invasive detection of LA in sweat. The electrocatalytic properties of AuNP/Cu-TCPP(Fe) hybrid nanosheets, which were prepared by an optimised synthetic method, were studied by CV and EIS electrochemical methods for the first time and the working electrode can be fabricated using a drip coating method. The lactate sensor was optimised and validated for usability, adoptability and interpretability. To the best of our knowledge, this was the fastest, lowest detection line and widest linear range method reported to date for the detection of lactate. It achieved the detection limit of 0.91 pM and a linear range from 0.013 nM to 100 mM. The dual catalytic effects of the hybrid NSs shortened the detection time by nearly two times and enhanced the sensitivity approximately two times, an accuracy unmatched until now. Furthermore, this sensor was employed for LA analysis and validated by high performance liquid chromatography (HPLC). The ePAD shows superior biocompatibility, accuracy, and high sensitivity and can be easily manufactured. Hence, it is applicable for the long-term monitoring of sweat LA concentrations in point-of-care testing, athletic testing of athletes and military personnel and other subjects in different extreme environments.

A fluorescence aptasensor based on hybridization chain reaction for simultaneous detection of T-2 toxins and zearalenone1.

It is extremely necessary to establish a rapid and high-throughput method to detect mycotoxins in food, because grains and cereals are greatly vulnerable to mycotoxins before and after harvest. In this study, we developed a portable aptasensor based on streptavidin magnetic microspheres (MMPs) and hybridization chain reaction (HCR) to simultaneously detect T-2 toxin and zearalenone (ZEN) in corn and oat flour. The MMPs compete with the aptamer for binding, which releases more H0 and triggers HCR with the H1 intermediate modified using 6-FAM and BHQ-1 and the unmodified H2. Subsequently, placing the HCR system corresponding to T-2 and ZEN in a constant-temperature fluorescence detector resulted in well-recovered fluorescence of the HCR products. T-2 and ZEN exhibited good fluorescence response in the dynamic range of 0.001-10 ng mL-1 and 0.01-100 ng mL-1 with detection limits of 0.1 pg mL-1 and 1.2 pg mL-1, respectively. In addition, this strategy achieved the selective detection of T-2 and ZEN in the spiked corn and oat flour samples. The results are also in good agreement with those obtained using commercial ELISA kits. This developed aptasensor with the characteristics of simple operation and portability has the application potential of establishing sensitive and portable field detection of various mycotoxins.

Sensitive colorimetric detection of antibiotic resistant Staphylococcus aureus on dairy farms using LAMP with pH-responsive polydiacetylene.

Rapidly and accurately detecting antibiotic-resistant pathogens in agriculture and husbandry is important since these represent a major threat to public health. While much attention has been dedicated to detecting now-common resistant bacteria, such as methicillin-resistant Staphylococcus aureus, fewer methods have been developed to assess resistance against macrolides in Staphylococcus aureus (SA). Here, we report a visual on-site detection system for macrolide resistant SA in dairy products. First, metagenomic sequencing in raw milk, cow manure, water and aerosol deposit collected from dairy farms around Tianjin was used to identify the most abundant macrolide resistance gene, which was found to be the macB gene. In parallel, SA housekeeping genes were screened to allow selective identification of SA, which resulted in the selection of the SAOUHSC_01275 gene. Next, LAMP assays targeting the above-mentioned genes were developed and interpreted by agarose gel electrophoresis. For on-site application, different pH-sensitive colorimetric LAMP indicators were compared, which resulted in selection of polydiacetylene (PDA) as the most sensitive candidate. Additionally, a semi-quantitative detection could be realized by analyzing the RGB information via smartphone with a LOD of 1.344 × 10-7 ng/µL of genomic DNA from a milk sample. Finally, the proposed method was successfully carried out at a real farm within 1 h from sample to result by using freeze-dried reagents and portable devices. This is the first instance in which PDA is used to detect LAMP products, and this generic read-out system can be expanded to other antibiotic resistant genes and bacteria.