Biosensors-Basel最新文献

Traditional inducible systems typically induce the simultaneous expression of all genes controlled by similar promoters, thereby limiting their use. In this study, we used two metabolite-inducible systems, MarR from the Escherichia coli mar operon and TtgR from the Pseudomonas putida ttg operon, to assess their use as gene regulation platforms beyond reporter assays. Ligand-dependent transcription was validated using eGFP. The reporter was replaced with two flavonoid O-methyltransferases (OMTs), ROMT-9 and SOMT-2, under transcription factor (TF)-specific promoters. In E. coli, both systems enabled in using HPLC. TF-based expression did not impact enzyme activity. Induction with salicylic acid (MarR) produced stronger gains than that with 4'-hydroxyflavanone (TtgR), although the overall fold-changes in product levels were regulated by basal (leaky) expression. Thus, although transcriptional control was robust, enzymatic regulation was less stringent, highlighting the necessity for genetic engineering of components, including TFs, promoters, transcription factor binding sites, and ribosome binding sites, to reduce leakiness and expand the dynamic range. Overall, these orthogonal and modular TF-based systems offer a framework for independent and inducible control of multiple genes, with potential applications in biosensing, metabolic engineering, and programmable pathway design.

The reduced scattering coefficient (μ_s'), measured using time-resolved near-infrared spectroscopy (TR-NIRS) has been linked to brain water diffusion assessed by diffusion tensor imaging, suggesting its potential as a bedside marker of cerebral microstructure. However, the physiological determinants of μ_s' and its early postnatal changes remain unclear. This study examined clinical associations with cerebral μ_s' in healthy term newborn infants during the first 2 postnatal days. Eighteen newborn infants underwent TR-NIRS at 6 and 36 h postnatally. Associations between μ_s' and 14 clinical variables were analysed using generalised estimating equations. Median μ_s' was 7.395 cm^-1 (IQR: 6.140-8.159) at 6 h and 7.112 cm^-1 (IQR: 6.473-7.410) at 36 h, with no significant difference (p = 0.327). Male sex was associated with higher μ_s' (regression coefficient = 0.895, p = 0.007), whereas caesarean delivery (regression coefficient = -0.969, p = 0.012) was associated with lower μ_s'. A significant interaction between caesarean delivery and postnatal age indicated that the negative effect diminished between 6 and 36 h after birth (difference = 0.057, p = 0.016). These findings suggest delivery mode transiently influences brain scattering, whereas the effect of sex remains stable, supporting further investigation of TR-NIRS as an acute-phase cerebral marker.

Sensors to monitor the immune status of an individual play a crucial role in understanding the acquired immunity or signs of a latent infection. Such sensors can be an effective tool to manage infection and to design treatment options in vulnerable populations. We demonstrate here highly sensitive detection of acquired immunity to Cytomegalovirus CMV by detection of anti-CMV antibodies using plasmon-enhanced fluorescence (PEF). The PEF sensors leverage plasmonic enhancement from a high density of intense electromagnetic hotspots in self-assembly-derived gold nanopillar arrays. Comparing PEF assays with assays on a planar surface plasmon resonance sensor shows the PEF sensors to be sensitive to a small fraction of the antibodies on the surface. The detection scheme deploys peptide monolayers with specific affinity to anti-CMV antibodies to capture them onto the sensor surfaces. The results of the assay on the PEF sensor reveal high promise for sensors with miniaturized sensing footprints, ease of spatial multiplexing, high sensitivity, and quick response times. The developments are readily applicable to a range of other diagnostic contexts where peptide-protein interactions and self-assembly-derived PEF sensors can be leveraged.

Natural dyes have emerged as a promising alternative to synthetic dyes for industrial applications due to their advantages, namely, easy availability, low cost, and environmental friendliness. In this sense, natural dyes, due to their potential to react over the pH range, could offer an alternative to conventional pH measuring techniques for industrial products, such as potentiometers, sensors, or indicator drops. Therefore, this project aims to evaluate the potential of several natural organic dyes in response to changes in pH and develop an indicator for determining pH grades. We extracted and analyzed the pigments of forty natural vegetable species using two extraction methods with a mixture of solvents, specifically 70% MeOH/30% H₂O. The results find that pigments of cabbage, hibiscus flower, radish, and turmeric in their dry state exhibit the best reaction over a broad pH range, and color can be easily distinguished according to its level. These findings demonstrate the potential of natural dyes as a novel approach for pH verification, providing a sustainable and cost-effective alternative to conventional techniques.

Regulatory agencies worldwide have implemented stringent measures to monitor and reduce Salmonella contamination in poultry products. Rapid quantitative detection methods enable producers to identify contamination early, implement corrective actions, and enhance food safety. This study aimed to develop and optimize a surface plasmon resonance (SPR) biosensor for the quantitative detection of Salmonella Typhimurium in ground chicken. The sensor surface was functionalized with a well-characterized monoclonal antibody specific to Salmonella flagellin, and an SPR workflow was established for quantitative analysis. Ground chicken samples were inoculated with four S. Typhimurium strains at contamination levels ranging from -0.5 to 3.5 Log CFU/g and enriched at 42 °C for 10 or 12 h prior to SPR analysis. Contamination levels were confirmed using the Most Probable Number (MPN) method. Linear regression analysis indicated that optimal quantification was achieved after 10 h of enrichment (R² ≥ 0.86), whereas extended enrichment (12 h) did not improve performance. The limit of quantification (LOQ) was below 1 CFU/g. A strong positive correlation (R² ≥ 0.85) was observed between SPR and MPN results, demonstrating consistency between the two methods. These findings highlight SPR as a rapid, reliable, and cost-effective alternative to conventional methods for Salmonella quantification. By delivering accurate results within a single day, SPR enhances testing efficiency and supports the production of safer poultry products, thereby reducing public health risks associated with Salmonella contamination.

Single exosomes are detected via surface-enhanced Raman scattering (SERS) due to electromagnetic field accumulation on a specially designed flexible metasurface. This metasurface is a modulated silver nanofilm deposited on a thin, flexible plastic substrate. An explicit Equation for calculating the local electric field is given. The field reaches extremely high values under plasmon resonance conditions and fills the depressions of the metasurface. The thin, flexible metasurface can be incorporated into automated Lab-On-Chip analytical systems and used for spectroscopic studies of exosomes. We propose a method to distinguish individual exosomes from the HEK293T cell line on the metasurface and then obtain and assign their SERS spectra. An important advantage of the plasmonic metasurface presented in this work is its spatial complementarity to exosomes and other vesicle-like objects. The plasmonic metasurface is fabricated using holographic lithography and further investigated using a correlation approach combining atomic force microscopy, scanning spreading resistance microscopy, and surface-enhanced spectroscopy.

Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen, responsible for severe gastrointestinal disease and hemolytic uremic syndrome (HUS). Here, we report Click Detect, a novel diagnostic platform that leverages click display to efficiently produce sensing probes for sandwich-style antigen detection. Click display is an in vitro protein display technology that generates uniform and covalently linked protein-cDNA conjugates in a simple one-pot reaction format within 2 h. The captured sensing probe can be quantified by standard nucleic acid amplification assays. Using click displayed DARPin (D_#20) as the sensing probe and a high-affinity nanobody (N_G1) as the capture reagent, Click Detect reliably detected Shiga toxin 2 (Stx2) at 600 fM by quantitative PCR (qPCR) and 6 pM by loop-mediated isothermal amplification (LAMP). The assay maintained comparable sensitivity in matrices containing up to 40% public swimming pool water or lettuce extract, highlighting robustness for real-world surveillance applications. Key advantages of Click Detect include simple, rapid, and cost-effective (~USD 0.04 per assay) sensing probe preparation, as well as a versatile plug-and-play probe format for detecting other targets. We believe that Click Detect has great potential as a novel sensing platform for food/environmental monitoring and point-of-care diagnostics, with potentially broad applicability to other toxins and protein targets.

The detection of environmental toxicants is transitioning from centralized laboratory methods to decentralized, point-of-care (POC) monitoring. A highly innovative approach in this field is the repurposing of commercially available, low-cost, and portable personal glucose meters (PGMs) as universal biosensing platforms. This strategy leverages the widespread availability and ease of use of PGMs to develop rapid, on-site detection methods for a wide array of non-glucose targets, significantly reducing both cost and development time. This systematic review comprehensively examines the various strategies employed to adapt PGMs for the detection of a wide array of ecotoxicants, including chemical targets (antibiotics, mycotoxins, pesticides, heavy metals, persistent organic pollutants) and biological ones (pathogenic bacteria, and viruses). The systematic review critically evaluates different sensor designs, highlighting that while aptamer-based and non-enzymatic biosensors offer advantages in stability and cost, antibody-based sensors provide high specificity. A significant finding is the persistent trade-off between analytical sensitivity and practical field deployment; many of the most sensitive assays require multi-step procedures, precise temperature control, magnetic separation, centrifugation, and the use of additional equipment, factors that undermine true POC utility. To address this gap, we propose four essential criteria for POC readiness: (i) ambient-temperature operation, (ii) no reliance on magnetic or centrifugal separation, (iii) total assay time, and (iv) robustness in complex environmental matrices. This systematic review confirms the feasibility of this approach across a broad spectrum of targets. However, the key challenge for future research lies in simplifying the assay protocols, eliminating cumbersome sample preparation steps, and enhancing robustness to make these biosensors truly practical for routine, on-site environmental monitoring.

CRISPR/Cas12a systems coupled with lateral flow tests (LFTs) are a promising route to rapid, instrument-free nucleic acid diagnostics due to conversion target recognition into a simple visual readout via cleavage of dual-labeled single-stranded DNA reporters. However, the conventional CRISPR/Cas12a-LFT system is constructed in a format where the intact reporter should block nanoparticle conjugate migration and can produce false-positive signals and shows strong dependence on component stoichiometry and kinetics. Here, we present the first combined experimental and theoretical analysis quantifying these limitations and defining practical solutions. The experimental evaluation included 480 variants of LFT configuration with reporters differing in the concentration of interacting components and the kinetic conditions of the interactions. The most influential factor leading to 100% false-positive results was insufficient interaction time between the components; pre-incubation of the conjugate with the reporter for 5 min eliminated these artifacts. Theoretical analysis of the LFT kinetics based on a mathematical model confirmed kinetic constraints at interaction times below a few minutes, which affect the detectable signal. Reporter concentration and conjugate architecture represented the second major factors: lowering reporter concentration to 20 nM and using smaller gold nanoparticles with multivalent fluorescent reporters markedly improved sensitivity. The difference in sensitivity between various LFT configurations exceeded 50-fold. The combination of identified strategies eliminated false-positive reactions and enabled the detection of up to 20 pM of DNA target (the hisZ gene of Erwinia amylovora, a bacterial phytopathogen). The strategies reported here are general and readily transferable to other DNA targets and CRISPR/Cas12a amplification-free diagnostics.

Zearalenone (ZEN), a stable mycotoxin with estrogenic activity produced by various Fusarium species, poses a serious food safety risk. To facilitate the rapid, sensitive, on-site detection of ZEN in maize and ensure consumer dietary safety, a colloidal gold immunochromatographic assay (CG-ICA) based on a monoclonal antibody was established. ZEN was converted via oxime derivatization into hapten ZAN-O, which was conjugated to a carrier protein to prepare an immunogen for producing a highly specific and sensitive monoclonal antibody. Then, the antibody was conjugated into colloidal gold nanoparticles (AuNPs) and used as capture bioprobes of the CG-ICA test strip. The highly sensitive and specific detection platform was established through systematic optimization of pH value, coating antigen concentration, antibody-labeling dosage, incubation time, and strip assembly conditions. Under optimized conditions, the strip exhibited a detection limit of 11.79 pg/mL and an IC₅₀ of 99.06 pg/mL, with a linear detection range of 13.40-732.48 pg/mL. In addition, the anti-interference capability assay demonstrated that the developed test strip possessed excellent specificity. In spiked maize samples, the CG-ICA test strip demonstrated recoveries ranging from 85.36% to 98.86%, with relative standard deviations (RSDs) below 10%. Thus, the CG-ICA strip provides a rapid, sensitive, and robust on-site tool for ZEN screening in maize, and can be adapted to other hazards by simply switching the antibody.