Over the past decade, the field of immunoassays and biosensing has undergone remarkable expansion, driven by the urgent demand for sensitive, rapid, and reliable detection technologies across biomedical, environmental, and food safety applications [...].
DNA nanoparticles have emerged as potent platforms for wearable and implantable biosensors owing to their molecular programmability, biocompatibility, and structural precision. This study delineates two principal categories of DNA-based sensing materials, DNA hydrogels and DNA origami, and encapsulates their fabrication methodologies, sensing mechanisms, and applications at the device level. DNA hydrogels serve as pliable, aqueous signal transduction mediums exhibiting stimulus-responsive characteristics, facilitating applications such as sweat-based cytokine detection with limits of detection as low as pg·mL-1 and microneedle-integrated hydrogels for femtomolar miRNA sensing. DNA origami offers nanometer-scale spatial precision that improves electrochemical, optical, and plasmonic biosensing, as shown by origami-facilitated luminous nucleic acid detection and ultrasensitive circulating tumor DNA assays with fM-level sensitivity. Emerging integration technologies, such as flexible electronics, microfluidics, and wireless readout, are examined, alongside prospective developments in AI-assisted DNA design and materials produced from synthetic biology. This study offers a thorough and practical viewpoint on the progression of DNA nanotechnology for next-generation wearable and implantable biosensing devices.
Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor's response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application.
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 (μs'), 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 μs' and its early postnatal changes remain unclear. This study examined clinical associations with cerebral μs' 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 μs' and 14 clinical variables were analysed using generalised estimating equations. Median μs' 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 μs' (regression coefficient = 0.895, p = 0.007), whereas caesarean delivery (regression coefficient = -0.969, p = 0.012) was associated with lower μs'. 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 (NG1) 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.
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