Sensitive Method Research Articles

A Compact Prism-Based Microscope for Highly Sensitive Measurements in Fluid Biopsy.

The increasing demand for sensitive, portable, and affordable disease detection methods has spurred the development of advanced biosensors for rapid early-stage diagnosis, population mass screening, and bed-monitoring. Current high-sensitivity devices face hurdles such as high production costs and challenges in multiplexed signal detection. To address these, we developed a prism-based total internal reflection system which, in combination with surface functionalization techniques of gold nanoparticles, enables evanescent wave scattering for highly sensitive and rapid detection of specific analytes in both synthetic and human liquid samples. To validate its efficacy, we conducted scattering experiments in synthetic and human serum samples, exploiting functionalized AuNPs to recognize bacterial lipopolysaccharides as biomarkers for sepsis disease. We demonstrate a remarkable sensitivity in the femtogram per mL concentration range for this specific pathological biomarker. Based on this result we envisage the potential adoption of our technique for liquid biopsy in the clinical scenario.

Development of recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid and simple detection of Tylenchulus semipenetrans in soil.

Tylenchulus semipenetrans, the causal agent of citrus slow decline disease, is one of the most destructive plant-parasitic nematodes in all citrus-growing regions of the world, causing significant reductions in citrus growth and yield. Accurate and rapid detection of T. semipenetrans is critical for the diagnosis and effective control of the disease. We developed a rapid, visual, isothermal detection method using recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay to detect T. semipenetrans in soil. The primers and a probe were designed based on sequence differences in the internal transcribed spacer region 1 (ITS1) of ribosomal DNA (rDNA) among T. semipenetrans and four other Tylenchulus species. The RPA reaction can be performed in 10-25 min at a constant temperature ranging from 30 to 45 °C, and the result can be read directly on the LFD within 3 min. Under the optimized conditions, the RPA-LFD assay could specifically detect T. semipenetrans with a sensitivity as low as 10-2 second-stage juveniles/0.5 g soil, which was 10-fold more sensitive than that of the conventional PCR assay. Furthermore, we combined a soil DNA extraction method with the RPA-LFD assay to achieve simple and rapid detection of T. semipenetrans in natural field soil samples within 1 h. The developed RPA-LFD assay is a simple, rapid, specific, sensitive and visual method for detecting T. semipenetrans. It shows great potential for on-site rapid detection applications, especially in resource-limited settings. © 2025 Society of Chemical Industry.

Partially purified Strongyloides ratti antigen improved the diagnostic performance of strongyloidiasis by enzyme-linked immunosorbent assay (ELISA) and immunochromatographic test (ICT).

Strongyloides stercoralis infection is a neglected tropical disease with a global distribution. Serodiagnosis is a sensitive method, but improving its performance and simplifying into a point-of-care test (POCT) are needed. This study aimed to improve the diagnostic performance of serological tests using partially purified Strongyloides ratti antigen in an enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT). Crude S. ratti antigen was purified by an IgG affinity column to partition the antigen into flow-through, washing fraction (WF), and elution fractions. Optimized ELISA and ICT using crude and antigen fractions were used to analyze sera from three groups of subjects. Group 1 comprised subjects with proven strongyloidiasis, Group 2 were subjects with other parasitic infections, and Group 3 were negative parasitic infections. The diagnostic performance and Kappa agreement of the serological tests were analyzed and compared, using larvae detection as the reference test (fecal examination). The results showed that the WF was the most efficient antigen in terms of sensitivity and specificity, as determined by the ELISA and ICT. Kappa's agreement between fecal examination and WF-ELISA was moderate (Kappa = 0.52), and WF-ICT was almost perfect (Kappa = 0.94). The WF antigen reduced cross-reactivity to other parasitic infections, that is, Opisthorchis viverrini, Taenia spp., and hookworms, compared to crude S. ratti antigen when assessed by ELISA and ICT. We concluded that the WF of purified S. ratti improved the ELISA and ICT diagnostic performance, and the latter assay format could be used as a POCT for screening and controlling strongyloidiasis.IMPORTANCEThis study aimed to improve the serological diagnosis of strongyloidiasis, a disease caused by infection with the intestinal nematode Strongyloides stercoralis, by evaluating the impact of Strongyloides ratti antigen purification using an IgG affinity column for detecting parasite-specific IgG in serum via enzyme-linked immunosorbent assay (ELISA) and immunochromatographic test (ICT) formats. Compared to crude S. ratti antigen, the washing fraction (WF) of the purified antigen demonstrated significantly improved sensitivity and specificity in both ELISA and ICT, achieving strong diagnostic concordance with the gold-standard fecal examination. Furthermore, the WF antigen fraction exhibited reduced cross-reactivity with coinfections caused by the liver fluke (Opisthorchis viverrini), tapeworms (Taenia spp.), and hookworms. These findings underscore antigen purification as a promising strategy to enhance the accuracy of strongyloidiasis serodiagnosis.

New method for cytological evaluation using direct nipple discharge without aspiration

Conventional smear cytology (CSC) is a specific method used for breast tumor diagnosis in patients with nipple discharge. However, CSC tends to miss diagnose or even misdiagnose due to contaminating blood cells and other impurities. Thus, it is critical to develop more accurate and sensitive methods for clinical evaluation. Using nipple discharge collected directly from patients without aspiration, we performed liquid-based cytology (LBC) to analyze 111 collected samples for cytological evaluation for the first time. Following centrifugation to remove blood cells and protein contaminants, the sample of each nipple discharge was analyzed by light microscopy. In parallel, CSC was performed for comparison. Our results showed LBC has better diagnostic sensitivity than CSC (40.00% vs. 22.22%, = 6.636, P = 0.01). The specificity was improved (LBC 100% vs. CSC 95.2%) and area under the curve was also enhanced (AUCLBC=0.700 vs. AUCCSC=0.587). Moreover, LBC images have a cleaner background, clearer field of view, uniform cell arrangement, and improved colour contrast, with an overall image quality significantly better than CSC. Therefore, LBC provides a new and better diagnostic method for early detection of breast tumors with nipple discharge.

MSH2, MSH6, MLH1, and PMS2 immunohistochemistry as highly sensitive screening method for DNA mismatch repair deficiency syndromes in pediatric high-grade glioma

Pediatric high-grade glioma (pedHGG) can occur as first manifestation of cancer predisposition syndromes resulting from pathogenic germline variants in the DNA mismatch repair (MMR) genes MSH2, MSH6, MLH1, and PMS2. The aim of this study was to establish a generalized screening for Lynch syndrome and constitutional MMR deficiency (CMMRD) in pedHGG patients, as the detection of MMR deficiencies (MMRD) may enable the upfront therapeutic use of checkpoint inhibitors and identification of variant carriers in the patients’ families. We prospectively enrolled 155 centrally reviewed primary pedHGG patients for MMR-immunohistochemistry (IHC) as part of the HIT-HGG-2013 trial protocol. MMR-IHC results were subsequently compared to independently collected germline sequencing data (whole exome sequencing or pan-cancer DNA panel next-generation sequencing) available in the HIT-HGG-2013, INFORM, and MNP2.0 trials. MMR-IHC could be successfully performed in 127/155 tumor tissues. The screening identified all present cases with Lynch syndrome or CMMRD (5.5%). In addition, MMR-IHC also detected cases with exclusive somatic MMR gene alterations (2.3%), including MSH2 hypermethylation as an alternative epigenetic silencing mechanism. Most of the identified pedHGG MMRD patients had no family history of MMRD, and thus, they represented index patients in their families. Cases with regular protein expression in MMR-IHC never showed evidence for MMRD in DNA sequencing. In conclusion, MMR-IHC presents a cost-effective, relatively widely available, and fast screening method for germline MMRD in pedHGG with high sensitivity (100%) and specificity (96%). Given the relatively high prevalence of previously undetected MMRD cases among pedHGG patients, we strongly recommend incorporating MMR-IHC into routine diagnostics.

Rapid On-Site and Sensitive Detection of Microplastics Using Zirconium(IV)-Assisted SERS Label.

Microplastics have emerged as significant pollutants in terrestrial and marine ecosystems, with their accumulation posing a threat to human health through biomagnification along the food chain. Developing a rapid, on-site, and sensitive method for detecting microplastics in agri-food and environmental systems is important for assessing and minimizing their potential risks. In this study, we developed a novel surface-enhanced Raman spectroscopy (SERS) technique for the rapid, on-site, and ultrasensitive detection of microplastics. Our innovative technique incorporated Zr4+-assisted SERS label strategies, utilizing rhodamine B as a Raman reporter to improve microplastics analysis. By utilizing Zr4+-assisted SERS label approaches, we can achieve qualitative and ultrasensitive quantification of 10 μm polystyrene microplastics (PSMPs) at concentrations as low as 0.1 ppm with a detection limit of 1 ppb. Furthermore, this approach allows for detecting microplastics in real-world scenarios, with recovery rates exceeding 90% for polystyrene microplastic concentrations ranging from 5 to 30 ppm in tap water systems. When integrated with a portable Raman spectrometer, this innovative approach showcases the rapid, on-site, accurate, and sensitive detection of microplastics and has great potential for analyzing various types of microplastics in agri-food and environmental systems.

Research advance of solid-phase microextraction based on covalent organic framework materials

Solid-phase microextraction (SPME) is a fast and simple sample preparation technique that enables the enrichment of analytes, and it is used in combination with other detection techniques to provide accurate and sensitive analytical methods. SPME is widely used in environmental monitoring, food safety, life analysis, biomedicine, and other applications. The extractive coating is the core of the SPME technique, and the properties of the extractive coating greatly influence extraction selectivity and efficiency, as well as the enrichment effect. Therefore, the development of new and efficient extractive coating materials remains a hot topic in the analytical chemistry and sample preparation fields. Covalent organic frameworks (COFs) are a kind of porous crystalline network polymer materials formed by covalent bonds. Owing to the advantages of large specific surface area, high porosity, good stability, high designability, simple synthesis and post-modification, etc., it has been widely used in gas adsorption, catalysis, sensing and drug delivery. In recent years, COFs have attracted much attention in the field of sample preparation. A variety of novel COF-based SPME materials had been developed for extracting and enriching various types of analytes through π-π interaction, hydrophilic/hydrophobic interaction, electrostatic adsorption, and hydrogen-bonding, as well as pore effects. In this paper, the research advances of COFs for using in fiber-, in tube-, and membrane-based SPME over the past three years were discussed. Fiber surfaces had been modified with functionalized COFs or COF-hybrid materials for use in SPME through physical coating, in-situ growth, and chemical-bonding approaches. The combination of SPME fiber and chromatographic analysis can be used to detect a variety of analytes such as polycyclic aromatic hydrocarbons, phthalates, polychlorinated biphenyls, and pesticides in environmental and food samples, with good enrichment effects, wide linear ranges, and high sensitivity. Based on in tube-SPME, COFs-based monolithic column and fiber-filled tube as the extraction tubes were combined with high performance liquid chromatography online to develop highly sensitive detection methods for synthetic phenolic antioxidants and bisphenol compounds, respectively. In addition, COFs had also been used in membrane SPME technique, showing high efficiency in the extraction of trace polychlorinated biphenyls in environmental water. Finally, the development trends of COFs in the field of SPME was prospected.

Applying Shapley Effect for Sensitivity Analysis During Reactor Transient

Sensitivity analysis is a critical tool in reactor safety assessments, as it evaluates the impact of uncertainties in input parameters, identifies key factors, and highlights potential safety risks and measures. Conventional sensitivity methods, such as Spearman, Pearson, or Kendall, while straightforward, are typically limited to linear relationships and independent input parameters. Shapley values offer a more advanced, model-agnostic approach to sensitivity analysis, making them particularly valuable in scenarios with dependent parameters or nonlinear systems. This study not only applies variance-based sensitivity methods, including Sobol indices and Shapley values, but also introduces the development of a reduced-order model (ROM) based on deep neural networks (DNNs) combined with Shapley values for time-dependent reactor simulations. This approach addresses the computational challenges of traditional methods, especially in cases involving correlated parameters, providing a more efficient and accurate sensitivity analysis. Sensitivity indices are calculated for the TWIGL benchmark, with two-group cross sections as the input parameters and core power during the ramp reactivity insertion transient as the output. The results demonstrate that Shapley values, combined with the DNN-based ROM, yield robust, accurate, and physically meaningful indices, especially in models with dependent parameters where Sobol indices may lead to over- or underestimation and might even result in negative indices. This highlights the advantages of Shapley values for comprehensive and reliable sensitivity analyses in complex reactor simulations.

Determination of glufosinate, glyphosate and their metabolites in sediment by ultra-high performance liquid chromatography-tandem mass spectrometry with pass-through solid-phase extraction

Glufosinate (GLUF) and glyphosate (GLY) are nonselective phosphorus-containing amino acid herbicides that are widely used in agricultural gardens and noncultivated areas. These herbicides give rise to a number of key metabolites, with 3-methyl phosphinicopropionic acid (MPPA), N-acetyl glufosinate (N-acetyl GLUF), aminomethyl phosphonic acid (AMPA), N-acetyl aminomethyl phosphonic acid (N-acetyl AMPA), N-acetyl glyphosate (N-acetyl GLY), N-methyl glyphosate (N-methyl GLY) as the major metabolites obtained from GLUF and GLY. Extensive use of these herbicides may lead to their increased presence in the environment, especially aquatic ecosystems. An increasing number of research studies into the toxicities of GLUF, GLY, and their metabolites have shown that these herbicides are potentially toxic to aquatic biota. GLUF and GLY, as well as their metabolites, are extremely polar and water-soluble, and they lack chromogenic and fluorescent groups; therefore, their concentrations are difficult to determine using conventional methods. Most analytical methods used to date have largely depended on derivatization procedures, leading to overall determination processes that are tedious and time-consuming. Therefore, establishing a quick and sensitive method that does not require derivatives for determining GLUF, GLY, and their metabolites in water environments, including surface water, sediments, and aquatic organisms, is an important endeavor. In this study, a new approach was developed based on pass-through solid-phase extraction coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to determine GLUF, GLY, and their metabolites, including MPPA, N-acetyl GLUF, AMPA, N-acetyl AMPA, N-acetyl GLY, and N-methyl GLY, in sediments. Samples were extracted with 4% (v/v) ammonia water and purified using PRiME HLB pass-through solid-phase extraction columns. The extracts were filtered through a polyethersulfone microfiltration membrane and analyzed by UHPLC-MS/MS. Compounds were separated on a Metrosep A Supp 5 column (150 mm×4.0 mm, 5 μm) using gradient elution with water and 200 mmol/L ammonium hydrogen carbonate solution containing 0.05% (v/v) ammonia water as the mobile phases. Analytes were detected using MS/MS with a negative electrospray ionization (ESI-) source in the multiple reaction monitoring (MRM) mode. A matrix-matched external-standard approach was used for quantitative analysis. GLUF, GLY, and their metabolites were detected within 15 min with good peak shapes and high responses. Calibration curves were linear in the range of 2.0-200 μg/L, with correlation coefficients exceeding 0.995. This method delivered limits of detection (LODs) and limits of quantification (LOQs) of 5 μg/kg and 20 μg/kg, respectively, for GLUF, MPPA, N-acetyl-GLUF, N-acetyl AMPA, N-acetyl GLY, and N-methyl GLY, and 10 μg/kg and 30 μg/kg, for GLY and AMPA, respectively. Average spiked recoveries at three levels (LOQ, 5LOQ, 10LOQ) in sediment with low organic matter content were in the range of 78.5%-107%, and the relative standard deviations (RSDs) were in the range of 1.32%-14.7% (n=6). Average spiked recoveries of 76.4%-113% were determined for three levels (LOQ, 5LOQ, and 10LOQ) in sediments with high organic matter contents, with RSDs of 2.60%-11.2% (n=6). The developed method was used to analyze GLUF, GLY, and their metabolites in ponds, lakes, reservoirs, and river sediments. No target compounds were detected in any sediment sample obtained from a lake, reservoir, or river; however, GLY and AMPA were detected in one pond-sediment sample at levels of 31.7 and 52.3 μg/kg, respectively. The developed approach is simple, fast, and green; moreover, it offers advantages, including high accuracies, high sensitivities, and good reproducibilities. Accordingly, the developed method is suitable for determining GLUF, GLY, and their metabolites in sediments and can provide technical support for studying residue characteristics and environmental behavior in sediments.

A sensitive geometric self-calibration method and stability analysis for multiview spaceborne SAR images based on the range-Doppler model

A sensitive geometric self-calibration method and stability analysis for multiview spaceborne SAR images based on the range-Doppler model

Development and validation of a rapid and accurate ultra performance liquid chromatography-photodiode array method for concurrent quantification of thirty-two polyphenols in edible fruit of Cordia myxa Linn.

The aim of current work was to develop a novel, simple, sensitive, and reliable method for screening and quantification of thirty-two polyphenol compounds from Cordia myxa (C. myxa) using Ultra Performance Liquid Chromatography Photodiode Array detector (UPLC-PDA). With the help of the quaternary solvent manager and a comparison study of seven different columns packed with silica particles that are less than two micron thick (1.8-2.5 μm), the separation was completed in <35 min. We successfully tested the newly created method, demonstrating its sufficient sensitivity within the standard range of 0.13-0.44 μg/mL for LOD and 0.53-1.67 μg/mL for LOQ. We proved the method's accuracy with 99.2-105.0 % of recovery analysis and demonstrated its precision with inter-day RSD (0.02-0.65 %) and intra-day RSD (0.07-0.72 %). All the regression equations show a favourable linear relationship (R2 = 0.9970 - 0.9997). The fully validated method was further applied for the quantification of polyphenols in the C. myxa. The result showed that the butanol fraction of ripen fruit extract contains higher phenolics and flavonoids, followed by the chloroform fraction of unripen fruit extract; on the other hand, the hexane fraction contains the least number of polyphenols. Among the identified phenolics and flavonoids, rosmarinic acid was abundantly present in all the extracts and fractions (1.97-187.15 µg/10 mg). Therefore, it appears that this new UPLC-PDA method is a useful tool for the simultaneous determination of selected 32 polyphenols in medicinal and aromatic plants, and it may find use in industrial applications in the future.

Highly Sensitive Wideband Measurement Method of DC Micro-Discharges Based on Optimized Subdivided Electrode System

Highly Sensitive Wideband Measurement Method of DC Micro-Discharges Based on Optimized Subdivided Electrode System

Rapid and facile detection of Mycoplasma pneumoniae 16S rRNA based on CHA-FICA system.

Mycoplasma pneumoniae (MP) is a major a rapid, sensitive and reliable method for the detection of 16S ribosomal RNA (16S rRNA) was developed based on catalytic hairpin assembly (CHA) technology and digoxine-biotin double-labeled fluorescence immunochromatography (FICA) strip. The signal amplification achieved by CHA avoids the dependence on expensive thermal cycling equipment. The method can obtain the results within 1h in the absence of an enzyme, and the minimum detection limit can reach 1pM. It has good specificity and can effectively distinguish target sequences from non-target sequences. After verification with adult samples and pediatric samples, it was found that CHA-FICA detection was highly consistent with those of PCR detection, indicating that it has clinical application potential and is expected to be used in clinical screening and treatment detection of mycoplasma pneumoniae (MP) infection.

Signal-off colorimetric and signal-on fluorometric dual-mode aptasensor for ultrasensitive detection of Salmonella Typhimurium using graphitic carbon nitride.

Food safety is severely burdened by the prevalence of foodborne pathogens and the diseases they cause, necessitating the development of rapid, easy-to-use, highly sensitive, and reliable detection methods. Here, a signal-off colorimetric and signal-on fluorometric dual-mode detection method for Salmonella Typhimurium (S. typhimurium) was developed based on its unique interaction with aptamer DNA and graphitic carbon nitride (GCN). In the absence of a target Salmonella species, 6-carboxyfluorescein (FAM)-labeled aptamers are adsorbed on the surface of GCN primarily via a π-π interaction, resulting in reduced fluorescence of FAM through GCN-mediated quenching as well as improved peroxidase-like activity of GCN to generate intense blue color through facilitated electrostatic attraction between the negatively charged aptamer and positively charged 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The introduction of S. typhimurium to the sample solution causes the detachment of the aptamer from GCN due to its higher affinity for S. typhimurium than GCN, thereby rapidly reducing the colorimetric signal and recovering the fluorescence. We successfully determined the number of S. typhimurium using this method in a remarkably short duration (10-30min), highlighting its rapidity. The limit of detection values for S. typhimurium were as low as 8 and 3CFU/mL when using colorimetric and fluorometric methods, respectively. Moreover, this method can be used to detect S. typhimurium spiked in real vegetable extract and milk with high reproducibility and reliability. This method serves as a convenient route to the rapid, sensitive, selective, and reliable detection of pathogens from complex food samples, with the potential to replace conventional yet laborious methods currently in use.

Environmental DNA/RNA metabarcoding for noninvasive and comprehensive monitoring and assessment of marine fishes.

Marine fisheries resources are under increasing threat, necessitating the development of new effective monitoring and management strategies. Environmental DNA (eDNA) and RNA (eRNA) metabarcoding has emerged as a non-invasive and sensitive alternative method for monitoring fish biodiversity and fisheries resources and assessing the fisheries impact of anthropogenic activities. Here, we summarize crucial technical details about eDNA metabarcoding for marine fish monitoring and provide meta-analytical trends in primer selection and sample size, assessment standards, fish and fisheries databases, reference fish genomic databases, and other relevant metrics. The pressing need for better reference databases and standardization methods is discussed. We further highlight the potency of emerging eDNA metabarcoding studies for monitoring global fish diversity and revealed regional study hotspots in South China, Atlantic and Mediterranean Seas. The innovative advances in using eDNA/eRNA metabarcoding for fish diversity monitoring and assessment from the detection of rare or invasive species to branching applications in biomass estimation, population genetics, food web analysis, fish migration and feeding studies were reviewed. We also explore the potential of eRNA metabarcoding as an upcoming extension of eDNA metabarcoding in marine fish monitoring and assessment with improved functional relevance. We envision the integration of eDNA/eRNA metabarcoding-based fish monitoring methods with traditional monitoring approaches to significantly improve marine fish surveillance, ecological research, and conservation efforts.

Biosensors for the detection of celiac disease.

Celiac disease (CeD) is an autoimmune disorder triggered by sensitivity to gluten, a protein complex found in wheat, barley, and rye. Gliadins, a component of gluten, are proteins that trigger an immune response in individuals with CeD, primarily affecting the small intestine's inner lining. Despite a 1-1.5% prevalence, only 24% of cases are diagnosed due to non-specific symptoms. Screening is advised for high-risk groups, including first-degree relatives and type 1 diabetes patients. The accurate diagnosis of this condition and the assessment of the patient's response to the current treatment - a lifelong gluten-free diet - necessitate using dependable, swift, sensitive, specific, uncomplicated, and affordable analytical methods. Detecting CeD biomarkers in whole blood, serum, or plasma provides a non-invasive approach that serves as an ideal initial diagnostic step. Biosensors offer a novel and alternative way for CeD detection, began emerging in 2007, and hold promise for clinical and point-of-care applications. This review explores the use of biomarker-based diagnostic approaches for CeD, with a focus on biosensors. It delves into the progress of biosensors for CeD diagnosis, identifying trends and challenges in this evolving field. Key biomarkers are highlighted, offering insights into the evolving landscape of biosensors in CeD detection.

The materials synthesized in deep eutectic solvents for the detection of food contaminants

Abstract The present review delves into the burgeoning field of deep eutectic solvents (DESs) and their application in the synthesis of materials for detecting food contaminants. DESs, as a green alternative to conventional solvents, offer a sustainable platform for material synthesis with unique properties such as low toxicity, biodegradability, and excellent solvation capabilities. This review comprehensively discusses the classification, preparation methods, and properties of the materials synthesized in DESs that make them ideal for the development of sensitive and selective analytical methods for food safety. It covers the synthesis of various functional materials in DESs, including noble metal nanoparticles, metal oxides, conductive polymers, hydrogels, carbon-based materials, molecularly imprinted polymers, chalcogenide materials, and metal-organic frameworks. These materials have demonstrated enhanced stability, selectivity, and sensitivity in the detection of food contaminants such as heavy metals, pesticides, and mycotoxins. The review also highlights the challenges and future directions in the application of DES-based materials, emphasizing the need for innovative materials to address the growing demand for rapid, accurate, and cost-effective food contaminant detection methods. By showcasing the latest research and developments, this review underscores the potential of materials synthesized with DESs in revolutionizing food safety monitoring through the detection and monitoring of contaminants in food products.

Simultaneous detection of multiple food allergens using high signal-to-background SERS probes.

Avoiding exposure to food allergens remains the most reliable way to protect allergic individuals. Therefore, it is essential to develop selective, sensitive, and rapid methods for detecting food allergens. Herein, we introduce a novel SERS-based sandwich immunoassay that utilizes three distinct types of SERS detection probes: Ag@CA NPs, AgAu@PB NPs, and Ag@MB NPs, along with magnetic capture probes, to simultaneously detect almond, lactoglobulin, and gliadin allergens. These SERS probes generate unique Raman peaks at 1987cm-1, 2151cm-1, and 2223cm-1 in the Raman-silent region (1800-2800cm-1), effectively avoiding interference from the Raman-fingerprint region (400-1800cm-1) of potential food matrix substrates. This design ensures high signal-to-background ratios and detection accuracy, achieving limits of detection (LODs) of 7.4pg/mL for almonds, 66pg/mL for lactoglobulin, and 0.36pg/mL for gliadin, with corresponding recoveries ranging from 83.7% to 118.8%, 98.9% to 112.2%, and 91.9% to 109.5%, respectively, demonstrating satisfactory analytical performance.

Development and Efficacy Evaluation of a Novel Nanoparticle-Based Hemagglutination Inhibition Assay for Serological Studies of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus (PEDV) is a major pathogen that causes serious economic losses to the swine industry. To aid PEDV clinical diagnosis and vaccine development, sensitive and precise serological methods are demanded for rapid detection of (neutralizing) antibodies. Aiming for the development of a novel virus-free hemagglutination inhibition (HI) assay, the N-terminal region of the PEDV S1 subunit, encompassing the sialic acid-binding motif, was first expressed as an Fc-fusion protein with a C-terminal Spy Tag (S10A-Spy). The S10A-Spy protein was then presented on SpyCatcher-mi3 nanoparticles, forming virus-like particles designated S10A-NPs. Electron microscopy and dynamic light scattering analysis confirmed its topology, and the hemagglutination assay showed that S10A-NPs can efficiently agglutinate red blood cells. The HI assay based on S10A-NPs was then validated with PEDV-positive and -negative samples. The results showed that the HI assay had high specificity for the detection of PEDV antibodies. Next, a total of 253 clinical serum samples were subjected to the HI testing along with virus neutralization (VN) assay. The area under the receiver operating characteristic curve with VN was 0.959, and the kappa value was 0.759. Statistical analysis of the results indicated that the HI titers of the samples tested exhibited high consistency with the VN titers. Taken together, a novel virus-free HI assay based on the multivalent display of a chimeric PEDV spike protein upon self-assembling nanoparticles was established, providing a new approach for PEDV serological diagnosis.

Evaluation of a next generation sequencing assay for Hepatitis B antiviral drug resistance on the oxford nanopore system.

Next-generation sequencing (NGS) for Hepatitis B virus (HBV) antiviral resistance (AVR) testing is a highly sensitive diagnostic method, able to detect low-level mutant subpopulations. Our clinical virology laboratory previously transitioned from DNA hybridization (INNO-LiPA) to NGS, initially with the GS Junior System and subsequently the MiSeq. The Oxford Nanopore Technology (ONT) sequencing system was evaluated for HBV resistance testing, with regards to sequencing accuracy and turn-around time. We performed amplicon sequencing of the HBV polymerase gene from patient plasma and external quality assessment (EQA) samples on the MiSeq Reagent Nano Kit v2 and GridION ONT with R10.4.1 flowcells. Mutational analysis and genotyping were performed by DeepChek®Assay-HBV (version 2.0). A total of 49 patient samples and 15 EQA samples were tested on both the MiSeq and ONT. There was high agreement for both patient and EQA samples between the MiSeq and ONT systems, with regards to total drug resistance mutations detected and total patient sample agreement, 68/70 (97 %) and 47/49 (96 %), respectively. The ONT NGS platform provided accurate HBV AVR results, with improved turn-around times. Sequencing error rates at AVR codons were below 1 %.