A Novel Electrochemical Sensor Based on MnO2/Sepiolite Nanocomposite for the Detection of Hydrogen Peroxide in Human Serum Samples

Background: The reliable and easy-to-operate detection of hydrogen peroxide (H2O2) has attracted extensive attention in the fields of biomedicine, food security, and environmental analysis. Objectives: In this work, a novel electrochemical method was proposed for H2O2 monitoring using a carbon paste electrode (CPE) modified with MnO2/sepiolite nanocomposite. Methods: MnO2/sepiolite material was characterized by transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDS), and X-ray diffraction (XRD) technique. The modified CPE was employed for the amperometric monitoring of H2O2 in human serum samples. Results: Electrochemical data showed that the MnO2/sepiolite-CPE displays a high peak current towards H2O2 oxidation. A linear range from 5 to 700 μM and a low detection limit of 0.8 μM for H2O2 were obtained with the proposed sensor. Besides, the electrode depicted excellent reproducibility and anti-interferant ability, promising the applicability of this electrochemical method in practical analyses. Conclusions: This work introduced a new and effective enzyme-less H2O2 sensor based on the MnO2/sepiolite nanocomposite modified CPE. The suggested sensor showed good sensitivity for the rapid detection of H2O2 in a wide linear range with a low detection limit and satisfactory reproducibility, which made it practical for the analysis of hydrogen H2O2 in real samples.

Novel Method Development for Extraction and Analysis of Pesticide Residues in Human Serum Samples

In recent years, exposure to pesticides has gained widespread attention due to their adverse health effects. Long-term exposure to pesticides has shown hazardous effects on vital functions of the human nervous and reproductive systems. Therefore, it is crucial to determine the extent of pesticide exposure in humans. Primarily, it is quite challenging to determine trace levels of pesticide residues in biological matrices. Hence, a quick, multi-residue extraction procedure was experimented for pesticide residue analysis in human serum. Herein, the original QuEChERS extraction method was modified for achieving the best possible recoveries. A total of 15 representative pesticides from each class were selected and fortified into the human serum samples. The extraction was performed by employing acidified solution containing acetonitrile and ethyl acetate followed by vortex and centrifugation. The obtained aqueous layer was collected and vapourised to dryness and d-SPE clean-up was conducted utilising PSA. The extracted sample was injected into the GC-MS/MS system under MRM mode. The method development parameters such as linearity, % RSD, accuracy, LOD, LOQ and % ME were assessed. The results obtained for the serum matrix were found to be within the criteria mentioned in European Union SANTE/12682/2019 guidelines for method validation. The developed solitary method is quick, simple and highly efficient for routine pesticide residue analysis. Hence, a wide spectrum of pesticides can be analysed utilising the proposed method for human serum.

Development of Array-Based Gold Nanoclusters for Discrimination of CA125 Overexpressed Serum Samples

Here, we reported a simple method for recognizing CA125 overexpressed serum samples using array-based fluorescent gold nanoclusters (AuNCs). The sensing array was fabricated by the combination of various CA125 aptamer functionalized AuNCs. The fluorescence of different aptamer-capped AuNCs was quenched to a different extent in the presence of CA125. By comprehensive analysis of these fluorescence changes, unique patterns were formed when CA125 was overexpressed in serum samples. This strategy is successfully used for discriminating CA125 overexpressed human serum samples, which have great importance for the diagnosis of ovarian cancer in the early stage.

A method to reduce ELISA serial dilution assay workload applied to SARS-CoV-2 and seasonal HCoVs

Objectives Assays using ELISA measurements on serially diluted serum samples have been heavily used to measure serum reactivity to SARS-CoV-2 antigens and are widely used in virology and elsewhere in biology. We test a method to reduce the workload of these assays, and measure reactivity of SARS-CoV-2 and HCoV antigens to human serum samples collected before and during the COVID-19 pandemic. Methods We apply Bayesian hierarchical modelling to ELISA measurements of human serum samples against SARS-CoV-2 and HCoV antigens. Results Inflection titers for SARS-CoV-2 full-length spike protein (S1S2), spike protein receptor-binding domain (RBD), and nucleoprotein (N) inferred from three spread-out dilutions correlated with those inferred from eight consecutive dilutions with an R2 value of 0.97 or higher. We confirm existing findings showing a small proportion of pre-pandemic human serum samples contain cross-reactive antibodies to SARS-CoV-2 S1S2 and N, and that SARS-CoV-2 infection increases serum reactivity to the beta-HCoVs OC43 and HKU1 S1S2. Conclusions In serial dilution assays, large savings in resources and/or increases in throughput can be achieved by reducing the number of dilutions measured and using Bayesian hierarchical modelling to infer inflection or endpoint titers. We have released software for conducting these types of analysis.

A Paper-based Immunofluorescent Device for the Detection of SARS-CoV-2 Humanized Antibody

We report on an immunofluorescent paper-based assay for the detection of severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper-based device was fabricated by using lamination technique for easy and optimized handling. Our approach utilises a two-step strategy that involves (i) initial coating of the paper-electrode with recombinant SARS-CoV-2 nucleocapsid antigen to capture the target SARS-CoV-2 specific antibodies, and (ii) subsequent detection of SARS-CoV-2 antibodies using fluorophore-conjugated IgG antibody. The fluorescence readout was observed with fluorescence microscopy. The images were processed and quantified using a MATLAB program. The assay can selectively detect SARS-CoV-2 humanized antibodies spiked in PBS and healthy human serum samples with the relative standard deviation of approximately 6.4% (for n = 3). It has broad dynamic ranges (1 ng to 50 ng/µL in PBS and 5 to 100 ng/µL in human serum samples) for SARS-CoV-2 humanized antibodies with the detection limits of 2 ng/µL (0.025 IU/mL) and 10 ng/µL (0.125 IU/mL) in PBS and human serum samples, respectively. We believe that our assay has the potential to be used as a simple, rapid, and inexpensive paper-based diagnostic device with a portable fluorescent reader to provide point-of-care diagnosis. This assay can be used for rapid examination of a large batch of samples toward clinical screening of SARS-CoV-2 specific antibodies as a confirmed infected active case or to evaluate the immune response to a SARS-CoV-2 vaccine.

Single-step label-free nanowell immunoassay accurately quantifies serum stress hormones within minutes

A non-faradaic label-free cortisol sensing platform is presented using a nanowell array design, in which the two probe electrodes are integrated within the nanowell structure. Rapid and low volume (≤5 μl) sensing was realized through functionalizing nanoscale volume wells with antibodies and monitoring the real-time binding events. A 28-well plate biochip was built on a glass substrate by sequential deposition, patterning, and etching steps to create a stack nanowell array sensor with an electrode gap of 40 nm. Sensor response for cortisol concentrations between 1 and 15 μg/dl in buffer solution was recorded, and a limit of detection of 0.5 μg/dl was achieved. Last, 65 human serum samples were collected to compare the response from human serum samples with results from the standard enzyme-linked immunosorbent assay (ELISA). These results confirm that nanowell array sensors could be a promising platform for point-of-care testing, where real-time, laboratory-quality diagnostic results are essential.

Simultaneous measurement of 13 circulating vitamin D3 and D2 mono and dihydroxy metabolites using liquid chromatography mass spectrometry

Objectives Clinical evaluation of vitamin D status is conventionally performed by measuring serum levels of a single vitamin D metabolite, 25-hydroxyvitamin D predominantly by immunoassay methodology. However, this neglects the complex metabolic pathways involved in vitamin D bioactivity, including two canonical forms D3 and D2, bioactive 1,25-dihydroxy metabolites and inactive 24-hydroxy and other metabolites. Methods Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can measure multiple analytes in a sample during a single run with high sensitivity and reference level specificity. We therefore aimed to develop and validate a LC-MS/MS method to measure simultaneously 13 circulating vitamin D metabolites and apply it to 103 human serum samples. Results The LC-MS/MS method using a Cookson-type derivatization reagent phenyl-1,2,4-triazoline-3,5-dione (PTAD) quantifies 13 vitamin D metabolites, including mono and dihydroxy-metabolites, as well as CYP11A1-derived D3 and D2 metabolites in a single run. The lower limit of quantitation was 12.5 pg/mL for 1,25(OH)2D3 with accuracy verified by analysis of National Institute of Standards and Technology (NIST) 972a standards. Quantification of seven metabolites (25(OH)D3, 25(OH)D2, 3-epi-25(OH)D3, 20(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3 and 1,20S(OH)2D3) was consistently achieved in human serum samples. Conclusions This profiling method can provide new insight into circulating vitamin D metabolite pathways forming the basis for improved understanding of the role of vitamin D in health and disease.