Simultaneous Detection of SARS-CoV-2 Nucleoprotein and Receptor Binding Domain by a Multi-Area Reflectance Spectroscopy Sensor.

The COVID-19 pandemic has emphasized the urgent need for point-of-care methods suitable for the rapid and reliable diagnosis of viral infections. To address this demand, we report the rapid, label-free simultaneous determination of two SARS-CoV-2 proteins, namely, the nucleoprotein and the receptor binding domain peptide of S1 protein, by implementing a bioanalytical device based on Multi Area Reflectance Spectroscopy. Simultaneous detection of these two proteins is achieved by using silicon chips with adjacent areas of different silicon dioxide thickness on top, each of which is modified with an antibody specific to either the nucleoprotein or the receptor binding domain of SARS-CoV-2. Both areas were illuminated by a single probe that also collected the reflected light, directing it to a spectrometer. The online conversion of the combined reflection spectra from the two silicon dioxide areas into the respective adlayer thickness enabled real-time monitoring of immunoreactions taking place on the two areas. Several antibodies have been tested to define the pair, providing the higher specific signal following a non-competitive immunoassay format. Biotinylated secondary antibodies and streptavidin were used to enhance the specific signal. Both proteins were detected in less than 12 min, with detection limits of 1.0 ng/mL. The assays demonstrated high repeatability with intra- and inter-assay coefficients of variation lower than 10%. Moreover, the recovery of both proteins from spiked samples prepared in extraction buffer from a commercial self-test kit for SARS-CoV-2 collection from nasopharyngeal swabs ranged from 90.0 to 110%. The short assay duration in combination with the excellent analytical performance and the compact instrument size render the proposed device and assay suitable for point-of-care applications.

Integrated Plastic Microfluidic Device for Heavy Metal Ion Detection.

The presence of heavy metal ions in soil, air and water constitutes an important global environmental threat, as these ions accumulate throughout the food chain, contributing to the rise of chronic diseases, including, amongst others, cancer and kidney failure. To date, many efforts have been made for their detection, but there is still a need for the development of sensitive, low-cost, and portable devices able to conduct on-site detection of heavy metal ions. In this work, we combine microfluidic technology and electrochemical sensing in a plastic chip for the selective detection of heavy metal ions utilizing DNAzymes immobilized in between platinum nanoparticles (PtNPs), demonstrating a reliable portable solution for water pollution monitoring. For the realization of the microfluidic-based heavy metal ion detection device, a fast and easy-to-implement fabrication method based on the photolithography of dry photosensitive layers is proposed. As a proof of concept, we demonstrate the detection of Pb2+ ions using the prototype microfluidic device.

Simultaneous determination of procalcitonin and interleukin-6 in human serum samples with a point-of-care biosensing device.

The simultaneous determination of two inflammatory diseases biomarkers, namely procalcitonin (PCT) and interleukin-6 (IL-6), in human serum samples employing a Point-of-Care device based on Multi Area Reflectance Spectroscopy is presented. Dual-analyte detection was achieved using silicon chips with two silicon dioxide areas of different thickness, one functionalized with an antibody specific for PCT and the other with an antibody specific for IL-6. The assay included reaction of immobilized capture antibodies with mixtures of PCT and IL-6 calibrators with the biotinylated detection antibodies, streptavidin and biotinylated-BSA. The reader provided for the automated execution of the assay procedure, as well as for the collection and processing of the reflected light spectrum, the shift of which is correlated to analytes concentration in the sample. The assay was completed in 35 min and the detection limits for PCT and IL-6 were 2.0 and 0.01 ng/mL respectively. The dual-analyte assay was characterized by high reproducibility (the intra- and inter-assay coefficients of variation were less than 10% for both analytes) and accuracy (the percent recovery values ranged from 80 to 113% for both analytes). Moreover, the values determined for the two analytes in human serum samples with the assay developed were in good agreement with the values determined for the same samples by clinical laboratory methods. These results support the potential of the proposed biosensing device application for inflammatory biomarkers determination at the Point-of-Need.

AChE-based electrochemical biosensor for pesticide detection in vegetable oils: matrix effects and synergistic inhibition of the immobilized enzyme.

Enzyme-based electrochemical biosensors have been widely deployed for the detection of a range of contaminants in different food products due to their significant advantages over other (bio)sensing techniques. Nevertheless, their performance is greatly affected by the sample matrix itself or by the matrix they are presented with in pretreated samples, both of which can impact the accuracy as well as the sensitivity of the measurements. Therefore, and in order to acquire reliable and accurate measurements, matrix effects and their influence on sensor performance should be taken into consideration. Herein, acetylcholinesterase (AChE)-modified electrochemical sensors were employed for the detection of pesticides in vegetable oils. Sensor interrogation with pretreated oil samples, spiked with carbofuran, revealed the inhibitory potential of the extracted matrix varies between different types of vegetable oil and their fatty acid content. In addition, synergies between the extracted matrix from different types of vegetable oils and the carbamate pesticide, carbofuran, were observed, which led to significant deviations of the sensor's performance from its anticipated behavior in buffered solution. Taking the aforementioned into consideration, appropriate calibration curves for each type of vegetable oil were drafted, which allowed for the highly reproducible determination of different pesticide concentrations in pretreated real samples. Collectively, a better understanding of AChE inhibition by single or multiple contaminants present in vegetable oils was gained, which can find many applications in numerous fields, ranging from sensor development to the design of new pesticides and medicinal products.

Development of a Point-of-Care System Based on White Light Reflectance Spectroscopy: Application in CRP Determination.

The development of methods and miniaturized systems for fast and reliable quantitative determinations at the Point-of-Care is a top challenge and priority in diagnostics. In this work, a compact bench-top system, based on White Light Reflectance Spectroscopy, is introduced and evaluated in an application with high clinical interest, namely the determination of C-Reactive protein (CRP) in human blood samples. The system encompassed all the necessary electronic and optical components for the performance of the assay, while the dedicated software provided the sequence and duration of assay steps, the reagents flow rate, the real-time monitoring of sensor response, and data processing to deliver in short time and accurately the CPR concentration in the sample. The CRP assay included two steps, the first comprising the binding of sample CRP onto the chip immobilized capture antibody and the second the reaction of the surface immunosorbed CRP molecules with the detection antibody. The assay duration was 12 min and the dynamic range was from 0.05 to 200 µg/mL, covering both normal values and acute inflammation incidents. There was an excellent agreement between CRP values determined in human plasma samples using the developed device with those received for the same samples by a standard diagnostic laboratory method.