Functionalized N95 Face Mask with a Chemical-Free Paper-Based Collector for Exhaled Breath Analysis: SARS-CoV-2 Detection with a Printed Immunosensor as a Case Study.

Exhaled breath electrochemical sensing is a promising biomedical technology owing to its portability, painlessness, cost-effectiveness, and user-friendliness. Here, we present a novel approach for target analysis in exhaled breath by integrating a comfortable paper-based collector into an N95 face mask, providing a universal solution for analyzing several biomarkers. As a model analyte, we detected SARS-CoV-2 spike protein from the exhaled breath by sampling the target analyte into the collector, followed by its detection out of the N95 face mask using a magnetic bead-based electrochemical immunosensor. This approach was designed to avoid any contact between humans and the chemicals. To simulate human exhaled breath, untreated saliva samples were nebulized on the paper collector, revealing a detection limit of 1 ng/mL and a wide linear range of 3.7-10,000 ng/mL. Additionally, the developed immunosensor exhibited high selectivity toward the SARS-CoV-2 spike protein, compared to other airborne microorganisms, and the SARS-CoV-2 nucleocapsid protein. Accuracy assessments were conducted by analyzing the simulated breath samples spiked with varying concentrations of SARS-CoV-2 spike protein, resulting in satisfactory recovery values (ranging from 97 ± 4 to 118 ± 1%). Finally, the paper-based hybrid immunosensor was successfully applied for the detection of SARS-CoV-2 in real human exhaled breath samples. The position of the collector in the N95 mask was evaluated as well as the ability of this paper-based analytical tool to identify the positive patient.

Smartphone-assisted paper-based electrochemical immunosensor for SARS-CoV-2 detection in saliva.

Herein, we developed a new waste solution-free paper-based electrochemical immunosensor for SARS-CoV-2 detection in saliva, by combining vertical and lateral flow. In detail, the device was constituted of a reservoir containing all reagents for the construction of the immunological chain onto the magnetic beads and a lateral flow holder which contained a polyester-based electrode, a magnet, and an adsorbent pad. The measurement was carried out by adding the saliva sample into the reservoir, followed by the addition of this solution in the hole present in the lateral flow holder. The successive additions of washing buffer and TMB solution in the lateral flow holder allowed the detection of N protein in saliva in the range of 0.06 to 4 µg/mL with a detection limit equal to 30 ng/mL. The analysis of several saliva samples with the sensing tool and the reference method, demonstrated the effectiveness of this device, being able to identify positive patients with high values of CT e.g. 35. This new configuration paves the way for the realization of any magnetic beads-based immunosystem without waste solution production, enlarging the application of paper-based devices.

Nanomaterials and paper-based electrochemical devices: merging strategies for fostering sustainable detection of biomarkers.

In the last few decades, nanomaterials have made great advances in the biosensor field, thanks to their ability to enhance several key issues of biosensing analytical tools, namely, sensitivity, selectivity, robustness, and reproducibility. The recent trend of sustainability has boosted the progress of novel and eco-designed electrochemical paper-based devices to detect easily the target analyte(s) with high sensitivity in complex matrices. The huge attention given by the scientific community and industrial sectors to paper-based devices is ascribed to the numerous advantages of these cost-effective analytical tools, including the absence of external equipment for solution flow, thanks to the capillary force of paper, the fabrication of reagent-free devices, because of the loading of reagents on the paper, and the easy multistep analyses by using the origami approach. Besides these features, herein we highlight the multifarious aspects of the nanomaterials such as (i) the significant enlargement of the electroactive surface area as well as the area available for the desired chemical interactions, (ii) the capability of anchoring biorecognition elements on the electrode surface on the paper matrix, (iii) the improvement of the conductivity of the cellulose matrix, (iv) the functionality of photoelectrochemical properties within the cellulose matrix, and (v) the improvement of electrochemical capabilities of conductive inks commonly used for electrode printing on the paper support, for the development of a new generation of paper-based electrochemical biosensors applied in the biomedical field. The state of the art over the last ten years has been analyzed highlighting the various functionalities that arise from the integration of nanomaterials with paper-based electrochemical biosensors for the detection of biomarkers.

Paper-based immunoassay based on 96-well wax-printed paper plate combined with magnetic beads and colorimetric smartphone-assisted measure for reliable detection of SARS-CoV-2 in saliva.

Coronavirus disease 2019 (COVID-19) has been recognized as a global pandemic outbreak, opening the most severe socio-economic crisis since World War II. Different scientific activities have been emerged in this global scenario, including the development of innovative analytical tools to measure nucleic acid, antibodies, and antigens in the nasopharyngeal swab, serum, and saliva for prompt identification of COVID-19 patients and to evaluate the immune response to the vaccine. The detection of SARS-CoV-2 in saliva remains a challenge for the lack of sufficient sensitivity. To address this issue, we developed a novel paper-based immunoassay using magnetic beads to support the immunological chain and 96-well wax-printed paper plate as a platform for color visualization by using a smartphone combined with Spotxel free-charge app. To assess the reliability of the measurement of SARS-CoV-2 in saliva, untreated saliva was used as a specimen and the calibration curve demonstrated a dynamic range up to 10 µg/mL, with a detection limit equal to 0.1 µg/mL. The effectiveness of this sustainable analytical tool in saliva was evaluated by comparing the data with the nasopharyngeal swab specimens sampled by the same patients and tested with Real-Time PCR reference method, founding 100% of agreement, even in the case of high Cycle Threshold (CT) numbers (low viral load). Furthermore, the positive saliva samples were characterized by the next-generation sequencing method, demonstrating the capability to detect the Delta variant, which is actually (July 2021) the most relevant variant of concern.

State of the Art on the SARS-CoV-2 Toolkit for Antigen Detection: One Year Later.

The recent global events of COVID-19 in 2020 have alerted the world to the risk of viruses and their impacts on human health, including their impacts in the social and economic sectors. Rapid tests are urgently required to enable antigen detection and thus to facilitate rapid and simple evaluations of contagious individuals, with the overriding goal to delimitate spread of the virus among the population. Many efforts have been achieved in recent months through the realization of novel diagnostic tools for rapid, affordable, and accurate analysis, thereby enabling prompt responses to the pandemic infection. This review reports the latest results on electrochemical and optical biosensors realized for the specific detection of SARS-CoV-2 antigens, thus providing an overview of the available diagnostics tested and marketed for SARS-CoV-2 antigens as well as their pros and cons.

Magnetic beads combined with carbon black-based screen-printed electrodes for COVID-19: A reliable and miniaturized electrochemical immunosensor for SARS-CoV-2 detection in saliva.

The diffusion of novel SARS-CoV-2 coronavirus over the world generated COVID-19 pandemic event as reported by World Health Organization on March 2020. The huge issue is the high infectivity and the absence of vaccine and customised drugs allowing for hard management of this outbreak, thus a rapid and on site analysis is a need to contain the spread of COVID-19. Herein, we developed an electrochemical immunoassay for rapid and smart detection of SARS-CoV-2 coronavirus in saliva. The electrochemical assay was conceived for Spike (S) protein or Nucleocapsid (N) protein detection using magnetic beads as support of immunological chain and secondary antibody with alkaline phosphatase as immunological label. The enzymatic by-product 1-naphtol was detected using screen-printed electrodes modified with carbon black nanomaterial. The analytical features of the electrochemical immunoassay were evaluated using the standard solution of S and N protein in buffer solution and untreated saliva with a detection limit equal to 19 ng/mL and 8 ng/mL in untreated saliva, respectively for S and N protein. Its effectiveness was assessed using cultured virus in biosafety level 3 and in saliva clinical samples comparing the data using the nasopharyngeal swab specimens tested with Real-Time PCR. The agreement of the data, the low detection limit achieved, the rapid analysis (30 min), the miniaturization, and portability of the instrument combined with the easiness to use and no-invasive sampling, confer to this analytical tool high potentiality for market entry as the first highly sensitive electrochemical immunoassay for SARS-CoV-2 detection in untreated saliva.

A challenge in biosensors: Is it better to measure a photon or an electron for ultrasensitive detection?

Biosensor development exploiting various transduction principles is characterized by a strong competition to reach high detectability, portability and robustness. Nevertheless, a literature-based comparison is not possible, as different conditions are employed in each paper. Herein, we aim at evaluating which measurement, photons or electrons, yields better biosensor performance. Upon outlining an update in recent achievements to boost analytical performance, amperometry and chemiluminescence (CL)-based biosensors are directly compared employing the same biospecific reagents and analytical formats. Horseradish peroxidase (HRP) and hydrogen peroxide concentrations were directly measured, while glucose and mouse IgG were detected employing an enzyme paper-based biosensor and an immunosensor, respectively. Detectability was down to picomoles of hydrogen peroxide (4 pmol for CL and 210 pmol for amperometry) and zeptomoles of HRP (45 zmol for CL and 20 zmol for amperometry); IgG was detected down to 12 fM (CL) and 120 fM (amperometry), while glucose down to 17 µM (CL) and 40 µM (amperometry). Results showed that amperometric and CL biosensors offered similar detectability and analytical performance, with some peculiarities that suggest complementary application fields. As they generally provided slightly higher detectability and wider dynamic ranges, CL-based biosensors appear more suitable for point-of-care testing of clinical biomarkers, where detectability is crucial. Nevertheless, as high detectability in CL biosensors usually requires longer acquisition times, their rapidity will allocate electrochemical biosensors in real-time monitoring and wearable biosensors. The analytical challenge demonstrated that these biosensors have competitive and similar performance, and between photons and electrons the competition is still open.

An electrochemical immunoassay for the screening of celiac disease in saliva samples.

A highly sensitive electrochemical immunoassay for the initial diagnosis of celiac disease (CD) in saliva samples that overcomes the problems related to its high viscosity and to the low concentration of anti-transglutaminase antigen (tTG) IgA in this medium has been developed for the first time. The system uses magnetic beads (MBs) covered with tTG, which reacts with the anti-tTG IgA antibodies present in positive saliva samples. An anti-human IgA, conjugated with alkaline phosphate (AP) enzyme, was used as the label and a strip of eight magnetized screen-printed electrodes as the electrochemical transducer. In particular, two different immunoassay approaches were optimized and blindly compared to analyze a large number of saliva samples, whose anti-tTG IgA levels were independently determined by the radioimmunoassay (RIA) method. The obtained results, expressed as Ab index, were used to perform a diagnostic test evaluation through the construction of receiver operating characteristic (ROC) curves. The approach, involving a pre-incubation between the anti-human IgA-AP and saliva samples prior to the addition of MBs-tTG, showed a cutoff of 0.022 with 95% clinical sensitivity and 96% clinical specificity. The area under the ROC curve is equal to 1, a result that classifies our test as "perfect." This study demonstrates that it is possible to perform the screening of CD with a rapid, simple, inexpensive, and sensitive method able to detect anti-tTG antibodies in saliva samples, which are easily obtained by non-invasive techniques. This aspect is of fundamental importance to screen a large number of subjects, especially in the pediatric age.