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.

Paper as smart support for bioreceptor immobilization in electrochemical paper-based devices.

The use of paper as a smart support in the field of electrochemical sensors has been largely improved over the last 15 years, driven by its outstanding features such as foldability and porosity, which enable the design of reagent and equipment-free multi-analysis devices. Furthermore, the easy surface engineering of paper has been used to immobilize different bioreceptors, through physical adsorption, covalent bonding, and electrochemical polymerization, boosting the fine customization of the analytical performances of paper-based biosensors. In this review, we focused on the strategies to engineer the surface of the paper for the immobilization of (bio)recognition elements (eg., enzymes, antibodies, DNA, molecularly imprinted polymers) with the overriding goal to develop accurate and reliable paper-based electrochemical biosensors. Furthermore, we highlighted how to take advantage of paper for designing smart configurations by integrating different analytical processes in an eco-designed analytical tool, starting from the immobilization of the (bio)receptor and the reagents, through a designed sample flow along the device, until the analyte detection.

Paper card-like electrochemical platform as a smart point-of-care device for reagent-free glucose measurement in tears.

This communication describes the development of polyvinyl chloride electrochemical system in which a paper layer loaded with reagents is inserted into the device, demonstrating a new concept of a paper card-like pad for a reagent-free and easy measurement of the target analyte in solution. This device detects glucose in artificial tears in the range of 0.2-2 mM with a detection limit of 50 µM by simply adding the artificial tears to the paper card-like pad. The novel configuration goes beyond the state of the art, widening the application range of paper in the design of smart analytical devices.

Development of an optical immunoassay based on peroxidase-mimicking Prussian blue nanoparticles and a label-free electrochemical immunosensor for accurate and sensitive quantification of milk species adulteration.

In contrast to reported enzyme-based immunoassays, an enzyme-free immunoassay (optical and electrochemical) is presented here for the first time that can be used as point-of-need detection bioplatforms of bovine IgG as goat milk adulterant. In the first format, Prussian blue nanoparticles (PBNPs) were used as antibody catalytic labels in a competitive colorimetric microplate immunoassay. Absorbance measurement was performed photometrically at 450 nm. After in-depth optimization, excellent sensitivity was achieved (0.01% cow/goat volume ratio), which is 100 times lower than the limit allowed by the European legislation (EL) (1% v/v), thanks to the high catalytic activity of PBNPs compared with natural peroxidase. Moreover, the antibody-PBNPs bioconjugates showed excellent stability over 4 weeks (> 94% of the initial response) confirming the successful anchoring of the antibodies to the surface of the PBNPs. On the other hand, a label-free voltammetric immunoassay for the detection of bovine IgG was developed. The sensing principle was based on the hindrance of charge transfer between ferri-ferrocyanide redox couple and the screen-printed gold electrodes modified with bovine IgG antibody. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the step-by-step modification of the electrode surface. Under optimal conditions, this single-step electrochemical analysis achieved a high sensitivity of 0.1% (cow/goat) when monitoring the ferrocyanide oxidation at + 0.092 V (vs. Ag/AgCl) using differential pulse voltammetry (DPV). The selectivity of the developed immunoassays was evaluated for different species of milk of similar composition, and both immunoassays exhibited a selective response only to bovine IgG. Unlike conventional immunoassays, the developed enzyme-free immunoassays have many attractive features for the detection of milk adulteration, whether they are used in quality control laboratories for routine milk analysis (optical immunoassay) or at on-site checkpoints (electrochemical immunoassay) using wireless electrochemical detectors. The sensors provide high sensitivity (≤ 0.1%), excellent precision (RSD < 6%), low cost (no enzyme is required) and ease of operation, including handling of milk samples.

Smartphone-based competitive immunoassay for quantitative on-site detection of meat adulteration.

Rapid, sensitive, and portable analytical methods for on-site inspection of food fraud are now an urgent requirement to ensure food quality and satisfy the ethnic considerations of consumers. Hence, for the first time, a colorimetric smartphone-based immunoassay was developed for the on-site detection of pork adulteration in meat. In detail, the immunoassay was based on a competitive strategy in which immobilized standard porcine IgG competed with the target porcine IgG extracted in a single step from meat samples. The parameters involved in each step of the immunoassay conception and the digital colorimetric detection were carefully investigated and optimized. Using polystyrene microplates as ready-to-use stable and portable immunoplatforms, TMB as chromogenic substrate, smartphone as signal readout, and Image J software for image processing; the developed immunoassay was able to detect as low as 0.01% of pork in meat mixtures in a total assay time of 30 min. The selectivity of the immunoassay was evaluated for different meat species, and it was shown to selectively respond only to pork. Furthermore, excellent stability of the prepared immunological platform was demonstrated under extreme temperature conditions (50 °C), which confirms its high portability potential for in situ quantification of pork, while being relatively cost effective and non-laborious. The developed method also provides great precision (RSD < 6%) and accuracy (relative error< 6%). Given the universal use of smartphones as portable and affordable devices, such format of immunoassay could be a promising approach for rapid and sensitive real-time monitoring of food fraud.

Enzyme immunoassay (ELISA/immunosensor) for a sensitive detection of pork adulteration in meat.

ELISA/immunosensor were developed for a sensitive detection of pork adulteration in meat. Two formats of ELISA were performed. First, an extracted IgG was directly immobilized in the microplate. This assay allowed an identification of 0.01% as level of pork adulteration in 14 h15 min. In order to decrease the time of the assay, a competitive ELISA was developed by immobilizing IgG standard, which compete with the extracted IgG. This assay allowed a detection of 0.1% of pork adulteration in 45 min. Furthermore, two formats of electrochemical immunosensors were elaborated using the electro-entrapment of IgG in polymer modified graphite paste electrode. First, a direct immunosensor was capable of identifying 0.1 and 1% in raw and cooked meat respectively in 2 h. The second format was based on a competitive immunosensor, which was able to detect 0.01% of pork adulteration within 20 min. Both competitive immunoassays revealed high sensitivity, good specify and reduced analysis time.