Paper-Based Biosensors for COVID-19: A Review of Innovative Tools for Controlling the Pandemic

Abstract

The appearance and quick spread of the new severe acute respiratory syndrome coronavirus disease, COVID-19, brought major societal challenges. Importantly, suitable medical diagnosis procedures and smooth clinical management of the disease are an emergent need, which must be anchored on novel diagnostic methods and devices. Novel molecular diagnostic tools relying on nucleic acid amplification testing have emerged globally and are the current gold standard in COVID-19 diagnosis. However, the need for widespread testing methodologies for fast, effective testing in multiple epidemiological scenarios remains a crucial step in the fight against the COVID-19 pandemic. Biosensors have previously shown the potential for cost-effective and accessible diagnostics, finding applications in settings where conventional, laboratorial techniques may not be readily employed. Paper- and cellulose-based biosensors can be particularly relevant in pandemic times, for the renewability, possibility of mass production with sustainable methodologies, and safe environmental disposal. In this review, paper-based devices and platforms targeting SARS-CoV-2 are showcased and discussed, as a means to achieve quick and low-cost PoC diagnosis, including detection methodologies for viral genomic material, viral antigen detection, and serological antibody testing. Devices targeting inflammatory markers relevant for COVID-19 are also discussed, as fast, reliable bedside diagnostic tools for patient treatment and follow-up.

Figure 1

(A)Targets for SARS-CoV-2 diagnosis include viral genomic material and specific viral antigens, namely, the spike and nucleocapsid proteins, as well as organism response markers to the presence of the virus, through antibody production and inflammatory response. Respiratory samples or serological samples are used for identification of these markers, with complementary laboratory techniques or alternative diagnostic tools and point of care devices, where paper-based alternatives present themselves as attractive, affordable and more accessible tools. (B) Statistics on number of publications related to biosensing developed for SARS-CoV-2 and COVID-19, retrieved from PubMed database (query: (COVID-19 OR SARS-CoV-2) AND biosensors). Data were retrieved from the date June 15th 2021.

Figure 2

Viral genetic material identification and diagnosis methods in paper-based formats. (A) Workflow of sample collection and treatment for subsequent detection methodologies for RNA-based diagnosis. (B) CRISPR-based SARS-CoV-2 RNA identification with paper-based, LFA signal readout. Adapted with permission from ref (69). Copyright 2020 Springer Nature. (C) Fluorescence LFA format device for amplification-free RNA detection. Adapted with permission from ref (73). Copyright 2020 Springer Nature. (D) RT-LAMP RNA detection in a microfluidics-based, multifunctional device, carrying multiple tasks of the molecular diagnosis assay. Adapted from ref (75), copyright Garneret et al. (E) Electrochemical, paper-based platform for amplification-free, viral genomic identification and diagnosis, using ssDNA probes and graphene/Au electrodes modified with conjugated AuNPS. Adapted with permission from ref (88). Copyright 2020 American Chemical Society.

Figure 3

SARS-CoV-2 spike antigen detection employing paper-based platforms. (A) Spike antigen detection in LFA format, employing ACE2 as capture molecule for flowing immunocomplexes. Adapted with permission from ref (96). Copyright 2021 Elsevier. (B) Spike antigen detection in LFA format, using the natural binding path of this antigen to sialic acid, removing the need for antibodies and immunoassay-based detection of this antigen. Adapted with permission from ref (97). Copyright 2020 American Chemical Society. (C) LFA device for spike antigen detection, employing nanozymes and chemiluminescence signaling, for portable reading using smartphone readout. Adapted with permission from ref (98). Copyright 2021 Elsevier.

Figure 4

SARS-CoV-2 nucleocapsid antigen detection or simultaneous spike and nucleocapsid antigen detection employing paper-based platforms. (A) Nucleocapsid antigen detection using LFA system built using fusion antibodies produced from cloned phages. Adapted with permission from ref (100). Copyright 2021 Elsevier (B) Spike and nucleocapsid simultaneous antigen detection using LFA system with up-converting nanoparticles and a 5G-enabled fluorescence sensor for IoT applications. Adapted with permission from ref (101). Copyright 2021 Elsevier. (C) High-throughput, proteomic detection of SARS-CoV-2 S and N antigen and their subunits, by printing of cloned phages expressed antibodies targeting the antigens, in nitrocellulose substrate. Adapted with permission from ref (102). Copyright 2020 Elsevier.

Figure 5

LFA test strip systems for detection of IgG and/or IGM antibodies. (A) Anti-nucleocapsid antigen IgG antibody detection employing lanthanide-doped polystyrene nanoparticles for highly sensitive luminescence signaling. Adapted with permission from ref (130). Copyright 2020 American Chemical Society. (B) Simultaneous detection of IgG and IgM antibodies using conjugated AuNPs-based immunoassay. Adapted with permission from ref (132). Copyright 2020 Wiley. (C) Simultaneous IgG and IgM antibodies against spike antigen, employing an alternative optical transduction method with SiO₂@Au@QD for fluorescence immunocomplex formation signaling. Adapted with permission from ref (133) Copyright 2020 American Chemical Society. (D) SERS-based LFA system for highly-sensitive anti-spike antigen IgG and IgM antibodies, employing SiO₂@Ag reporter for immunocomplex signal enhancement, down to pg/mL levels. Adapted with permission from ref (135). Copyright 2020 Elsevier.

Figure 6

Alternative paper-based assay formats for anti-SARS-CoV-2 antibody detection. (A) Fabrication and preparation process of ELISA paper-based device for SARS-CoV-2 humanized antibody. Adapted with permission from ref (136). Copyright 2020 Royal Society of Chemistry. (B) Paper-based electrode fabrication for electrochemical impedance spectroscopy detection of anti-spike antigen IgG antibodies. Adapted with permission from ref (138). Copyright 2020 Elsevier. (C) Origami-style, paper-based electrochemical biosensor for IgG and IgM anti-spike antigen antibodies detection and quantification. Adapted with permission from ref (120). Copyright 2021 Elsevier.

Figure 7

Paper-based biosensors for intereukin-6 detection. (A) Regenerated cellulose membranes used for concentration of the analyte applied towards sensitive electrochemical detection. Adapted with permission from ref (159). Copyright 2018 Royal Society of Chemistry. (B) LFA system for IL-6 detection at bedside, using gold-based immunoassay. Adapted from ref (160). Copyright 2020 Maples. (C) Paper-based device using a folding approach targeting IL-6 in COVID-19 patients for disease progression monitoring. Adapted with permission from ref (163). Copyright 2021 Elsevier.

Figure 8

Paper-based biosensors for C-reactive protein detection. (A) Paper-based electrochemical biosensor using AuNPs modified electrodes, using the affinity of CRP to Ca²⁺ for binding of the analyte and subsequent current transduction. Adapted with permission from ref (167). Copyright 2019 Springer Nature. (B) Vertical flow system for AuNPs-based immunoassay, replacing conventional horizontal flow. Adapted with permission from ref (170). Copyright 2013 Royal Society of Chemistry. (C) LFA system using an asymmetric polysulfone membrane for delayed release, paired with chemiluminescence signaling for improved sensitivity. Adapted with permission from ref (172). Copyright 2014 Elsevier.

Figure 9

Ferritin and PCT paper-based detection approaches. (A) Electrochemical detection of ferritin using paper-based electrodes using immunocomplex formation causing alterations in current signals correlated to the presence of ferritin. Adapted with permission from ref (177). Copyright 2020 Royal Society of Chemistry. (B) LFA system for PCT detection, using UCP conjugated with anti-PCT antibodies, for sensitive fluorescence detection. Adapted with permission from ref (182). Copyright 2017 Wiley.