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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., The authors acknowledge funding through projects Eco2Covid (POCI-01-02B7-FEDER-068174) and TecniCov (POCI-01-02B7-FEDER-069745), co-funded by FEDER through COMPETE2020 and Lisboa2020. T.P., A.R.C. and A.C.M. acknowledge funding to National Foundation for Science and Technology, I.P., FCT, through their PhD grants, references DFA/BD/8606/2020, SFRH/BD/130107/2017 and SFRH/BD/115173/2016, respectively, info:eu-repo/semantics/publishedVersion

The plasmonic properties of gold nanoparticles (AuNPs) are a promising tool to develop sensing alternatives to traditional, enzyme-catalyzed reactions. The need for sensing alternatives, especially in underdeveloped areas of the world, has given rise to the application of nonenzymatic sensing approaches paired with cellulosic substrates to biochemical analysis. Herein, we present three individual, low-step, wet-chemistry, colorimetric assays for three target biomarkers, namely, glucose, uric acid, and free cholesterol, relevant in diabetes control and their translation into paper-based assays and microfluidic platforms for multiplexed analysis. For glucose determination, an in situ AuNPs synthesis approach was applied into the developed μPAD, giving semiquantitative measures in the physiologically relevant range. For uric acid and cholesterol determination, modified AuNPs were used to functionalize paper with a gold-on-paper approach with the optical properties changing based on different aggregation degrees and hydrophobic properties of particles dependent on analyte concentration. These paper-based assays show sensitivity ranges and limits of detection compatible for target analyte level determination and detection limits comparable to those of similar enzymatic, colorimetric systems, relying only on plasmonic transduction without the need for enzymatic activity or other chromogenic substrates. The resulting paper-based assays were integrated into a single 3D, multiplex paper-based device using paper microfluidics, showing the capability for performing different colorimetric assays with distinct requirements in terms of sample flow and sample uptake in test zones using a combination of both horizontal and vertical flows inside the same device. The presented device allows for multiparametric, colorimetric measures of different metabolite levels from a single complex sample matrix drop using digital color analysis, showing the potential for development of low-cost, low-complexity tools for diagnostics toward the point-of-care.

The development of accurate, reliable, inexpensive and fully recyclable analytical platforms is of utmost relevance to several fields from medical diagnosis to environmental screening. Surface-enhanced Raman spectroscopy (SERS) is a compelling detection method with high specificity and sensitivity. In this work, a microwave-assisted synthesis method was used for fast and uniform in situ growth of gold nanoparticles (AuNPs) onto nanocellulose (NC) membranes, through a seedmediated process. The as-prepared membranes were fully optimized and its application as SERS platforms was demonstrated. A direct comparison with other cellulose-based substrates showed the superior characteristics of NC such as high mechanical strength, high surface area and lower porous content. An Enhancement Factor (EF) up to 106 was obtained using rhodamine 6G (R6G) 10-6 M as probe molecule and a remarkable shelf life of at least 7 months was achieved, with no special storage required. Preliminary results on the detection of label-free spike protein present in SARSCoV-2 virus are shown, through direct measurements on the optimized SERS membrane. We believe that this work evidences the effectiveness of in situ seed-mediated microwave-assisted synthesis as a fabrication method, the high stability of AuNPs and the superior characteristics of NC substrates to be used as SERS platforms., This work is funded by National Funds through FCT I.P., under the scope of the project UIDB/50025/2020-2023. The authors acknowledge the ERC AdG project DIGISMART ref. 787410 and EC project SYNERGY H2020-WIDESPREAD-2020-5, CSA, proposal n° 952169. Also, the authors thank funding co-financed by the Operational Programme for Competitiveness and Internationalisation (COMPETE 2020) and Lisbon Regional Operational Programme (Lisboa 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) under the projects ECO2COVID ref. 68174 and TecniCOV ref. 69745. A. C. Marques acknowledges funding from FCT I.P., through the PhD Grant SFRH/BD/115173/2016. The authors acknowledge Professor Pedro Costa from KAUST for the TEM imaging., info:eu-repo/semantics/publishedVersion

Laser irradiation to induce networks of graphene-based structures toward cost-effective, flexible device fabrication is a highly pursued area, with applications in various polymeric substrates. This work reports the application of this approach toward commonly available, eco-friendly, low-cost substrates, namely, chromatographic and office papers. Through an appropriate chemical treatment with sodium tetraborate as a fire-retardant agent, photothermal conversion to porous laser-induced graphene (LIG) on paper is achieved. Raman peaks are identified, with I2D/IG and ID/IG peak ratios of 0.616 ± 0.095 and 1.281 ± 0.173, showing the formation of multilayered graphenic material, exhibiting sheet resistances as low as 56.0 sq1. Coplanar, LIG-based, three-electrode systems (working, counter and reference electrodes) are produced and characterized, showing high current Faradaic oxidation and reduction peaks, translating in high electrochemical active area, doubling the geometric area. Good electron transfer kinetics performed exclusively with on-chip measurements are reached, with k0 values as high as 7.15 × 104 cm s1. Proof-of-concept, amperometric, enzymatic glucose biosensors are developed, exhibiting good analytical performance in physiologically relevant glucose levels, with results pointing to the applicability of paper-based LIG toward efficient, disposable electrochemical sensor development, increasing their sustainability and accessibility, while simplifying their production and reducing their cost., This work was funded by National Funds through FCT I.P. under the scope of the project UIDB/50025/2020-2023. The authors acknowledge the ERC AdG project DIGISMART ref. 787410 and EC project SYNERGY H2020-WIDESPREAF-2020-5, CSA, proposal number 952169. T. Pinheiro acknowledges funding from FCT I.P. through the Ph.D. Grant DFA/ BD/8606/2020. S. Silvestre acknowledges funding from FCT I.P. through the Ph.D. Grant SFRH/BD/149751/2019. A.C. Marques acknowledges funding from FCT I.P. through the Ph.D. Grant SFRH/BD/115173/2016, info:eu-repo/semantics/publishedVersion

Due to its properties, paper represents an alternative to perform point-of-care tests for colorimetric determination of glucose levels, providing simple, rapid, and inexpensive means of diagnosis. In this work, we report the development of a novel, rapid, disposable, inexpensive, enzyme-free, and colorimetric paper-based assay for glucose level determination. This sensing strategy is based on the synthesis of gold nanoparticles (AuNPs) by reduction of a gold salt precursor, in which glucose acts simultaneously as reducing and capping agent. This leads to a direct measurement of glucose without any enzymes or depending on the detection of intermediate products as in conventional enzymatic colorimetric methods. Firstly, we modelled the synthesis reaction of AuNPs to determine the optical, morphological, and kinetic properties and their manipulation for glucose sensing, by determining the influence of each of the reaction precursors towards the produced AuNPs, providing a guide for the manipulation of nucleation and growth. The adaptation of this synthesis into the developed paper platform was tested and calibrated using different standard solutions with physiological concentrations of glucose. The response of the colorimetric signals obtained with this paper-based platform showed a linear behavior until 20 mM, required for glycemic control in diabetes, using the Red &times, Value/Grey feature combination as a calibration metric, to describe the variations in color intensity and hue in the spot test zone. The colorimetric sensor revealed a detection limit of 0.65 mM, depending on calibration metric and sensitivity of 0.013 AU/mM for a linear sensitivity range from 1.25 to 20 mM, with high specificity for the determination of glucose in complex standards with other common reducing interferents and human serum.