Direct immunoassay on a polyester microwell plate for colorimetric detection of the spike protein in swab and saliva samples.

This study presents the development of a polyester microplate for detecting the S-protein of the SARS-CoV-2 virus in saliva and nasopharyngeal swab samples using direct enzyme-linked immunosorbent assay (ELISA) technology. The polyester microplate was designed to contain 96 zones with a 3 mm diameter each, and a volume of 2-3 µL. The experimental conditions including reagent concentration and reaction time were optimized. The microplate image was digitized and analyzed using graphical software. The linear range obtained between protein S concentrations and pixel intensity was 0-10 µg mL-1, with a correlation coefficient of 0.99 and a limit of detection of 0.44 µg mL-1. The developed methodology showed satisfactory intraplate and interplate repeatability with RSD values lower than 7.8%. The results achieved through immunoassay performed on polyester microplates were consistent with those of the RT-PCR method and showed a sensitivity of 100% and 90% and specificity of 85.71% and 100% for saliva and nasopharyngeal samples, respectively. The proposed direct immunoassay on polyester microplates emerges as an alternative to conventional immunoassays performed on commercial polystyrene plates, given the low cost of the device, low consumption of samples and reagents, lower waste generation, and shorter analysis time. Moreover, the immunoassay has shown great potential for diagnosing COVID-19 with precision and accuracy.

"Do it yourself" protocol to fabricate dual-detection paper-based analytical device for salivary biomarker analysis.

This paper describes the design and construction of dual microfluidic paper-based analytical devices (dual-µPADs) as a lab-on-paper platform involving a "do-it-yourself" fabrication protocol. The device comprises a colorimetric and electrochemical module to obtain a dual-mode signal readout sensing strategy. A 3D pen polymeric resin was used to prepare graphite carbon-based electrodes and hydrophobic barriers on paper substrates. The proposed carbon-based ink was employed to manufacture electrodes on paper based on a stencil-printing approach, which were further characterized by electrochemical and morphological analyses. The analytical performance of the dual-µPADs was simultaneously evaluated for lactate, pH, nitrite, and salivary amylase (sAA) analysis. To demonstrate the proof-of-concept, saliva samples collected from both healthy individuals and those with periodontitis were successfully tested to demonstrate the feasibility of the proposed devices. Samples collected from individuals previously diagnosed with periodontitis showed high levels of nitrite and sAA (> 94 µmol L-1 and > 610 U mL-1) in comparison with healthy individuals (≤ 16 µmol L-1 and 545 U mL-1). Moreover, periodontitis saliva resulted in acid solution and almost null lactate levels. Notably, this protocol supplies a simple way to manufacture dual-µPADs, a versatile platform for sensitive detecting of biomarkers in saliva playing a crucial role towards the point-of-care diagnosis of periodontal disease.

Simultaneous analysis of multiple adulterants in milk using microfluidic paper-based analytical devices.

This study reports the simultaneous colorimetric detection of urea, H2O2, and pH in milk samples using microfluidic paper-based analytical devices (µPADs) fabricated through a craft cutter printer. Paper-based devices were designed to contain three detection zones interconnected to a sampling zone by microfluidic channels. Colorimetric analysis was performed using images digitalized through an office scanner. The volumes of chromogenic and sample solutions were optimized, and the best colorimetric performance was achieved by adding 0.5 and 10 µL into detection and sampling zones, respectively. Simultaneous assays were then carried out, and the recorded responses revealed a linear behavior in the concentration ranges from 0-30.0 mmol L-1, 0-10.0 mmol L-1 and 6.0-9.0 for urea, H2O2 and pH, respectively. The limit of detection values obtained for urea and H2O2 were 2.4 mmol L-1 and 0.1 mmol L-1, respectively. For pH measurements, colorimetric assay allowed the monitoring of solution pH with a resolution of 0.25 units. The use of µPADs to detect target adulterants exhibited suitable reproducibility (RSD ≤ 6.0%), accuracy (91-102%) and no cross-reaction occurrence. When compared to reference techniques, colorimetric assays did not reveal a significant difference at a confidence level of 95%. As a proof-of-concept, the feasibility of the proposed approach was successfully demonstrated through the analysis of potential adulterants in sixteen milk samples, which were tested without any pretreatment requirement. Based on the achievements, µPADs in conjunction with colorimetric measurements emerge as a powerful tool for rapid screening of potential adulterants in milk.

Fabrication of microwell plates and microfluidic devices in polyester films using a cutting printer.

This study reports, for the first time, the possibility to manufacture analytical devices on polyester substrates using a cutting printer. The protocol involves the design of a layout in a graphical software, the cut into polyester films and the lamination against one or multiple polyester films coated with a thermosensitive layer. The feasibility of the proposed approach was demonstrated through the fabrication of 96-microwell plates, 3D microfluidic mixing and distance-based microfluidic devices. The printer has enabled cutting microchannels wider than 300 µm on polyester films and a thickness of 250 µm. Urea and glucose assays were performed on microwell plates aiming for their quantification in artificial urine and serum samples. The presented results revealed good agreement with the expected values. The complexation reaction between Fe2+ and o-phenanthroline was selected as model to investigate the feasibility of the 3D mixing device. Absorbance measurements were recorded for the reaction product performed in both on and off-chip modes. Considering the achieved data, the on-chip mixing exhibited similar behavior when compared to off-chip reaction, thus demonstrating to be efficient to perform mixtures due to the turbulence generated inside three-dimensional channels. Lastly, a distance-based device was designed to detect H2O2 based on the displacement of a dye plug promoted by the oxygen generation using a copper-modified paper sheet. The distance-based peroxymeter revealed a linear behavior in the concentration range between 1 and 5% (v/v) and a LOD equal to 0.5% (65.2 mM). Based on the results herein reported, the proposed method represents a simple and alternative protocol to produce microdevices, using affordable and inexpensive raw materials, within 10 min, and at a cost lower than US$ 0.10 per unit.