Improving design features and air bubble manipulation techniques for a single-step sandwich electrochemical ELISA incorporating commercial electrodes into capillary-flow driven immunoassay devices.

Integrating electrochemistry into capillary-flow driven immunoassay devices provides unique opportunities for quantitative point-of-care testing. Although custom electrodes can be inexpensive and are tunable, they require skilled fabrication. Here, we report the incorporation of a commercial electrode into a capillary-flow driven immunoassay (iceCaDI) device for a single end-user step sandwich electrochemical enzyme-linked immunosorbent assay (ELISA). The iceCaDI device is a pump-free portable microfluidic device with an integrated commercial screen-printed electrode and flow driven by capillary action. The iceCaDI device is composed of alternating polyester transparency film and double-sided adhesive film layers that are patterned with a laser cutter. This platform was designed to address known limitations of laminated device fabrication methods and operation. First, we developed a foldable laminated device fabrication using hinges for easy assembly and precise alignment. Second, reagent dispersing was achieved by incorporating a 1 mm wide arrow-shaped notch in the middle of the channel that trapped an air bubble and formed a baffle that facilitated reagent spreading to cover the detection area. Third, small vent holes were added to the top layer of the channels to prevent air bubbles from blocking flow. Finally, we fabricated a CRP immunosensor with a detection range of 0.625 to 10.0 µg mL-1 as a proof-of-concept to demonstrate an automatically driven sandwich electrochemical ELISA using the iceCaDI device. Three concentrations of CRP were successfully measured under flow conditions within 8 min. Our proposed device is a promising approach and a step forward in the development of point-of-care (POC) devices for techniques that traditionally require multiple user steps.

Electrochemical Immunoassay for the Detection of SARS-CoV-2 Nucleocapsid Protein in Nasopharyngeal Samples.

Point-of-care (POC) methods currently available for detecting SARS-CoV-2 infections still lack accuracy. Here, we report the development of a highly sensitive electrochemical immunoassay capable of quantitatively detecting the presence of the SARS-CoV-2 virus in patient nasopharyngeal samples using stencil-printed carbon electrodes (SPCEs) functionalized with capture antibodies targeting the SARS-CoV-2 nucleocapsid protein (N protein). Samples are added to the electrode surface, followed by horseradish peroxidase (HRP)-conjugated detection antibodies also targeting the SARS-CoV-2 N protein. The concentration of the virus in samples is quantified using chronoamperometry in the presence of 3,3'5,5'-tetramethylbenzidine. Limits of detection equivalent to less than 50 plaque forming units/mL (PFU/mL) were determined with virus sample volumes of 20 µL. No cross-reactivity was detected with the influenza virus and other coronavirus N proteins. Patient nasopharyngeal samples were tested as part of a proof-of-concept clinical study where samples were also tested using the gold-standard real-time quantitative polymerase chain reaction (RT-qPCR) method. Preliminary results from a data set of 22 samples demonstrated a clinical specificity of 100% (n = 9 negative samples according to RT-qPCR) and a clinical sensitivity of 70% for samples with RT-PCR cycle threshold (Ct) values under 30 (n = 10) and 100% for samples with Ct values under 25 (n = 5), which complies with the World Health Organization (WHO) criteria for POC COVID-19 diagnostic tests. Our functionalized SPCEs were also validated against standard plaque assays, and very good agreement was found between both methods (R2 = 0.9993, n = 6), suggesting that our assay could be used to assess patient infectivity. The assay currently takes 70 min from sampling to results.

Electrochemical Capillary Driven Immunoassay for Detection of SARS-CoV-2.

The COVID-19 pandemic focused attention on a pressing need for fast, accurate, and low-cost diagnostic tests. This work presents an electrochemical capillary driven immunoassay (eCaDI) developed to detect SARS-CoV-2 nucleocapsid (N) protein. The low-cost flow device is made of polyethylene terephthalate (PET) and adhesive films. Upon addition of a sample, reagents and washes are sequentially delivered to an integrated screen-printed carbon electrode for detection, thus automating a full sandwich immunoassay with a single end-user step. The modified electrodes are sensitive and selective for SARS-CoV-2 N protein and stable for over 7 weeks. The eCaDI was tested with influenza A and Sindbis virus and proved to be selective. The eCaDI was also successfully applied to detect nine different SARS-CoV-2 variants, including Omicron.