Toward Rapid and Automated Immunoassays: Using a Localized Electrochemical pH Modulation Platform to Perform a Single-Step Immunoassay.

An electrochemical platform for generating and controlling a localized pH microenvironment on demand is proposed by employing a closed-loop control algorithm based on an iridium oxide pH sensor input. We use a combination of solution-borne quinones and galvanostatic excitation on a prepatterned indium tin oxide (ITO) working electrode to modulate pH within a very well confined, small volume of solution close to the electrode surface. We demonstrate that the rate of pH change can be controlled at up to 2 pH s-1 with an excellent repeatability (±0.004). The desired pH microenvironment can be stably maintained for longer than 2 h within ±0.0012 pH. As a high-impact application of the platform technology, we propose a single-step immunoassay and demonstrate its utility in measuring C-reactive protein (CRP), a critical inflammatory marker in various conditions such as myocardial infarction and even SARS-Cov-2. Utilizing pH modulation technology along with pH-sensitive fluorescence dye simplifies the immunoassay process into a single-step, where a mixture of all of the reagents is incubated only for 1 h without any washing steps or the need to change solution. This simplified immunoassay process minimizes the hands-on time of the end-user and thus decreases technician-driven errors. Moreover, the absence of complicated liquid-handling hardware makes it more suitable and attractive for an ultracompact platform to ultimately be used in a point-of-care diagnostic assay.

Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants.

The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. We further developed an mCARMEN panel to enable the identification of 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples. The mCARMEN platform enables high-throughput surveillance of multiple viruses and variants simultaneously, enabling rapid detection of SARS-CoV-2 variants.

Local and systemic responses to SARS-CoV-2 infection in children and adults.

It is not fully understood why COVID-19 is typically milder in children1-3. Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children.