Multiplexed biosensor for point-of-care COVID-19 monitoring: CRISPR-powered unamplified RNA diagnostics and protein-based therapeutic drug management.

In late 2019 SARS-CoV-2 rapidly spread to become a global pandemic, therefore, measures to attenuate chains of infection, such as high-throughput screenings and isolation of carriers were taken. Prerequisite for a reasonable and democratic implementation of such measures, however, is the availability of sufficient testing opportunities (beyond reverse transcription PCR, the current gold standard). We, therefore, propose an electrochemical, microfluidic multiplexed polymer-based biosensor in combination with CRISPR/Cas-powered assays for low-cost and accessible point-of-care nucleic acid testing. In this study, we simultaneously screen for and identify SARS-CoV-2 infections (Omicron-variant) in clinical specimens (Sample-to-result time: ∼30 min), employing LbuCas13a, whilst bypassing reverse transcription as well as target amplification of the viral RNA (LODs of 2,000 and 7,520 copies/µl for the E and RdRP genes, respectively, and 50 copies/ml for combined targets), both of which are necessary for detection via PCR and other isothermal methods. In addition, we demonstrate the feasibility of combining synthetic biology-driven assays based on different classes of biomolecules, in this case protein-based ß-lactam antibiotic detection, on the same device. The programmability of the effector and multiplexing capacity (up to six analytes) of our platform, in combination with a miniaturized measurement setup, including a credit card sized near field communication (NFC) potentiostat and a microperistaltic pump, provide a promising on-site tool for identifying individuals infected with variants of concern and monitoring their disease progression alongside other potential biomarkers or medication clearance.

Designing electrochemical microfluidic multiplexed biosensors for on-site applications.

Clinical assessment based on a single biomarker is in many circumstances not sufficient for adequate diagnosis of a disease or for monitoring its therapy. Multiplexing, the measurement of multiple analytes from one sample and/or of the same target from different samples simultaneously, could enhance the accuracy of the diagnosis of diseases and their therapy success. Thus, there is a great and urgent demand for multiplexed biosensors allowing a low-cost, easy-to-use, and rapid on-site testing. In this work, we present a simple, flexible, and highly scalable strategy for implementing microfluidic multiplexed electrochemical biosensors (BiosensorX). Our technology is able to detect 4, 6, or 8 (different) analytes or samples simultaneously using a sequential design concept: multiple immobilization areas, where the assay components are adsorbed, followed by their individual electrochemical cells, where the amperometric signal readout takes place, within a single microfluidic channel. Here, first we compare vertical and horizontal designs of BiosensorX chips using a model assay. Owing to its easier handling and superior fluidic behavior, the vertical format is chosen as the final multiplexed chip design. Consequently, the feasibility of the BiosensorX for multiplexed on-site testing is successfully demonstrated by measuring meropenem antibiotics via an antibody-free ß-lactam assay. The multiplexed biosensor platform introduced can be further extended for the simultaneous detection of other anti-infective agents and/or biomarkers (such as renal or inflammation biomarkers) as well as different (invasive and non-invasive) sample types, which would be a major step towards sepsis management and beyond.

Biosensor-Enabled Multiplexed On-Site Therapeutic Drug Monitoring of Antibiotics.

Personalized antibiotherapy ensures that the antibiotic concentration remains in the optimal therapeutic window to maximize efficacy, minimize side effects, and avoid the emergence of drug resistance due to insufficient dosing. However, such individualized schemes need frequent sampling to tailor the blood antibiotic concentrations. To optimally integrate therapeutic drug monitoring (TDM) into the clinical workflow, antibiotic levels can either be measured in blood using point-of-care testing (POCT), or can rely on noninvasive sampling. Here, a versatile biosensor with an antibody-free assay for on-site TDM is presented. The platform is evaluated with an animal study, where antibiotic concentrations are quantified in different matrices including whole blood, plasma, urine, saliva, and exhaled breath condensate (EBC). The clearance and the temporal evaluation of antibiotic levels in EBC and plasma are demonstrated. Influence of matrix effects on measured drug concentrations is determined by comparing the plasma levels with those in noninvasive samples. The system's potential for blood-based POCT is further illustrated by tracking ß-lactam concentrations in untreated blood samples. Finally, multiplexing capabilities are explored successfully for multianalyte/sample analysis. By enabling a rapid, low-cost, sample-independent, and multiplexed on-site TDM, this system can shift the paradigm of "one-size-fits-all" strategy.