The proposed multiplexed microfluidic biosensor (BiosensorX) for the POC scenario, monitoring the treatment of bacterial co- or superinfection in COVID-19 patients. BiosensorX is capable of harboring both the CRISPR-powered assays for the detection of multiple COVID-19-specific RNA sequences, derived from nasal swabs, and a protein-based assay for ß-lactam antibiotic monitoring in serum samples. Simultaneous detection of the analytes is enabled by immobilization of different assays (light blue to black) onto the sequentially arranged incubation areas of the single-channel microfluidic chip.High-throughput testing is a central part of controlling the spread of SARS-CoV-2 infections. While PCR (polymerase chain reaction) testing remains the gold standard for accuracy and sensitivity, it is not ideal for fast, high-throughput testing or resource-limited environments. Conversely, while easy and cheap lateral flow device (LFD) rapid antigen testing is widely used for home testing, only a high viral load produces a positive result. Now a team from the University of Freiburg in Germany has designed an electrochemical, microfluidic polymer-based biosensor that uses CRISPR/Cas-powered assays to offer quick, low-cost, accurate point-of-care testing for SARS-CoV-2 Omicron infections [Johnston et al., Materials Today 61 (2022) 129-138, https://doi.org/10.1016/j.mattod.2022.11.001].
The approach is based on CRISPR (clustered regularly interspaced short palindromic repeats), a family of DNA sequences found in bacteria that identify and destroy matching sequences associated with the infection. Combined with Cas9 (CRISPR-associated protein 9), an enzyme that uses CRISPR to help recognize and remove matching sequences, CRISPR is a powerful method of editing DNA sequences. Here, a CRISPR-Cas assay is combined with a novel microfluidic electrochemical biosensing device, BiosensorX, to detect two genetic sequences characteristic of SARS-CoV-2.
“Similar to rapid tests performed at home or in testing centers, a nasal or oral swab sample solution is added to a reaction mix. If the sample contains the RNA snippet of interest, the CRISPR-associated protein (Cas13a) is activated and cleaves the reporter RNA provided within the reaction mix,” explains Midori Johnson, first author of the work. “The signal readout is performed electrochemically using shelf-stable, nontoxic, and inexpensive reagents.”
Since RNA is susceptible to degradation, contamination can produce false negative results, which is potentially disastrous in vulnerable settings like hospitals, nursing homes, or schools. The new approach, however, includes both positive and negative controls that can determine whether the assay is working to its full capacity and rule out contamination. Consequently, the team were able to test clinical samples and confirm that patients were not infected with SARS-CoV-2 and rule out contamination.
The design of the microfluidic multiplexed platform offers a range of options and combinations of different diagnostic assays for simultaneous screening of biological molecules. The team included an assay to measure ß-lactam antibiotic concentration, since bacterial infections often occur with viral infections and are treated with generic antibiotics.
“Our method’s short sample-to-result time (approximately 30 minutes) and high sensitivity without any nucleic-acid amplification could bridge the gap between existing test methods after further optimization and clinical validation,” says Can Dincer, who led the work.