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.

Transcriptional characterization of the glial response due to chronic neural implantation of flexible microprobes.

Long term implantation of (micro-)probes into neural tissue causes unique and disruptive responses. In this study, we investigate the transcriptional trajectory of glial cells responding to chronic implantation of 380 µm flexible micro-probes for up to 18 weeks. Transcriptomic analysis shows a rapid activation of microglial cells and a strong reactive astrocytic polarization, both of which are lost over the chronic of the implant duration. Animals that were implanted for 18 weeks show a transcriptional profile similar to non-implanted controls, with increased expression of genes associated with wound healing and angiogenesis, which raises hope of a normalization of the neuropil to the pre-injury state when using flexible probes. Nevertheless, our data shows that a subset of genes upregulated after 18 weeks belong to the family of immediate early genes, which indicates that structural and functional remodeling is not complete at this time point. Our results confirm and extend previous work on the molecular changes resulting from the presence of neural probes and provide a rational basis for developing interventional strategies to control them.

A CRISPR/Cas13a-powered catalytic electrochemical biosensor for successive and highly sensitive RNA diagnostics.

Rapid and specific quantitation of a variety of RNAs with low expression levels in early-stage cancer is highly desirable but remains a challenge. Here, we present a dual signal amplification strategy consisting of the CRISPR/Cas13a system and a catalytic hairpin DNA circuit (CHDC), integrated on a reusable electrochemical biosensor for rapid and accurate detection of RNAs. Signal amplification is accomplished through the unique combination of the CRISPR/Cas13a system with CHDC, achieving a limit of detection of 50 aM within a readout time of 6 min and an overall process time of 36 min, using a measuring volume of 10 µL. Enzymatic regeneration of the sensor surface and ratiometric correction of background signal allow up to 37 sequential RNA quantifications by square-wave voltammetry on a single biosensor chip without loss of sensitivity. The reusable biosensor platform could selectively (specificity = 0.952) and sensitively (sensitivity = 0.900) identify low expression RNA targets in human serum, distinguishing early-stage patients (n = 20) suffering from non-small-cell lung carcinoma (NSCLC) from healthy subjects (n = 30) and patients with benign lung disease (n = 12). Measurement of six NSCLC-related RNAs (miR-17, miR-155, TTF-1 mRNA, miR-19b, miR-210 and EGFR mRNA) shows the ability of the electrochemical CRISPR/CHDC system to be a fast, low-cost and highly accurate tool for early cancer diagnostics.

CRISPR-powered electrochemical microfluidic multiplexed biosensor for target amplification-free miRNA diagnostics.

Recently the use of microRNAs (miRNAs) as biomarkers for a multitude of diseases has gained substantial significance for clinical as well as point-of-care diagnostics. Amongst other challenges, however, it holds the central requirement that the concentration of a given miRNA must be evaluated within the context of other factors in order to unambiguously diagnose one specific disease. In terms of the development of diagnostic methods and devices, this implies an inevitable demand for multiplexing in order to be able to gauge the abundance of several components of interest in a patient's sample in parallel. In this study, we design and implement different multiplexed versions of our electrochemical microfluidic biosensor by dividing its channel into subsections, creating four novel chip designs for the amplification-free and simultaneous quantification of up to eight miRNAs on the CRISPR-Biosensor X ('X' highlighting the multiplexing aspect of the device). We then use a one-step model assay followed by amperometric readout in combination with a 2-min-stop-flow-protocol to explore the fluidic and mechanical characteristics and limitations of the different versions of the device. The sensor showing the best performance, is subsequently used for the Cas13a-powered proof-of-concept measurement of two miRNAs (miRNA-19b and miRNA-20a) from the miRNA-17-92 cluster, which is dysregulated in the blood of pediatric medulloblastoma patients. Quantification of the latter, alongside simultaneous negative control measurements are accomplished on the same device. We thereby confirm the applicability of our platform to the challenge of amplification-free, parallel detection of multiple nucleic acids.

Quantitative synchrotron X-ray tomography of the material-tissue interface in rat cortex implanted with neural probes.

Neural probes provide many options for neuroscientific research and medical purposes. However, these implantable micro devices are not functionally stable over time due to host-probe interactions. Thus, reliable high-resolution characterization methods are required to understand local tissue changes upon implantation. In this work, synchrotron X-ray tomography is employed for the first time to image the interface between brain tissue and an implanted neural probe, showing that this 3D imaging method is capable of resolving probe and surrounding tissue at a resolution of about 1 micrometer. Unstained tissue provides sufficient contrast to identify electrode sites on the probe, cells, and blood vessels within tomograms. Exemplarily, we show that it is possible to quantify characteristics of the interaction region between probe and tissue, like the blood supply system. Our first-time study demonstrates a way for simultaneous 3D investigation of brain tissue with implanted probe, providing information beyond what was hitherto possible.