Point-of-care CRISPR-Cas-assisted SARS-CoV-2 detection in an automated and portable droplet magnetofluidic device

Abstract

In the fight against COVID-19, there remains an unmet need for point-of-care (POC) diagnostic testing tools that can rapidly and sensitively detect the causative SARS-CoV-2 virus to control disease transmission and improve patient management. Emerging CRISPR-Cas-assisted SARS-CoV-2 detection assays are viewed as transformative solutions for POC diagnostic testing, but lack of streamlined sample preparation and full integration within an automated and portable device hamper their potential for POC use. We report herein POC-CRISPR – a single-step CRISPR-Cas-assisted assay that leverages facile magnetic concentration and transport of nucleic acid-binding magnetic beads to incorporate sample preparation with minimal manual operation. Moreover, POC-CRISPR has been adapted into a compact thermoplastic cartridge within a palm-sized yet fully-integrated and automated device. During analytical evaluation, POC-CRISPR was able detect 1 genome equivalent/μL SARS-CoV-2 RNA from a sample volume of 100 μL in < 30 min. Finally, when evaluated with 27 unprocessed clinical nasopharyngeal swab eluates that were pre-typed by standard RT-qPCR (C q values ranged from 18.3 to 30.2 for the positive samples), POC-CRISPR achieved 27 out of 27 concordance and could detect positive samples with high SARS-CoV-2 loads (C q < 25) in 20 min.

CRISPR-Powered Microfluidics in Diagnostics: A Review of Main Applications

“…Therefore, the CRISPR-powered microfluidic biosensors can perform better in sensitivity, selectivity, stability, and simultaneous detection of multiple oligoes as well as the possibility of integration with or without amplification [25][26][27][28]. The system can be cheaper and sometimes automated, portable, and compatible with PoC applications [29,30]. These platforms have also been shown to not only increase the expression of Cas9 mRNA considerably but also in certain cases enhance the gene-editing efficiency of the system [31].…”

“…A novel SARS-CoV-2 POC, introduced by Chen et al, composed of miniaturized magnetic arms, fluorescent detector, and heating module (Figure 2A) [29]. The sensor known as POC-CRISPR worked in three main steps.…”

“…Albeit the sensitive and smart idea behind the miSHERLOCK, the need for 4 mL of saliva sample and the 55-min reaction time were among its shortcomings. [29]. (B) An electrokinetic microfluidic chip that creates isotachophoresis mobility in the microfluidic device for RNA separation and sensitivity enhancement [34].…”

“…Figure 2. (A) POC-CRISPR assay with droplet microfluidics, in which RNAs are separated and concentrated using electrostatistical attachment to magnetic beads and fluorescent signals again using Cas12 trans cleavage[29]. (B) An electrokinetic microfluidic chip that creates isotachophoresis mobility in the microfluidic device for RNA separation and sensitivity enhancement[34].…”

Filtration-assisted magnetofluidic cartridge platform for HIV RNA detection from blood

“…We have previously demonstrated use of droplet magnetofluidics to enable automation of nucleic acid purification coupled to nucleic acid amplification directly from clinical samples in a point-of-care format. 19–24 In contrast to traditional microfluidic techniques that require precise fluidic controls and microchannel fabrication, magnetofluidics leverages transfer of magnetic beads through discrete droplets of reagents for fully integrated sample-to-answer bioassays using simple low-power actuation of permanent magnets. 25,26 Due to the simplicity of our cartridge construction, each cartridge can be fabricated for around $2 to provide an affordable alternative to currently available commercial products.…”