CRISPR/Cas13a Powered Portable Electrochemiluminescence Chip for Ultrasensitive and Specific MiRNA Detection

Abstract

MicroRNAs (miRNAs) have been widely investigated as potential biomarkers for early clinical diagnosis of cancer. Developing an miRNA detection platform with high specificity, sensitivity, and exploitability is always necessary. Electrochemiluminescence (ECL) is an electrogenerated chemiluminescence technology that greatly decreases background noise and improves detection sensitivity. The development of a paper‐based ECL biosensor further makes ECL suitable for point‐of‐care detection. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a as high‐fidelity, efficient, and programmable CRISPR RNA (crRNA) guided RNase has brought a next‐generation biosensing technology. However, existing CRISPR/Cas13a based detection often faces a trade‐off between sensitivity and specificity. In this research, a CRISPR/Cas13a powered portable ECL chip (PECL‐CRISPR) is constructed. Wherein target miRNA activates Cas13a to cleave a well‐designed preprimer, and triggers the subsequent exponential amplification and ECL detection. Under optimized conditions, a limit‐of‐detection of 1 × 10 −15 m for miR‐17 is achieved. Through rationally designing the crRNA, the platform can provide single nucleotide resolution to dramatically distinguish miRNA target from its highly homologous family members. Moreover, the introduction of “light‐switch” molecule [Ru(phen) 2 dppz] 2+ allows the platform to avoid tedious electrode modification and washing processes, thereby simplifying the experimental procedure and lower testing cost. Analysis results of miRNA from tumor cells also demonstrate the PECL‐CRISPR platform holds a promising potential for molecular diagnosis.

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

“…Zhou et al used Cas13a to detect the presence of miRNA from tumor cells ( Zhou et al, 2020 ). The presence of the miRNA which induces collateral cleavage of an EXPAR specific pre-trigger primer.…”

“…Their sensing method was translated to a paper-based bipolar electrode electrochemiluminescence (pBPE-ECL) biosensing platform, consisting of a simple paper based device, on which an electrochemical reaction was driven by a portable DC voltage supply. Their luminescence approach makes specific detection possible without the need of extensive washing procedures, which improves reproducibility and simplifies the experimental procedure, which is of great interest in POC sensing ( T. Zhou et al, 2020 ).…”

Enzyme kinetics of CRISPR molecular diagnostics

“…Subsequently, Shan et al 15 reported a k cat of 4854 per second and K M of 4.45 × 10 −6 M using a 5 nt (of rU) ssRNA fluorescent reporter in an application ofLbuCas13a for MicroRNA (miRNA) detection. Later, Zhou et al 13 showed for LbuCas13a that a shorter reporter molecule which was composed of 2 nt (of rU) lowered the k cat to 204 per second and K M of 2.62 × 10 −6 M. Most recently, Fozouni et al 1 presented a study of SARS-CoV-2 viral RNA detection using LbuCas13a and a set of 3 gRNAs, and used a model to fit their experimental data which used parameters of k cat = 600 per second and K M between 1 × 10 −6 M and 3 × 10 −6 M.…”

“…We note the latter value of k cat is a factor 340 times lower than the second-lowest k cat ever reported for any CRIPSR associated enzyme (a study on LbuCas13a; Table 1 ). 13…”

Biological characteristics and biomarkers of novel SARS-CoV-2 facilitated rapid development and implementation of diagnostic tools and surveillance measures

“…RPA is also compatible to integrate with CRISPR technology for the reason that both requires identical optimal reaction temperature conditions. Patchsung et al has previously reported a Cas13-RPA-mediated CRISPR approach, named “specific highly-sensitive enzymatic reporter unlocking system” (SHERLOCK) ( Patchsung et al, 2020 ), and demonstrated its detailed protocol and application for the detection of SARS-CoV-2( Zhou et al, 2020d ). The detection limit for SHERLOCK was reported about 10 copy/μL was demonstrated for the extracted RNA specimens and both amplification and detection protocols can be performed within about 1 h ( Guo et al, 2020b ).…”