Exploring T7 RNA polymerase-assisted CRISPR/Cas13a amplification for the detection of BNP via electrochemiluminescence sensing platform.

ABSTRACT

Brain natriuretic peptide (BNP) is considered to be an important biomarker of heart failure (HF) attracting attention. However, its low concentration and short half-life in blood lead to a low-sensitivity detection of BNP, which is a challenge that has to be overcome. In this work, we propose a highly specific, highly sensitive T7 RNA polymerase-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system to detect BNP via an electrochemiluminescence (ECL) sensing platform and incorporate exonuclease III (Exo III)-hairpin and dumbbell-shaped hybridization chain reaction (HCR) technologies. In this detection scheme, the ECL sensing platform possesses low background signal and high sensitivity. Firstly, the T7 promoter-initiated T7 RNA polymerase acts as a signal amplification technique to generate large amounts of RNAs that can activate CRISPR/Cas13a activity. Secondly, CRISPR/Cas13a is able to trans-cleave the surrounding trigger strand to produce DNA1. Thirdly, DNA1 is involved in the co-amplification reaction of Exo III and hairpin DNA, which subsequently triggers a dumbbell-shaped HCR technology. Eventually, a large number of Ru (II) molecules are inserted into the interstitial space of the dumbbell-shaped HCR to generate a strong ECL signal. The CRISPR/Cas13a possesses outstanding specificity for a single base and increased sensitivity. The tightly conformed dumbbell-shaped HCR provides higher sensitivity than the traditional linear HCR amplification technique. Ultimately, the clever combination of several amplification reactions enables the limit of detection (LOD) as low as 3.2 fg/mL. It showed promise for clinical sample testing, with recovery rates ranging from 98.4% to 103% in 5% human serum samples. This detection method offered a valuable tool for early HF detection, emphasizing the synergy of amplification strategies and specificity conferred by CRISPR/Cas13a technology.