Liquid biopsy is a diagnostic technique that involves analyzing biomarkers from serum samples, which is highly valuable for disease diagnosis due to its convenience and noninvasive nature 1, 2. MicroRNAs (miRNAs) are small, noncoding RNA molecules that are naturally present in cells. They are composed of approximately 18–22 nucleotides and have significant effects on a wide range of biological processes 3, 4. For example, miRNAs have the capacity to regulate cell proliferation, differentiation, apoptosis, and metabolism 5, 6. Moreover, there is compelling evidence suggesting that miRNAs have a pivotal function in controlling gene expression, such as neuronal growth 7, synapse formation 8, and synaptic plasticity 9. Furthermore, these miRNAs have been linked to the control of neurobiological systems implicated in the processing of anxiety in the brain 10. Nevertheless, due to its concise sequence, very low expression level, and susceptibility to degradation, the precise identification of miRNAs from intricate biological samples is exceedingly difficult.
Conventional detection techniques, such as northern blotting, qRT-PCR, and oligonucleotide microarrays, are occasionally constrained by intricate procedures and the possibility of false positive outcomes 11, 12. Recently, different techniques that exhibit a higher efficiency and simplicity have been devised, including signal-based sensors, colorimetry 13–15, fluorescence 16–18, and electrochemistry. In contrast to the prevalent electrochemical or fluorescence sensors, colorimetric sensors are distinguished by their rapidity, low instrument cost, and simplicity. Lately, there has been significant interest in a unique bio-catalytic DNAzyme known as G-quadruplex (GQ)/hemin complex 19. A G-quadruplex is a DNA structure composed of four strands, with each strand containing guanine (G)-rich regions. When it binds with hemin, it displays a characteristic similar to that of horseradish peroxidase. When hydrogen peroxide is present, the GQ/hemin DNAzyme has the ability to accelerate the oxidation of particular substances, such as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), resulting in noticeable alterations in both color and absorption spectra 20, 21. Nevertheless, these solutions necessitated the extraction of miRNAs from samples and the reverse transcription of miRNAs, which might potentially lead to issues such as contamination, loss of target miRNAs, and intricate procedures. Hence, there is still a requirement for a precise and expeditious detection technique for miRNAs to be investigated. CRISPR/Cas systems, including as CRISPR/Cas9, CRISPR/Cas12a, and CRISPR/Cas13a, have demonstrated a wide range of potential applications in the development of biosensors 13, 22. This is due to their ability to accurately recognize genes and efficiently amplify signals. CRISPR/Cas13a systems have the ability to precisely identify target RNA molecules and then trigger a trans-cleavage reaction, achieving a minimum of 104 turnovers per recognized target RNA.
Through a catalytic chain assembly-assisted GQ/hemin DNAzyme-catalyzed color reaction, we present in this study a CRISPR-Cas13a-triggered method for the simple yet precise colorimetric detection of miRNAs in complex biological samples. This technique involves the utilization of CRISPR RNA (crRNA) to direct the Cas13a protein to selectively identify target miRNA, hence triggering its trans-cleavage activity (Fig. 1). Substrate with bulge structures are specifically created by the process of hybridization between the S chain and T chain. The bulge structure has seven bases, with two consecutive Uracil ribonucleotides (rU) present in the S chain, serving as cleavage sites for Cas13a. Upon activation, the Cas13a protein exhibits trans-cleavage characteristics, resulting in the non-specific cleavage of the surrounding substrate@MB. Subsequently, the Cas13a-crRNA13a complex initiates the cleavage of rUrU within the bulge structure, resulting in a substantial decrease in the melting temperature (Tm) of TS. Consequently, the S chain has the ability to separate itself from the surface of MBs, resulting in the formation of T@MB. Following magnetic enrichment and separation, the T@MB was combined with the H1 and H2 probe. The T chain initiates the catalytic chain assembly between the H1 probe and H2 probe by unfolding the H1 probe, thereby exposing the G-rich portion of the H2 probe. A region rich in guanine (G) nucleotides has the ability to form a secondary structure known as a G-quadruplex. Complexes formed by HRP-mimicking G-quadruplex/hemin catalyze the conversion of ABTS to green ABTS·–. This enables the colorimetric measurement of miRNA by signal amplification mediated by DNAzyme. The measurement can be done visually or using UV-vis spectrophotometry.