CRISPR/Cas12a and primer-assisted rolling circle amplification integrated ultra-sensitive dual-signal sensing platform for EGFR 19 detection.

Herein, we integrated CRISPR/Cas12a with primer-assisted rolling circle amplification (PARCA) to specifically detect EGFR 19 from the genome. We fused the method into fluorescent and electrochemical detection systems forming a stable and sensitive dual-signal sensing platform. The fluorescent detection system stably detected EGFR 19 in a linear range from 500 fM to 10 nM with an ultra-low background signal. The electrochemical detection system possessed a detection limit as low as 42 aM due to the introduction of nanomaterial UIO-66-NH2. The dual-signal sensing platform showed superior performance in complex serum samples and real cell genomes and provided a flexible and dynamic approach for the ultra-sensitive detection of EGFR 19.

Highly sensitive and facile microRNA detection based on target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a.

MicroRNAs (miRNAs) play a crucial role in the regulation of gene expression and have been implicated in many diseases. Herein, we develop a target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a (T-ERCA/Cas12a) system, which can achieve the ultrasensitive detection with simple operation and no annealing procedure. In this assay, T-ERCA combines the exponential amplification with rolling-circle amplification by introducing a dumb-bell probe with two enzyme recognition sites. miRNA-155 targets are activators that trigger exponential rolling circle amplification to produce large amounts of ssDNA, which is then recognized by CRISPR/Cas12a for further amplification. Compared with single EXPAR or RCA combined with CRISPR/Cas12a, this assay shows higher amplification efficiency. Therefore, benefiting from the excellent amplification effect of T-ERCA and the high recognition specificity of CRISPR/Cas12a, the proposed strategy shows a wide detection range from 1 fM to 5 nM with a LOD (limit of detection) down to 0.31 fM. Moreover, it shows good application ability for assessing miRNA levels in different cells, indicating that the T-ERCA/Cas12a may provide a new guidance for molecular diagnosis and clinical practical application.

A fluorescent biosensor based on exponential amplification reaction-initiated CRISPR/Cas12a (EIC) strategy for ultrasensitive DNA methyltransferase detection.

DNA methyltransferase (DNA MTase) catalyzes the process of DNA methylation, and the aberrant DNA MTase activity is closely associated with cancer incidence and progression. Inspired by the exponential amplification reaction (EXPAR) characteristics, we developed an EXPAR-initiated CRISPR/Cas12a (EIC) strategy for sensitively detecting DNA MTase activity. A hairpin probe (HP) was designed with a palindromic sequence in the stem as substrate and NH2-modified 3' end to prevent nonspecific amplification. HP could be methylated by DNA adenine methyltransferase (Dam MTase) and then digested by DpnI to generate an oligonucleotide that can serve as an EXPAR primer. With the assistance of Nt.BstNBI nicking enzyme and Vent(exo-) polymerase, this primer bound to template and induced EXPAR. Interestingly, the product of Cycle 1 in EXPAR can function as primer to initiate Cycle 2. Both EXPAR products can further activate the collateral cleavage of CRISPR/Cas12a-crRNA, resulting in the fragmentation of fluorescence reporters and fluorescence recovery. Due to the highly efficient amplification (about 5 times signal-to-noise of SDA) and the robust trans-cleavage of CRISPR/Cas12a, the EIC system owned an extreme limit of detection (LOD) of 2 × 10-4 U/mL and a broad detection range from 2 × 10-4 to 10 U/mL for Dam MTase. In addition, this method has succeeded in inhibitor screening and evaluation, showing magnificent promise in drug discovery and cancer therapy.

Ultrasensitive fluorescent biosensor for detecting CaMV 35S promoter with proximity extension mediated multiple cascade strand displacement amplification and CRISPR/Cpf 1.

A novel fluorescent biosensor was proposed for detecting the CaMV 35S promoter in genetically modified organisms (GMOs). It was based on a proximity extension mediated multiple cascade strand displacement amplification connected with CRISPR/Cpf 1 (termed PE-MC/SDA-CRISPR/Cpf1). In this protocol, the CaMV 35S was recognized by proximity reaction in the presence of two adjacent primer probes. The proximity extension further triggered the multiple cascade strand displacement amplification (MC/SDA), generating a mass of ssDNA. The products compelled the trans-cleavage activity of CRISPR/Cpf 1, so as to cleave nearby ssDNA-FQ reporters and generate a strong fluorescent signal. The ingenious three-link combination design allowed the CaMV 35S a low background interference. And the MC/SDA combined with CRISPR/Cpf 1 dramatically improved the detection sensitivity. Under optimized conditions, the detection linear range of ultrasensitive fluorescent biosensor for CaMV 35S was from 50 fM to10 pM and 10 pM-500 pM, along with the limit of detection (LOD) down to 14.4 fM. The sensing platform also had excellent performance in the assay of selectivity and real samples. Therefore, the method earned great application potential for transgenic crops.

A novel methyl-dependent DNA endonuclease GlaI coupling with double cascaded strand displacement amplification and CRISPR/Cas12a for ultra-sensitive detection of DNA methylation.

Detection of methylation changes associated with oncogenic transformation is essential for early screening and treatment of cancer. Herein, we propose a novel DNA methylation detection assay based on the methyl-dependent DNA endonuclease GlaI coupling with double cascaded strand displacement amplification and CRISPR/Cas12a (GlaI-DC-SDA-CRISPR/Cas12a). The GlaI enables highly specific recognition and digestion of methylated target site (dsDNA) but leaves unmethylated target intact. Therefore, only methylated DNA can be digested to produce two free 3'-OH terminus for triggering the next SDA-CRISPR/Cas12a. Compared with the fluorescence response under single amplification template, DC-SDA with double amplification templates shows higher sensitivity. Benefiting from the high specificity of GlaI and the cascaded amplification effect of DC-SDA combined with CRISPR/Cas12a, the proposed method shows excellent performance for DNA methylation detection with low LOD (1.28 × 10-13 M), ultra-low background interference and wide detection range (2 × 10-13 to 4 × 10-11, 4 × 10-11 to 1 × 10-8 M). 0.1% of DNA methylation can be discriminated from the mixture with a mass of unmethylated DNA. Most importantly, the proposed assay can be applied to the actual detection of human serum and genomic DNA, as well as to distinguish normal cells from cancer cells. It can also quantify DNA methylation in genomic DNA (HCT116) with a LOD of 37.95 ng, indicating its great potential in early clinical cancer screening.

A three-dimensional hydrogel-modified indium tin oxide electrode with enhanced performance for in situ electrochemical detection of extracellular H2O2.

Two different electrochemical sensors (Hemin-G4/Au/GCE and Hemin-G4/Au/ITO) were developed and applied to explore the electrocatalytic capacity of H2O2 reduction. Due to the excellent catalytic activity of Hemin-G4 and high conductivity of gold nanoparticles, both electrodes show excellent electrochemical performances towards H2O2 with a low LOD (0.67 µM for Hemin-G4/Au/GCE and 0.65 µM for Hemin-G4/Au/ITO), rapid response (<4 s), and high selectivity and sensitivity (314.33 µA mM-1 cm-2 for Hemin-G4/Au/GCE and 322.22 µA mM-1 cm-2 for Hemin-G4/Au/ITO). The two electrodes allow sensitive capture of H2O2 produced by A549 cells. Compared with the conventional method of detection in cell suspensions, an ITO electrode with a large specific surface area and good biocompatibility can provide a promising platform for cell adhesion, so as to realize real-time and in situ detection of extracellular H2O2. The experimental results show that A549 cells can adhere to the surface of the Hemin-G4/Au/ITO electrode and grow well. This is benefitted from the three-dimensional structure of the Hemin-G4/Au hydrogel, which provides a suitable microenvironment for cell adhesion and growth. Furthermore, the in situ detection shows a faster response time than that of in-solution detection. This is because the H2O2 generated by the cells can be directly captured by the ITO electrode, which avoids diffusion from the solution to the electrode. These results indicate that the self-supporting hydrogel modified ITO electrode has great application prospects in basic biomedical research and continuous dynamic surveillance of diseases.