Multiprobe Assay for Clinical SEPT9 Methylation Based on the Carbon Dot-Modified Liquid-Exfoliated Graphene Field Effect Transistor with a Potential to Present a Methylation Panorama

Abstract

The hypermethylation in the promoter region of the SEPT9 gene is associated with the development of colorectal cancer (CRC). Although its clinical significance for early diagnosis and screening of CRC has been demonstrated, the tedious operations in the conventional DNA methylation (DNAm) detection hinder its wide application. Herein, an electronic method for determining SEPT9 methylation in CRC patients is proposed by using the carbon dot-modified liquid exfoliated graphene field effect transistor (CDs-LEG-FET) as the DNAm sensor, the specifically designed probes to capture the SEPT9 gene and the immunologic recognition to recognize 5-methylcytosine (5mC) positions on the anchored sequences. The identification and nanomorphology of the as-prepared materials and devices are executed first by the characterizations of UV−vis, Raman, atomic force microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and electronic measurements. Then, the role of CDs in enhancing DNAm sensitivity of CD-LEG-FET is manifested by comparing it with that of CD-free LEG-FET. Third, the captured SEPT9 genes on CD-LEG-FETs by different probes are evaluated, and the optimized temperature for hybridizing the target ssDNA sequences is determined to be 48 °C. Furthermore, the detection sensitivity for the low-quantity of DNA samples is demonstrated to be as low as 2 ng. Finally, the methylation degree of the tumor and corresponding noncancerous tissue DNA samples were examined by the proposed electric method and methylight assay in parallel. The diagnostic value of the electrical assay is confirmed by using the receiver operating characteristic curves; meanwhile, the superiority of the CD-LEG-FET platform is found to present a methylation panorama of the target gene.

Dual-modality loop-mediated isothermal amplification for pretreatment-free detection of Septin9 methylated DNA in colorectal cancer

“…3D, E, and F)-indicating a potential of this method in quantification detection through real-time fluorescence LAMP. According to the result, as few as 0.25 ± 0.025 ng of mDNA (0.02 ng/μL in LAMP system with a total volume of 12.5 μL) could be detected by LAMP owing to its high sensitivity, excellent amplification efficiency, and good anti-interference, and the detection limit is much lower than that of the recently reported electrochemical sensing strategy [33] and field-effect transistor [41]. The distinct colorimetric discrimination (Fig.…”

“…Septin9 is a tumor suppressor gene; the hypermethylation of CpG islands in the promoter of Septin9 can repress the promoter activity and lose its tumor suppressor function [38]. Consequently, hypermethylated Septin9 serves as a biomarker for early cancer detection and monitoring treatment [39][40][41], which has been approved by the US FDA for colorectal cancer (CRC) screening [42]. Here, five pairs of LAMP primers (1, 2, 3, 4, 5) were designed according to the Septin9 gene (Table S1).…”

A disposable DNA methylation sensor based on the printable graphene field effect transistor

“…Under the action of methyltransferase, methyl is covalently bound to the fifth carbon atom of CpG dinucleotide cytosine to form 5methylcytosine (5mC) [1]. It has been believed an efficient method for the DNA methylation detection is of significance to understand the internal mechanism of gene expression, and improve the level of clinical screening, diagnosis, treatment, and prognosis of related diseases, so lots of efforts had been made in the disciplines of biology, chemistry and electronics [2,3,4,5].…”

“…The emergence of graphene field effect transistor (GFET) brought vitalities to almost all kinds of biosensors due to graphene's high electron mobility and specific surface area [6,7]. In our previous works, we tried to exploit it as the sensors for DNA methylation for DNA extracts from tumor cells [3] and tumor tissues [5]. To exploit it as a low-cost and practicable device for the clinical and biological applications, we would like to utilize the printing technique, which is one of the most popular electronic fabrication technologies.…”

“…The interdigital electrodes of source and drain were printed on the specified locations to form the basic devices of GFET. According to the previously experiments [5], the decoration of CDs was conducted to enhance the amount of single strand DNA (ssDNA) which were waiting to be tested. At last, the determination of the 5mC positions on the tested ssDNA are realized by using the similar immune strategy in our previous works [3,5], i.e., the antibodies of 5mC (anti-5mC) were incubated on the ssDNA anchored GFETs, the changed currents were recorded to evaluate the sensitivity for DNA methylation.…”