Rolling circle amplification and graphene-based sensor-on-a-chip for sensitive detection of serum circulating miRNAs

• Published in: Analytical biochemistry 2019-07, Vol.577, p.89-97
• Main Authors: Treerattrakoon, Kiatnida, Jiemsakul, Thanakorn, Tansarawiput, Chookiat, Pinpradup, Preedee, Iempridee, Tawin, Luksirikul, Patraporn, Khoothiam, Krissana, Dharakul, Tararaj, Japrung, Deanpen
• Format: Article
• Language: English
• Subjects: DNA, Single-Stranded - chemistry; Graphene oxide; HeLa Cells; Humans; Isothermal amplification; MicroRNAs - analysis; miRNAs; Neoplasms - diagnosis; Nucleic Acid Amplification Techniques - methods; Oligonucleotide Array Sequence Analysis - methods; Proof of Concept Study; Rolling circle amplification; Spectrometry, Fluorescence - methods; Tuberculosis - diagnosis
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2019.04.016

In this study, we developed a simple multiplex miRNA detection platform based on rolling circle amplification and the fluorescence quenching property of reduced graphene oxide. The detection platform could be applied on a microfluidics chip with a mobile system controller to eliminate contamination and to facilitate potential use in remote areas. As a proof of concept, two fluorescence-labeled ssDNA tags were used for detection of miR-29a and miR-144*, two miRNAs that are highly expressed in the blood circulation of some patients with cancer or tuberculosis. The circular ssDNA probes in this study were designed to have an advantage over padlock probes as they can be prepared in advance. Our multiplex miRNA detection platform exhibited high sensitivity and selectivity, with a limit of detection of 0.05 pmol. In addition, our platform could detect target miRNAs from the total miRNA population extracted from human serum or a cancer cell line. These results indicated that our miRNA sensor has the potential to provide simple and high throughput miRNA analysis for disease diagnosis and prognosis. •Graphene based detection of multiplex miRNAs.•Isothermal amplification of multiplex miRNAs.•LOD of 0.1 pmole for both targets, miR-29a and miR-144*.•Application to analysis of total miRNA extracted from human serum and cancer cell line.•Sensor-on-a-chip for detection of serum circulating miRNAs.