A multiplex and fast detection platform for microRNAs based on a self-priming microfluidic chip and duplex-specific nuclease.

MicroRNA expression levels highly correlate with the occurrence, diagnosis and prognosis of disease. However, challenges remain in establishing a multiplex and fast detection method. Here, we developed a multiplex and fast detection platform for microRNAs based on a self-priming microfluidic chip and duplex-specific nuclease. It can detect three types of miRNAs, including miR-100, miR-155, and Let-7a, simultaneously at 50 °C within 1 h. The probes are pre-introduced into the chip using the self-priming method and cross-contamination can be avoided by using a screw valve. The reagent consumption and cost have been largely reduced in this work compared to the traditional detection method. This chip also exhibits good quantitative performance and specificity. As a proof of concept, we detect three types of microRNAs from different cancer cell lines, which demonstrates its potential in real sample analysis. In summary, this microfluidic chip shows great advantages in multiplex, fast and simple detection of microRNAs, and possesses great potential in the early diagnosis of miRNA-related diseases, especially for point-of-care application.

[1]  Daniel B. Martin,et al.  Circulating microRNAs as stable blood-based markers for cancer detection , 2008, Proceedings of the National Academy of Sciences.

[2]  Stijn van Dongen,et al.  miRBase: microRNA sequences, targets and gene nomenclature , 2005, Nucleic Acids Res..

[3]  Zhiqiang Gao,et al.  A highly sensitive and selective electrochemical biosensor for direct detection of microRNAs in serum. , 2013, Analytical chemistry.

[4]  T. Clasen,et al.  Field Assessment of a Novel Household-Based Water Filtration Device: A Randomised, Placebo-Controlled Trial in the Democratic Republic of Congo , 2010, PloS one.

[5]  Pier Paolo Pompa,et al.  Absolute and direct microRNA quantification using DNA-gold nanoparticle probes. , 2014, Journal of the American Chemical Society.

[6]  Zhiqiang Gao,et al.  Detection of MicroRNAs using target-guided formation of conducting polymer nanowires in nanogaps. , 2007, Journal of the American Chemical Society.

[7]  M. Tewari,et al.  MicroRNA profiling: approaches and considerations , 2012, Nature Reviews Genetics.

[8]  Yu-Qiang Liu,et al.  One-step, multiplexed fluorescence detection of microRNAs based on duplex-specific nuclease signal amplification. , 2012, Journal of the American Chemical Society.

[9]  I. W. Nilsen,et al.  The Enzyme and the cDNA Sequence of a Thermolabile and Double-Strand Specific DNase from Northern Shrimps (Pandalus borealis) , 2010, PloS one.

[10]  Muneesh Tewari,et al.  Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR). , 2010, Methods.

[11]  S. Lukyanov,et al.  A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas. , 2002, Genome research.

[12]  Sichun Zhang,et al.  Homogeneous multiplexed digital detection of microRNA with ligation-rolling circle amplification. , 2020, Chemical communications.

[13]  R. Yu,et al.  Duplex-specific nuclease-mediated target recycling amplification for fluorescence detection of microRNA , 2019, Analytical Methods.

[14]  Jing Zhang,et al.  An immobilization-free electrochemical impedance biosensor based on duplex-specific nuclease assisted target recycling for amplified detection of microRNA. , 2016, Biosensors & bioelectronics.

[15]  Ning Gan,et al.  Ratiometric biosensor array for multiplexed detection of microRNAs based on electrochemiluminescence coupled with cyclic voltammetry. , 2016, Biosensors & bioelectronics.

[16]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[17]  D. Haber,et al.  Dual Role for Argonautes in MicroRNA Processing and Posttranscriptional Regulation of MicroRNA Expression , 2007, Cell.

[18]  Lin He,et al.  MicroRNAs: small RNAs with a big role in gene regulation , 2004, Nature reviews genetics.

[19]  Jin-Woo Choi,et al.  Point-of-care testing (POCT) diagnostic systems using microfluidic lab-on-a-chip technologies , 2015 .

[20]  K. Livak,et al.  Real-time quantification of microRNAs by stem–loop RT–PCR , 2005, Nucleic acids research.

[21]  Steven J. M. Jones,et al.  Comprehensive molecular characterization of human colon and rectal cancer , 2012, Nature.

[22]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[23]  P. Doyle,et al.  Microparticle parking and isolation for highly sensitive microRNA detection. , 2017, Lab on a chip.

[24]  E. Voigtman,et al.  Reduction of electronic noise in inductively coupled plasma atomic emission and fluorescence spectrometric measurements , 1983 .

[25]  Tianlun Jiang,et al.  Duplex-specific nuclease-mediated bioanalysis. , 2015, Trends in biotechnology.