A microfluidic chip‐based fluorescent biosensor for the sensitive and specific detection of label‐free single‐base mismatch via magnetic beads‐based “sandwich” hybridization strategy

A novel microfluidic chip‐based fluorescent DNA biosensor, which utilized the electrophoretic driving mode and magnetic beads‐based “sandwich” hybridization strategy, was developed for the sensitive and ultra‐specific detection of single‐base mismatch DNA in this study. In comparison with previous biosensors, the proposed DNA biosensor has much more robust resistibility to the complex matrix of real saliva and serum samples, shorter analysis time, and much higher discrimination ability for the detection of single‐base mismatch. These features, as well as its easiness of fabrication, operation convenience, stability, better reusability, and low cost, make it a promising alternative to the SNPs genotyping/detection in clinical diagnosis. By using the biosensor, we have successfully determined oral cancer‐related DNA in saliva and serum samples without sample labeling and any preseparation or dilution with a detection limit of 5.6 × 10−11 M, a RSD (n = 5) < 5% and a discrimination factor of 3.58–4.54 for one‐base mismatch.

[1]  S. Mangru,et al.  Dynamic DNA hybridization on a chip using paramagnetic beads. , 1999, Analytical chemistry.

[2]  Michael J Sailor,et al.  A stable, label-free optical interferometric biosensor based on TiO2 nanotube arrays. , 2010, ACS nano.

[3]  Sylvia Kwakye,et al.  Electrochemical microfluidic biosensor for nucleic acid detection with integrated minipotentiostat. , 2006, Biosensors & bioelectronics.

[4]  Kevin W Plaxco,et al.  Biosensors based on binding-modulated donor-acceptor distances. , 2005, Trends in biotechnology.

[5]  Luke P. Lee,et al.  Innovations in optical microfluidic technologies for point-of-care diagnostics. , 2008, Lab on a chip.

[6]  Guonan Chen,et al.  Electrochemical genotyping and detection of single-nucleotide polymorphisms based on junction-probe containing 2'-deoxyinosine. , 2010, Chemical communications.

[7]  Mehmet Ozsoz,et al.  Electrochemical DNA biosensor for detecting cancer biomarker related to glutathione S-transferase P1 (GSTP1) hypermethylation in real samples. , 2012, Biosensors & bioelectronics.

[8]  Hsueh-Chia Chang,et al.  A rapid field-use assay for mismatch number and location of hybridized DNAs. , 2010, Lab on a chip.

[9]  Seokheun Choi,et al.  Microfluidic-based biosensors toward point-of-care detection of nucleic acids and proteins , 2010, Microfluidics and nanofluidics.

[10]  Ultrasensitive immunoassay of 7-aminoclonazepam in human urine based on CdTe nanoparticle bioconjugations by fabricated microfluidic chip. , 2009, Biosensors & bioelectronics.

[11]  S. Parveen,et al.  Ultrasensitive signal-on DNA biosensor based on nicking endonuclease assisted electrochemistry signal amplification. , 2011, Biosensors & bioelectronics.

[12]  Chun-Yang Zhang,et al.  Improved sensitivity for the electrochemical biosensor with an adjunct probe. , 2010, Analytical chemistry.

[13]  P. Bartlett,et al.  A label-free, electrochemical SERS-based assay for detection of DNA hybridization and discrimination of mutations. , 2012, Journal of the American Chemical Society.

[14]  E. Ohtsuka,et al.  Studies on nucleic acid interactions. I. Stabilities of mini-duplexes (dG2A4XA4G2-dC2T4YT4C2) and self-complementary d(GGGAAXYTTCCC) containing deoxyinosine and other mismatched bases. , 1986, Nucleic acids research.

[15]  Yi Xiao,et al.  i-Motif quadruplex DNA-based biosensor for distinguishing single- and multiwalled carbon nanotubes. , 2009, Journal of the American Chemical Society.

[16]  Jinghua Chen,et al.  A novel micro-fluidic biosensor for the rapid and sequence-specific detection of DNA with electrophoretic driving mode and laser-induced fluorescence detector , 2013 .

[17]  I. Tinoco,et al.  Base pairing involving deoxyinosine: implications for probe design. , 1985, Nucleic acids research.

[18]  Navid Nasirizadeh,et al.  Introduction of hematoxylin as an electroactive label for DNA biosensors and its employment in detection of target DNA sequence and single-base mismatch in human papilloma virus corresponding to oligonucleotide. , 2011, Biosensors & bioelectronics.

[19]  Guonan Chen,et al.  Electrochemical biosensor for detection of BCR/ABL fusion gene using locked nucleic acids on 4-aminobenzenesulfonic acid-modified glassy carbon electrode. , 2008, Analytical chemistry.

[20]  M. Navazesh,et al.  Methods for Collecting Saliva , 1993, Annals of the New York Academy of Sciences.

[21]  Ren Sun,et al.  Genetic analysis of H1N1 influenza virus from throat swab samples in a microfluidic system for point-of-care diagnostics. , 2011, Journal of the American Chemical Society.

[22]  M. Heller,et al.  Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Zongwei Cai,et al.  An end-channel amperometric detector for microchip capillary electrophoresis. , 2004, Talanta.

[24]  Hsueh-Chia Chang,et al.  Shear and AC Field Enhanced Carbon Nanotube Impedance Assay for Rapid, Sensitive, and Mismatch-Discriminating DNA Hybridization. , 2009, ACS nano.

[25]  Anne-Laure Gassner,et al.  Magnetic forces produced by rectangular permanent magnets in static microsystems. , 2009, Lab on a chip.

[26]  Antje J Baeumner,et al.  Electrochemical microfluidic biosensor for the detection of nucleic acid sequences. , 2006, Lab on a chip.

[27]  M. Heller,et al.  Electric field directed nucleic acid hybridization on microchips. , 1997, Nucleic acids research.

[28]  Fake Li,et al.  Detection of single-nucleotide polymorphisms with novel leaky surface acoustic wave biosensors, DNA ligation and enzymatic signal amplification. , 2012, Biosensors & bioelectronics.

[29]  Elena E. Ferapontova,et al.  Unmediated by DNA electron transfer in redox-labeled DNA duplexes end-tethered to gold electrodes. , 2012, Journal of the American Chemical Society.

[30]  N. Sugimoto,et al.  Facilitation of RNA enzyme activity in the molecular crowding media of cosolutes. , 2009, Journal of the American Chemical Society.

[31]  Martin A M Gijs,et al.  Microfluidic applications of magnetic particles for biological analysis and catalysis. , 2010, Chemical reviews.

[32]  C. Ulrich,et al.  Polymorphisms in DNA repair genes and associations with cancer risk. , 2002, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[33]  Kin Fong Lei,et al.  Colorimetric immunoassay chip based on gold nanoparticles and gold enhancement , 2009 .

[34]  A. Bard,et al.  DNA analysis by application of Pt nanoparticle electrochemical amplification with single label response. , 2012, Journal of the American Chemical Society.

[35]  Y. Sugiyama,et al.  Genetic polymorphisms of uptake (OATP1B1, 1B3) and efflux (MRP2, BCRP) transporters: implications for inter-individual differences in the pharmacokinetics and pharmacodynamics of statins and other clinically relevant drugs , 2009, Expert opinion on drug metabolism & toxicology.

[36]  Chunhai Fan,et al.  Sequence-specific detection of femtomolar DNA via a chronocoulometric DNA sensor (CDS): effects of nanoparticle-mediated amplification and nanoscale control of DNA assembly at electrodes. , 2006, Journal of the American Chemical Society.

[37]  Su Jin Lee,et al.  ssDNA aptamer-based surface plasmon resonance biosensor for the detection of retinol binding protein 4 for the early diagnosis of type 2 diabetes. , 2008, Analytical chemistry.