Ion sensing (EIS) real-time quantitative monitorization of isothermal DNA amplification.

Field-effect-based devices are becoming a basic structural element in a new generation of microbiosensors. Reliable molecular characterization of DNA and/or RNA is of paramount importance for disease diagnostics and to follow up alterations in gene expression profiles. The use of such devices for point-of-need diagnostics has been hindered by the need of standard or real-time PCR amplification procedures. The present work focuses on the development of a tantalum pentoxide (Ta2O5) based sensor for the real-time label free detection of DNA amplification via loop mediated isothermal amplification (LAMP) allowing for quantitative analysis of the cMYC proto-oncogene. The strategy based on the field effect sensor was tested within a range of 1 × 10(8)-10(11) copies of target DNA, and a linear relationship between the log copy number of the initial template DNA and threshold time was observed allowing for a semi-quantitative analysis of DNA template. The concept offers many of the advantages of isothermal quantitative real-time DNA amplification in a label free approach and may pave the way to point-of-care quantitative molecular analysis focused on ease of use and low cost.

[1]  Yasuyoshi Mori,et al.  Real-time turbidimetry of LAMP reaction for quantifying template DNA. , 2004, Journal of biochemical and biophysical methods.

[2]  T. F. Smith,et al.  Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing , 2006, Clinical Microbiology Reviews.

[3]  D. Ginzinger.  Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. , 2002, Experimental hematology.

[4]  L. Blum,et al.  DNA biosensors and microarrays. , 2008, Chemical reviews.

[5]  Piet Bergveld,et al.  Thirty years of ISFETOLOGY ☆: What happened in the past 30 years and what may happen in the next 30 years , 2003 .

[6]  F. Battaglini,et al.  Disposable Gold Electrode Array for Simultaneous Electrochemical Studies , 2008 .

[7]  Manmohan Parida,et al.  Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases , 2008, Reviews in medical virology.

[8]  S. Takashiba,et al.  Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method. , 2005, FEMS immunology and medical microbiology.

[9]  Francine Kivlehan,et al.  Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids. , 2011, The Analyst.

[10]  Elvira Fortunato,et al.  Real-time monitoring of PCR amplification of proto-oncogene c-MYC using a Ta₂O₅ electrolyte-insulator-semiconductor sensor. , 2011, Biosensors & bioelectronics.

[11]  Juan F Medrano,et al.  Real-time PCR for mRNA quantitation. , 2005, BioTechniques.

[12]  Bernard P. Puc,et al.  An integrated semiconductor device enabling non-optical genome sequencing , 2011, Nature.

[13]  C. Toumazou,et al.  Simultaneous DNA amplification and detection using a pH-sensing semiconductor system , 2013, Nature Methods.

[14]  H. D. Vanguilder,et al.  Twenty-five years of quantitative PCR for gene expression analysis. , 2008, BioTechniques.

[15]  Kathryn A. O’Donnell,et al.  The c-Myc target gene network. , 2006, Seminars in cancer biology.

[16]  Y Husimi,et al.  Real-time monitoring of DNA polymerase reactions by a micro ISFET pH sensor. , 1992, Analytical chemistry.

[17]  Satoshi Tsuneda,et al.  Real-time quantitative LAMP (loop-mediated isothermal amplification of DNA) as a simple method for monitoring ammonia-oxidizing bacteria. , 2006, Journal of biotechnology.

[18]  Omowunmi A Sadik,et al.  Status of biomolecular recognition using electrochemical techniques. , 2009, Biosensors & bioelectronics.

[19]  T. Notomi,et al.  Loop-mediated isothermal amplification of DNA. , 2000, Nucleic acids research.

[20]  Shingo Inoue,et al.  Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of West Nile Virus , 2004, Journal of Clinical Microbiology.