Quantitative real-time monitoring of RCA amplification of cancer biomarkers mediated by a flexible ion sensitive platform

• Published in: Biosensors & bioelectronics 2017-05, Vol.91, p.788-795
• Main Authors: Veigas, Bruno, Pinto, Joana, Vinhas, Raquel, Calmeiro, Tomás, Martins, Rodrigo, Fortunato, Elvira, Baptista, Pedro Viana
• Format: Article
• Language: English
• Subjects: Biomarkers, Tumor - genetics; Biosensing Techniques - instrumentation; Cancer; Cell Line, Tumor; Cmisfet; Electrodes; Equipment Design; Field-effect sensors; Fusion Proteins, bcr-abl - genetics; Gene expression analysis; Gene Expression Profiling - instrumentation; Genes, myc; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive - diagnosis; Leukemia, Myelogenous, Chronic, BCR-ABL Positive - genetics; Neoplasms - diagnosis; Neoplasms - genetics; Nucleic Acid Amplification Techniques - instrumentation; Rca
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2017.01.052

Ion sensitive field-effect transistors (ISFET) are the basis of radical new sensing approaches. Reliable molecular characterization of specific detection of DNA and/or RNA is vital for disease diagnostics and to follow up alterations in gene expression profiles. Devices and strategies for biomolecular recognition and detection should be developed into reliable and inexpensive platforms. Here, we describe the development of a flexible thin-film sensor for label free gene expression analysis. A charge modulated ISFET based sensor was integrated with real-time DNA/RNA isothermal nucleic acid amplification: Loop-mediated isothermal amplification (LAMP) and Rolling Circle Amplification (RCA) techniques for c-MYC and BCR-ABL1 genes, allowing for the real-time quantification of template. Also, RCA allowed the direct quantification of RNA targets at room temperature, eliminating the requirement for external temperature controllers and overall complexity of the molecular diagnostic approach. This integration between the biological and the sensor/electronic approaches enabled the development of an inexpensive and direct gene expression-profiling platform. This work focused on the development of ion sensitive solid state sensors produced on flexible and transparent substrates for gene expression analysis. The optimized charge modulated ion sensitive field effect device was coupled with isothermal nucleic acid amplification techniques allowing direct detection and quantification of BCR-ABL RNA targets at room temperature, reducing the overall complexity of the molecular diagnostic approach. [Display omitted] •Isothermal amplification of DNA and RNA on a charge modulated ion sensitive flexible sensor.•Quantitative real-time monitoring of LAMP and RCA via an ion sensitive filed effect sensor.•Sensor for quantitative real-time isothermal amplification (qRT-LAMP) and (qRT-RCA) comparable to standard qRT-PCR.•Direct and quantitative real-time monitoring of RNA amplification at room temperature.