Electric chips for rapid detection and quantification of nucleic acids

Abstract

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.

New Micro‐ and Nanotechnologies for Electrochemical Biosensor Development

“…This was achieved by integrating an interdigitated ultramicroelectrode array in the glass chip, downstream of the capturing magnet and labeling the hybrid with liposomes fi lled with an electrochemical marker, which could be oxidized or reduced at the electrode surface. A similar approach, based on silicon electrical chip arrays coupled to interdigitated ultramicroelectrodes, was reported by Gabig et al [181] for the recognition of the RNA of infectious bacteria. In this case, the DNA probe was captured on streptavidin -coated beads and labeled with a secondary probe functionalized with digoxigenin.…”

Electrochemical Detection of RNA

“…The resultant voltammograms display current peaks, resulting from the oxidation of guanine, whose height is proportional to the amount of RNA hybridized to DNA capture oligonucleotides. This technique allowed the detection of down to 10 7 colony-forming units (cfu) E. coli within 4 h. Gabig-Ciminska et al (2004) applied silicon-based biochips possessing interdigitated electrodes in combination with bead-based sandwich hybridization (BBSH) for 16S rRNA analysis. A sandwich hybridization assembly between a capture probe immobilized on paramagnetic beads, a single-stranded target RNA, and a DIG-labeled detector probe was produced.…”

Recent developments in the monitoring, modeling and control of biological production systems

“…Designs that utilize electronics for detection in combination with miniaturization are being developed in several labs that are investigating cultivation problems [8,9]. Interesting demonstrations of how the microsystems can be extended to encompass the entire fluidic path from pretreatment to final detection of complex biological samples have been shown [10,11].…”