Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection

Abstract

Microfabricated silicon/glass-based devices with functionalities of simultaneous polymerase chain reaction (PCR) target amplification and sequence-specific electrochemical (EC) detection have been successfully developed. The microchip-based device has a reaction chamber (volume of 8 µl) formed in a silicon substrate sealed by bonding to a glass substrate. Electrode materials such as gold and indium tin oxide (ITO) were patterned on the glass substrate and served as EC detection platforms where DNA probes were immobilized. Platinum temperature sensors and heaters were patterned on top of the silicon substrate for real-time, precise and rapid thermal cycling of the reaction chamber as well as for efficient target amplification by PCR. DNA analyses in the integrated PCR-EC microchip start with the asymmetric PCR amplification to produce single-stranded target amplicons, followed by immediate sequence-specific recognition of the PCR product as they hybridize to the probe-modified electrode. Two electrochemistry-based detection techniques including metal complex intercalators and nanogold particles are employed in the microdevice to achieve a sensitive detection of target DNA analytes. With the integrated PCR-EC microdevice, the detection of trace amounts of target DNA (as few as several hundred copies) is demonstrated. The ability to perform DNA amplification and EC sequence-specific product detection simultaneously in a single reaction chamber is a great leap towards the realization of a truly portable and integrated DNA analysis system.

Flow through PCR module of BioBriefcase

“…[34] This group demonstrated successful and sensitive detection of target DNA template with two electrochemical hybridization transduction methods. [34] Although, sample preparation is not incorporated into this microfabricated device. Some developments have been made to incorporate an ECD with other forms of analytical techniques.…”

“…In addition, ECD has the ability to obtain sequence information by manipulation of the hybridization between the target analyte and a DNA probe, which is immobilized on an electrode transducer surface. [34] This hybridization characteristic relies on external electrochemically-active indicators. [34] A few groups have used this ECD technique for nucleic acid amplification product detection.…”

“…[34] This hybridization characteristic relies on external electrochemically-active indicators. [34] A few groups have used this ECD technique for nucleic acid amplification product detection. [34][35][36][37] In 2003, Lee et al reported the first integrated microchip-based PCR with ECD functionality.…”

“…[55] In 2003, one group demonstrated the capability of a microfabricated silicon and glass based device to perform DNA amplification and in situ PCR product detection. [34] Heaters and temperature sensors are patterned on top of the silicon reaction chamber for precise thermal cycling. [34] Although, this integrated system did not include a sample preparation and purification step.…”

Amperometric Enzyme-based Biosensors for Lowering the Interferences

“…Nevertheless, they miniature the size and have the properties of fast response, less sample and low cost (Auroux et al, 2002) and this kind of sensing chip is also called Lab-on-a-chip. For example, the biosensors based on the field effect transistor (FET) made by MEMS immobilize anti-PSA on the carbon nanotubes (CNTs) (Kojima et al, 2005), liquid-chromatography-based biochip detects peptide mixture (Xie et al, 2005), and the biochip combines PCR-based DNA amplification and electrochemical detection (Lee et al, 2003) have been reported. Other few examples include antibody-based chips for determining protein isoform (Loonberg & Carlsson, 2006), liquid-chromatography-based chips for detecting peptide mixture (Xie et al, 2005), and electrophoresis-based chips for sensing catechol and dopamine (Schoning et al, 2005).…”