Polymerase Chain Reaction-Capillary Electrophoresis Genetic Analysis Microdevice with In-Line Affinity Capture Sample Injection

Abstract

An integrated polymerase chain reaction (PCR)-capillary electrophoresis (CE) microdevice with an efficient in-line affinity-based injector has been developed for genetic analysis. Double stranded DNA PCR amplicons generated in an integrated 250 nL PCR reactor are captured, purified, and preconcentrated by an oligonucleotide probe immobilized in an in situ polymerized gel matrix followed by thermal release and injection into the CE-separation channel. This in-column injector employs a photopolymerized oligonucleotide-modified acrylamide capture gel to eliminate band broadening and increase the injection efficiency to 100%. The on-chip generated PCR amplicons processed on this microdevice exhibit a 3-5 fold increase in signal intensities and improved resolution compared to our previous T-shaped injector. Multiplex analysis of 191-bp amplicons from Escherichia coli O157 and 256-bp amplicons from E. coli K12 is achieved with a 6-fold increase in resolution. These advances are exploited to successfully detect E. coli O157 in a 500-fold higher background of E. coli K12. This microdevice with in-line affinity capture gel injection provides an improved platform for low-volume, high sensitivity, fully integrated genetic analysis.

Multiplexing Capabilities of Biosensors for Clinical Diagnostics

“…Multiplex PCR is one of the most common techniques used in clinical diagnostics because the technology has matured significantly since its invention almost three decades ago. This is also rather easy to implement on biosensors, as the process can be carried out in microchambers (Merritt , 2010), or coupled to a capillary electrophoretic module (Thaitrong, 2009). The ability to perform multiplexed detection in PCR results from (a) the unique feature in PCR that allows primers to be designed to amplify fragments of different sizes, (b) the ability of the gel electrophoresis or real-time PCR system to differentiate the fragments by size as a result of their difference in electrophoretic mobility or melting temperature, and (3) the ability to differentiate the probes through color-emitting dyes.…”

Recent Progress of On-line Sample Preconcentration Techniques in Microchip Electrophoresis

“…Besides this, some novel sample preconcentration techniques utilizing specific characteristics of microchips have appeared in MCE. 8 The on-line sample preconcentration techniques for MCE have four classifications: preconcentration by analyte velocity change in two or three discontinuous solutions system, focusing, [9][10][11][12][13][14][15][16][17][18][19][20] solid phase extraction, [21][22][23] and electrokinetic trapping (filtering). [24][25][26][27] Among them, novel focusing and electrokinetic trapping techniques have been exhaustively investigated in the recent decade.…”

“…Physically and electrokinetically preconcentration techniques using the nanostructures are called solid phase extraction [21][22][23] and electrokinetic trapping, 24-27 respectively. These approaches can give extremely high preconcentration efficiency, especially for biomacromolecules.…”

An automated instrument for human STR identification: Design, characterization, and experimental validation

“…This approach uses different strategies to cleanup the PCR product and improve the sensitivity of the CE analysis [6,7] but requires the extraction and purification of the DNA analyte to be performed externally on the bench. Other approaches have focused on improving CE analysis [8,9] or using fast multiplex PCR to accelerate the STR cycling protocol [10] but require several bulky instruments operating separately and have, so far, not been fully integrated to perform an automated analysis.…”