Integrated continuous flow polymerase chain reaction and micro‐capillary electrophoresis system with bioaffinity preconcentration

Abstract

An integrated and modular DNA analysis system is reported that consists of two modules: (i) A continuous flow polymerase chain reaction (CFPCR) module fabricated in a high T(g) (150°C) polycarbonate substrate in which selected gene fragments were amplified using biotin and fluorescently labeled primers accomplished by continuously shuttling small packets of PCR reagents and template through isothermal zones as opposed to heating and cooling large thermal masses typically performed in batch-type thermal reactors. (ii) μCE (micro-capillary electrophoresis) module fabricated in poly(methylmethacrylate) (PMMA), which utilized a bioaffinity selection and purification bed (2.9 μL) to preconcentrate and purify the PCR products generated from the CFPCR module prior to electrophoretic sorting. Biotin-labeled CFPCR products were hydrostatically pumped through the streptavidin-modified bed, where they were extracted onto the surface of micropillars. The affinity bed was also fabricated in PMMA and was populated with an array of microposts (50 μm width; 100 μm height) yielding a total surface area of ∼117 mm(2). This solid-phase extraction (SPE) process demonstrated high selectivity for biotinylated amplicons and utilized the strong streptavidin/biotin interaction (K(d) = 10(-15) M) to generate high recoveries. The SPE selected CFPCR products were thermally denatured and single-stranded DNA released for injection into a 7-cm-long μCE channel for size-based separations and fluorescence detection. The utility of the system was demonstrated using Alu DNA typing for gender and ethnicity determinations as a model. Compared with the traditional cross-T injection procedure typically used for μCE, the affinity pre-concentration and injection procedure generated signal enhancements of 17- to 40-fold, critical for CFPCR thermal cyclers due to Taylor dispersion associated with their operation.

Immobilization of Lambda Exonuclease onto Polymer Micropillar Arrays for the Solid-Phase Digestion of dsDNAs

“…48 Details on the device fabrication are provided in the Supporting Information. Parts A and B of Figure S1 in the Supporting Information show the CAD drawing and SEM image of the device, respectively.…”

Advances in Microfluidic Materials, Functions, Integration, and Applications

“…121 Biotinylated PCR products were selectively extracted on a streptavidin-modified bed fabricated in a PMMA microdevice; the purified material was subsequently eluted by thermal denaturation and analyzed by μCE. 122 Aptamers, which are short nucleic acid sequences that bind to target molecules with high specificity, are alternatives to antibody-based extraction. A PDMS-glass multilayer microfluidic device (Figure 14) packed with aptamer-functionalized microbeads was used to selectivity extract and concentrate arginine vasopressin, with its temperature-dependent binding and release controlled by an integrated microheater and temperature sensor.…”

Microfluidic chips with reversed-phase monoliths for solid phase extraction and on-chip labeling

“…The most common SPE modes in microfluidics are affinity [4,17,18] and reversed-phase [6,19,20]. Affinity SPE in microchips has been used to extract and quantify four cancer biomarkers in blood [4], to preconcentrate and purify PCR products [17], and to extract thiazole orange-conjugated adenosine monophosphate [18]. Reversed-phase columns are useful in the extraction of non-polar to moderately polar compounds.…”