Immobilized molecular beacons: a new strategy using UV-activated poly(methyl methacrylate) surfaces to provide large fluorescence sensitivities for reporting on molecular association events.

We have designed appropriately prepared solid supports consisting of poly(methyl methacrylate) (PMMA) that provide enhanced performance levels for molecular beacons (MBs) that are used for recognizing and reporting on signature DNA sequences in solution. The attachment of primary amine-containing MBs to the PMMA surface was carried out by UV activating the PMMA to produce surface-confined carboxylate groups, which could then be readily coupled to the MBs using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) chemistry. The fluorescence properties of the MBs covalently attached onto this UV-activated PMMA surface were evaluated and compared with the same MBs immobilized onto glass supports. We observed improved limits of detection for the solution complement to the MBs when immobilized onto PMMA, and this was attributed to both the lower autofluorescence levels exhibited by PMMA at the detection wavelengths used and the improved quenching efficiency of the MBs when in their closed hairpin configuration when strapped to a PMMA surface as opposed to glass. As an example of the utility of the PMMA-based immobilization strategies developed for MBs, we report on the analysis of complementary DNAs specific for fruitless (fru) and Ods-site homeobox (OdsH) genes extracted from Drosophila melanogaster fruit flies. The fru gene functions in the central nervous system, where it is necessary for sex determination and male courtship behavior, whereas the OdsH gene is involved in the regulation of transcription.

Merging microfluidics with microarray-based bioassays.

Microarray technologies provide powerful tools for biomedical researchers and medicine, since arrays can be configured to monitor the presence of molecular signatures in a highly parallel fashion and can be configured to search either for nucleic acids (DNA microarrays) or proteins (antibody-based microarrays) as well as different types of cells. Microfluidics on the other hand, provides the ability to analyze small volumes (micro-, nano- or even pico-liters) of sample and minimize costly reagent consumption as well as automate sample preparation and reduce sample processing time. The marriage of microarray technologies with the emerging field of microfluidics provides a number of advantages such as, reduction in reagent cost, reductions in hybridization assay times, high-throughput sample processing, and integration and automation capabilities of the front-end sample processing steps. However, this potential marriage is also fraught with some challenges as well, such as developing low-cost manufacturing methods of the fluidic chips, providing good interfaces to the macro-world, minimizing non-specific analyte/wall interactions due to the high surface-to-volume ratio associated with microfluidics, the development of materials that accommodate the optical readout phases of the assay and complete integration of peripheral components (optical and electrical) to the microfluidic to produce autonomous systems appropriate for point-of-care testing. In this review, we provide an overview and recent advances on the coupling of DNA, protein and cell microarrays to microfluidics and discuss potential improvements required for the implementation of these technologies into biomedical and clinical applications.

Fabrication of DNA microarrays onto polymer substrates using UV modification protocols with integration into microfluidic platforms for the sensing of low-abundant DNA point mutations.

We describe the microfabrication and operational characteristics of a simple flow-through biochip sensor capable of detecting low abundant point mutations in K-ras oncogenes from genomic DNA, which carry high diagnostic value for colorectal cancers. The biochip consisted of an allele-specific ligase detection reaction (LDR) coupled to a universal array for interrogating multiple mutations simultaneously from a clinical sample. The integrated sensing platform was micro-manufactured from two different polymers, polycarbonate, PC, which was used for the LDRs, and poly(methyl methacrylate), PMMA, which was used to build the microarray. Passive elements were hot embossed into the PC and PMMA microchips and then, the chips assembled into a three-dimensional architecture with the interconnect fabricated from an elastomer, poly(dimethylsiloxane), PDMS, to produce a leak-free connection between the biochips. The array in PMMA was produced using a photomodification process, which involved three steps; (1) UV (254 nm) exposure of the polymer surface; (2) EDC coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond) and; (3) washing of the surface. The LDR/hybridization flow-through biochip performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time=19.1 min) and could screen multiple mutations simultaneously for high throughput applications at a level of one mutant sequence in 100 wild-type sequences.

Fabrication of DNA microarrays onto poly(methyl methacrylate) with ultraviolet patterning and microfluidics for the detection of low-abundant point mutations.

We have developed a simple ultraviolet (UV)-photomodification protocol using poly(methyl methacrylate) and polycarbonate to produce functional scaffolds consisting of carboxylic groups that allow covalent attachment of amine-terminated oligonucleotide probes to these surface groups through carbodiimide coupling. Use of the photomodification procedure coupled to microfluidics allowed for the rapid generation of medium-density DNA microarrays. The method reported herein involves the use of poly(dimethylsiloxane) microchannels reversibly sealed to photomodified poly(methyl methacrylate) surfaces to serve as stencils for patterning the oligonucleotide probes. After array construction, the poly(dimethylsiloxane) stencil is rotated 90 degrees to allow interrogation of the array using microfluidics. The photomodification process for array fabrication involves only three steps: (1) broadband UV exposure of the polymer surface, (2) carbodiimide coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond), and (3) washing of the surface. The density of probes attached to this activated surface was found to be approximately 41pmolcm(-2), near the steric-saturation limit for short oligonucleotide probes. We demonstrate the use of this procedure for screening multiple KRAS2 mutations possessing high diagnostic value for colorectal cancers. A ligase detection reaction/universal array assay was carried out using parallel detection of two different low-abundant DNA point mutations in KRAS2 oncogenes with the allelic composition evaluated at one locus. Four zip code probes immobilized onto the poly(methyl methacrylate) surface directed allele-specific ligation products containing mutations in the KRAS2 gene (12.2D, 12.2A, 12.2V, and 13.4D) to the appropriate address of a universal array with minimal amounts of cross-hybridization or misligation.