DNA Hybridization and Discrimination of Single-Nucleotide Mismatches Using Chip-Based Microbead Arrays

Abstract

The development of a chip-based sensor array composed of individually addressable agarose microbeads has been demonstrated for the rapid detection of DNA oligonucleotides. Here, a "plug and play" approach allows for the simple incorporation of various biotinylated DNA capture probes into the bead-microreactors, which are derivatized in each case with avidin docking sites. The DNA capture probe containing microbeads are selectively arranged in micromachined cavities localized on silicon wafers. The microcavities possess trans-wafer openings, which allow for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microbeads. Collectively, these features allow the identification and quantitation of target DNA analytes to occur in near real time using fluorescence changes that accompany binding of the target sample. The unique three-dimensional microenvironment within the agarose bead and the microfluidics capabilities of the chip structure afford a fully integrated package that fosters rapid analyses of solutions containing complex mixtures of DNA oligomers. These analyses can be completed at room temperature through the use of appropriate hybridization buffers. For applications requiring analysis of < or = 10(2) different DNA sequences, the hybridization times and point mutation selectivity factors exhibited by this bead array method exceed in many respects the operational characteristics of the commonly utilized planar DNA chip technologies. The power and utility of this microbead array DNA detection methodology is demonstrated here for the analysis of fluids containing a variety of similar 18-base oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 10000 and limit of detection values of approximately 10(-13) M are obtained readily with this microbead array system.

Preparation of Carbodiimide-terminated Dithiolane Self-Assembly Monolayers as a New DNA-Immobilization Method

“…DNA microarray or DNA chip based on a DNA-immobilized substrate have been developed to enable a high-throughput analysis of many genes simultaneously. [1][2][3][4][5][6][7][8][9] Many kinds of DNA fragments, called a capture DNA probe, are immobilized on the substrate of a DNA microarray or DNA chip. A proper DNA fragment in a sample can hybridize with the complementary capture DNA probe on the substrate, and its hybrid can be detected by the combination of DNA detecting reagents.…”

“…Such a system has been applied to a study of gene expression and mutation analysis including single nucleotide polymorphism (SNP) analysis. [1][2][3][6][7][8][9] A goldcovered substrate, having conductivity sufficient for electrochemical DNA detection, is also a good candidate for a DNA chip of the next generation from the viewpoint of developing a simple, quick, and miniaturized detection system and in fact, many papers have dealt with this subject. [10][11][12][13][14][15] To construct such a system, it is important to develop a reproducible method to immobilize a capture DNA probe on the gold surface, and many kinds of immobilization methods have been reported.…”

Programmable Bio-NanoChip Technology for the Diagnosis of Cardiovascular Disease at the Point of Care

“…toward this goal, we have worked to improve the current state of PoC ivd through the development, validation, and implementation of P-BnCs ( Figure 1a). [17][18][19][20][21][22][23][24] the "programmability" feature of the system refers to the capacity of the sensor ensemble to function as a standard platform that can be reprogrammed to serve a new application by inserting a molecular-level code (i.e., the biomarker-specific reagents). the "bio" terminology refers to the capacity to measure and extract the bio-signatures associated with the disease progression.…”

Multiplexing Capabilities of Biosensors for Clinical Diagnostics

“…Unlike 2-D microarrays, the high surface-to-volume ratio of beads allows a larger amount of probes to be immobilized to improve the detection signals and signal-to-noise ratios. The small size of beads can further reduce the reaction volume, and the use of microfluidics in bead arrays can shorten the hybridization time to < 10 min, a 50 to 70-fold reduction as compared to conventional microarrays (Ali et al, 2003). Unlike 2-D or the 3-D microarrays discussed, probes are usually conjugated onto the beads prior to them being immobilized onto the microarrays.…”