Performance improvement of label-free biosensors and their applications in DNA and protein microarrays.

摘要

Microarray technologies have provided powerful tools in modern biotechnology to decipher the complex interconnectivity of genetic and regulatory networks. Label-free biosensors have emerged as competitive technologies because they offer more economic and simple procedures, allow molecular interactions in their native state, and provide interaction kinetics, compared to the traditional luminescence-based sensors. While these advantages enable better understandings of the intricate biomolecular interactions, label-free biosensors have yet to be widely adapted in biological and medical research.;We present two approaches to develop successful, high-throughput, label-free biosensors by using Interferometric Reflectance Imaging Sensor (IRIS). First, we offer strategies to improve the performance of label-free biosensors by increasing sensitivity and accuracy. Sensitivity enhancement is achieved by utilizing mass tags. Accuracy improvement is accomplished through extensive characterization of stability of DNA and protein microarrays. Following stability characterization of the surface chemistry, the substrate, and the immobilized biomolecules, microarray fabrication methods and normalization techniques are developed to reduce error, hence, increasing the accuracy of quantitative analysis. The degradations of the sensor surface that we discover from stability characterization are susceptible to other label-free biosensors. Thus, the correction strategies that we present can be utilized for accurate quantitative analysis of a variety of label-free biosensors. Second, we develop four applications for IRIS. 1) We present applications for quantitative gene expression analysis and disease screening using DNA microarrays by demonstrating quantitative analysis of DNA hybridization and successful detection of single nucleotide polymorphism. 2) We present an application for quantitative analysis of transcription factors using ssDNA and dsDNA microarrays. We discover a new binding motif for TATA-binding protein and propose alternative models for eukaryotic transcription initiation. 3) We present an application to study immune response using antibody microarrays by demonstrating dynamic detection interleukin-6 with ∼ ng/mL sensitivity and successfully detecting the small macromolecule in biologically complex fluid. 4) We propose an application to investigate cellular response to external stimuli, such as drugs, toxins, and pathogens, using patterned cell-protein microarrays. We present strategies of fabricating cell-protein microarrays and demonstrate interleukin-8 detection with ∼ pg/mL sensitivity using gold nanoparticles. Improved performance and diverse biological applications of IRIS will help successful implementations of high-throughput label-free biosensors in healthcare.