Abstract-A biosensor application of vertically coupled glass microring resonators with Q ∼ 12 000 is introduced. Using balanced photodetection, very high signal to noise ratios, and thus high sensitivity to refractive index changes (limit of detection of 1.8 × 10
A primary advantage of label-free detection methods over fluorescent measurements is its quantitative detection capability, since an absolute measure of adsorbed material facilitates kinetic characterization of biomolecular interactions. Interferometric techniques relate the optical phase to biomolecular layer density on the surface, but the conversion factor has not previously been accurately determined. We present a calibration method for phase shift measurements and apply it to surface-bound bovine serum albumin, immunoglobulin G, and single-stranded DNA.Biomolecules with known concentrations dissolved in salt-free water were spotted with precise volumes on the array surface and upon evaporation of the water, left a readily calculated mass. Using our label-free technique, the calculated mass of the biolayer was compared with the measured thickness, and we observed a linear dependence over 4 orders of magnitude. We determined that the widely accepted conversion of 1 nm of thickness corresponds to ~1 ng/mm 2 surface density held reasonably well for these substances and through our experiments can now be further specified for different types of biomolecules. Through accurate calibration of the dependence of thickness on surface density, we have established a relation allowing precise determination of the absolute number of molecules for single-stranded DNA and two different proteins.
A novel method is proposed for direct detection of DNA hybridization on microarrays. Optical interferometry is used for label-free sensing of biomolecular accumulation on glass surfaces, enabling dynamic detection of interactions. Capabilities of the presented method are demonstrated by highthroughput sensing of solid-phase hybridization of oligonucleotides. Hybridization of surface immobilized probes with 20 base pair-long target oligonucleotides was detected by comparing the label-free microarray images taken before and after hybridization. Through dynamic data acquisition during denaturation by washing the sample with low ionic concentration buffer, melting of duplexes with a single nucleotide mismatch was distinguished from perfectly matching duplexes with high confidence interval (>97%). The presented technique is simple, robust, and accurate, and eliminates the need of using labels or secondary reagents to monitor the oligonucleotide hybridization.
Conformation of a three-dimensional polymeric coating (copoly(DMA-NAS-MAPS)), and immobilization and hybridization of DNA strands on the polymer coated surface are investigated. A conformational change, specifically the swelling of the surface adsorbed polymer upon hydration, is quantified in conjunction with the application of these polymer coatings for DNA microarray applications. Fluorescently labeled short DNA strands (23mer) covalently linked to the functional groups on the adsorbed polymer are used as probes to measure the swelling of the polymer. A fluorescence microscopy technique, Spectral Self-Interference Fluorescence Microscopy (SSFM), is utilized to directly measure the change in axial position of fluorophores due to swelling with subnanometer accuracy. Additionally, immobilization characteristics of single stranded DNA (ssDNA) and double stranded DNA (dsDNA) probes, as well as hybridization of ssDNA with target strands have been studied. The results show that ssDNA further away from the surface is hybridized more efficiently, which strengthens the earlier analysis of this polymeric coating as a simple but highlyefficient and robust DNA microarray surface chemistry.
A platform for in situ and real-time measurement of proteininduced conformational changes in dsDNA is presented. We combine electrical orientation of surface-bound dsDNA probes with an optical technique to measure the kinetics of DNA conformational changes. The sequence-specific Escherichia coli integration host factor is utilized to demonstrate protein-induced bending upon binding of integration host factor to dsDNA probes. The effects of probe surface density on binding/bending kinetics are investigated. The platform can accommodate individual spots of microarrayed dsDNA on individually controlled, lithographically designed electrodes, making it amenable for use as a high throughput assay.DNA conformation | high throughput | optical biosensor | protein-DNA U nderstanding conformational dynamics and orientation of biomolecules is critical in determining their function. For dsDNA, conformational changes result because of interactions with proteins such as histones, transcription factors, and DNAmodifying enzymes. Regulation of gene expression, for example, in both prokaryotes and eukaryotes involves formation of specialized nucleoprotein structures, or snups (1). Within these complexes, distant segments of DNA are brought into close proximity to each other and, frequently, protein-induced DNA bends or kinks are formed. Understanding the DNA conformational changes and forces responsible for bending DNA to form specific complexes are thus of considerable biological significance.Several platforms were recently developed to study protein-DNA interactions. The most popular assays, ChIP chips and DNA adenine methyltransferase identification (DamID) have proven useful for measuring protein-DNA complexes in vivo but do not have high throughput capability. For in vitro measurements, protein-binding microarrays (PBMs) provide a high throughput approach but require cloning and expression of the proteins with an epitope tag. None of the aforementioned approaches provide information indicating the conformational changes of DNA that can result upon protein binding. Methods such as FRET and atomic force microscopy are able to measure DNA conformational changes in protein-DNA complexes but do not allow for high throughput measurements. For platforms wherein probes are surface-bound, such as spectral self-interference fluorescent microscopy, the random orientation of probes limits conformational analyses (2).Here we introduce a platform for in situ real-time measurement of protein-induced conformational changes on immobilized DNA. The platform can accommodate individual spots of microarrayed dsDNA on individually controlled, lithographically designed pixels, making it amenable for use as a high throughput assay. We utilized the sequence-specific Escherichia coli integration host factor (IHF) to measure protein-induced dsDNA bending. IHF functions in many processes that depend on the formation of snups, e.g., DNA replication, transcriptional regulation, and site-specific recombination (3, 4). The primary function of IHF appears t...
We demonstrate an accurate, quantitative, and label-free optical technology for high-throughput studies of receptor-ligand interactions, and apply it to TATA binding protein (TBP) interactions with oligonucleotides. We present a simple method to prepare single-stranded and double-stranded DNA microarrays with comparable surface density, ensuring an accurate comparison of TBP activity with both types of DNA. In particular, we find that TBP binds tightly to single-stranded DNA, especially to stretches of polythymine (poly-T), as well as to the traditional TATA box. We further investigate the correlation of TBP activity with various lengths of DNA and find that the number of TBPs bound to DNA increases >7-fold as the oligomer length increases from 9 to 40. Finally, we perform a full human genome analysis and discover that 35.5% of human promoters have poly-T stretches. In summary, we report, for the first time to our knowledge, the activity of TBP with poly-T stretches by presenting an elegant stepwise analysis of multiple techniques: discovery by a novel quantitative detection of microarrays, confirmation by a traditional gel electrophoresis, and a full genome prediction with computational analyses.
The immune system is a network of cells in which the constitutive members interact through dense and sometimes overlapping connections. The extreme complexity of this network poses a significant challenge for monitoring pathological conditions (e.g., food allergies, autoimmunity, and other chronic inflammatory diseases) and for discovering robust signatures of immunological responses that correlate with or predict the efficacy of interventions. The diversity among immune cells found in clinical samples (variations in cellular functions, lineages, and clonotypic breadth) requires approaches for monitoring immune responses with single-cell resolution.In this chapter, we present an engineering approach for integrated single-cell analysis that uses interchangeable modular operations to provide a comprehensive characterization of the phenotypic, functional, and genetic variations for individual cells. We focus on the use of microfabricated devices to isolate and interrogate single cells, and on the analytical components that enable subsequent detection, correlation, and interpretation of multidimensional sets of data. We discuss specific challenges and opportunities in the realization of this concept, and review two examples where it has been implemented. The presented approach should provide a basis for the design and implementation of nonconventional bioanalytical processes for studying specific responses of an immune system.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.