Detection of Sequence-Specific Protein-DNA Interactions via Surface Enhanced Resonance Raman Scattering

Abstract

Practical and high-throughput assays for probing protein-ligand interactions are essential for proteomics and drug development. 1 For example, the analysis of multiprotein complexes involved in gene regulation is a combinatorial challenge with applications in medical diagnostics. 2 Here we describe an approach using surfaceenhanced resonance Raman scattering (SERRS) for protein sensing in a tightly controlled assembly of gold nanoparticles and DNA, which has great potential for high sensitivity with high-throughput multiplexing capacity. 3 SERRS techniques greatly enhance signal strength and sensitivity in many applications, with demonstrations of detection limits at the single-molecule level, 4,5 while offering other important benefits over fluorescent detection methods, including resistance to photobleaching and narrow emission peaks for spectral multiplexing. 6 However, the enhancement possible from SERRS is very dependent on the distance between, the surface morphology of, and the optical resonance of closely associated metal nanoparticles, making the design of controlled assemblies paramount to correctly position analytes for optimal detection. 7 We describe an effective architecture of DNA-bridged nanoparticle assemblies for binding and detecting sequence and concentration dependent protein-DNA interactions. Each short stretch of duplex DNA, which is to be bound by the analyte protein, is prepared with overhangs that hybridize and cross-link a generic set of gold nanoparticles (NPs) functionalized with complementary DNA. 8 This self-assembling scaffold allows control of the positioning of metallic NPs to directly surround a DNA sequence recognized by an analyte protein (tagged with a resonance Raman molecule). These NPs are subsequently grown using a silver plating step to decrease the distance between the surfaces and the analyte causing a large increase in SERRS signal, detected by a confocal Raman microprobe. 5,9 The assembly consists of a three-part oligonucleotide scaffolding tethering NPs as shown in Figure 1A. Double-stranded oligonucleotides C (oligo-C) of lengths 15 to 39 base pairs containing the protein binding site of interest were designed to generate appropriate spacing for protein access into the final assemblies, with 12 base pair single-stranded overhangs on each end that are complementary to surface-bound 22 base pair oligonucleotides A or B (oligo-A or oligo-B, DNA sequences in Supporting Information). Gold NPs diameter of ∼13 nm were prepared by citrate reduction of gold aurate, 11 and the resultant citrate shell was displaced by thiolmodified oligo-A or oligo-B 10 . Conjugates were determined to have 183 ( 20 oligonucleotides per particle (Supporting Information). Nanoparticles of this size have previously been used as seeds for silver plating and multiplexed detection by SERRS. 11 Upon annealing of the single-stranded overhangs, the three components condense into assemblies. 10 Variations of assemblies were formed with oligo-C containing the GCGC recognition site of M.HhaI, 12 the T...

Surface-Enhanced Raman Spectroscopy (SERS) Tracking of Chelerythrine, a Na+/K+Pump Inhibitor, into Cytosol and Plasma Membrane Fractions of Human Lens Epithelial Cell Cultures

“…are often limited by low detection limits, increased experimental duration and cost, or indirect observation of the species of interest via a (radio) chemical “label”. Recently, surface-enhanced Raman spectroscopy (SERS) has emerged as a powerful molecular sensing technique due to its high degree of molecular specificity, low detection limits, and multiplex detection capabilities [20,21], permitting accurate determination of distribution and amount of metabolites in biological systems with very short experimental durations, a conditio sine qua non for pharmacological studies.…”

A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G

“…Research developments in biosensor applications for surface enhanced Raman spectroscopy (SERS) have demonstrated a broad range of solutions for the sensitive and selective detection of nucleic acids (Cao et al, 2002;Hering et al, 2008;Harpster et al, 2009), proteins (Xu et al, 2005;Porter et al, 2008;Han et al, 2009a), protein-DNA interactions (Bonham et al, 2007) and in vivo tumor tissue (Qian et al, 2008). Although technical strategies can vary widely, SERS-based assays for analyte detection are largely predicated on the interdependence of target molecule capture and positioning of an appropriate Raman scattering label, at, or in close proximity to roughened noble metal surfaces.…”

Creating Well-Defined Hot Spots for Surface-Enhanced Raman Scattering by Single-Crystalline Noble Metal Nanowire Pairs

“…[3][4][5][6] Because of these advantages of SERS, it can be applied to noninvasive chemical detection and identification of biological molecules such as DNAs and proteins. [7][8][9][10] The strong enhancement of Raman signal has been attributed to "hot spots" that can be generated at the junction between metal surfaces upon irradiation with light. [11][12][13] Since the SERS intensity is primarily dominated by these hot spots, much of the effort to develop SERS into an effective analytical tool has been devoted to fabricate well-defined, reproducible, and controlled hot spot structures.…”