No 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...

Two double-cysteine mutants of a small protein judiciously modified so that the cysteines appear at axially opposite sides of the native fold were prepared such that different axes were defined in the two mutants. Upon reduction, the disulfide bonds are broken, and the proteins act as bifunctional ligands toward Ag nanoparticles, encouraging their assembly into nanoparticle dimers and small aggregates such that, when excited with laser light, the proteins are automatically located at electromagnetic hot spots within the aggregates. Because the protein molecules are small (~2.3 nm) and because the electromagnetic energy at a hot spot tends to increase as the size of the interparticle gap decreases, this nanoparticle-protein-nanoparticle geometry significantly enhances the Raman emission at the metallic surface. Exploiting this effect, we have recorded surface-enhanced Raman spectra (SERS) of the proteins at near-single-molecule level. The observed SERS spectra were dominated by the vibrations of molecular groups near the anchor points of the proteins.

A proposed tangential flow ultrafiltration method was compared to the widely used ultracentrifugation method for efficiency and efficacy in concentrating, size selecting, and minimizing the aggregation state of a silver nanoparticle (AgNP) colloid while probing the AgNPs' SERS-based sensing capabilities. The ultrafiltration method proved to be more efficient and more effective and was found to tremendously boost the SERS-based sensing capabilities of these AgNPs through the increased number of homogeneous SERS hot spots available for a biotarget molecule within a minimal focal volume. Future research studies and applications addressing the physiochemical properties or biological impact of AgNPs would greatly benefit from ultrafiltration for its ability to generate monodisperse colloidal nanoparticles, to eliminate excess toxic chemicals from nanoparticle synthesis, and to obtain minimum levels of aggregation during nanoparticle concentration.

R ecently, the National Science Foundation projected that the current 10-billion dollar nanotechnology sector will employ 2 million workers, including as many as 1 million workers in the United States. 1 It is expected that over 80% of the jobs created in this sector will require trained individuals in nanoscience. However, little training at the undergraduate level has been initiated to provide highly specialized scientists to this rapidly developing field. The proposed laboratory experiment, which was implemented for both undergraduate and graduate student laboratories in physical chemistry and nanotechnology, addresses the future projected demand.In 1998, G. C. Weaver and K. Norrod proposed an undergraduate laboratory to introduce the surface-enhanced Raman scattering (SERS) effect and to extend the scope of the Raman theory normally covered in physical chemistry courses. 2 A Raman-active molecule, pyridine, was adsorbed on colloidal silver nanoparticles (AgNPs) to demonstrate the large increase in Raman signal. Although successful, the SERS experiment did not estimate the analytical enhancement factor (AEF) and surface enhancement factor (SEF), the most important values for characterizing the SERS effect. 3,4 The Raman signal enhancement of 100À300 times was simply determined by calculating the ratio of integrated areas for specific vibrational modes of pyridine adsorbed on AgNPs and in solution. However, the pyridine concentrations (1.0 Â 10 À1 M for normal Raman and 6.25 Â 10 À3 M for SERS measurements) were extremely large when compared with the trace amounts of analyte that are now detected via SERS. In the following years, theoretical and experimental studies have demonstrated that single-molecule SERS-based detection and identification can be achieved under favorable circumstances. 5,6 Because of the enormous enhancement, SERS found numerous cutting-edge applications in medical, biological, chemical, military defense, homeland security, pharmacological, and environmental settings. 7À9 Most SERSbased detection and identification applications require an accurate determination of the magnitude of the signal enhancement.In this experiment, Raman and fluorescence spectrophotometers were employed to estimate the analytical and surface enhancement factors for rhodamine 6G adsorbed on a Creighton colloid. 10 Among the many kinds of SERS-active substrates, silver colloids are known to lead to huge enhancement factors and to enable single-molecule SERS experiments. 5À7,11 The Creighton method has been widely used for its simplicity, relative low cost, accessibility, and time efficiency. These parameters were critical in designing a feasible experiment for a laboratory. Not surprisingly, in 2007, Solomon et al. implemented the Creighton procedure for the synthesis of colloidal AgNPs as a new laboratory experiment for a general chemistry class. 12