Aaron D. Strickland

Detection of Carbendazim by Surface-Enhanced Raman Scattering Using Cyclodextrin Inclusion Complexes on Gold Nanorods

Surface-enhanced Raman scattering (SERS) was used for the detection of carbendazim using gold nanorods derivatized with a beta-cyclodextrin derivative to bind this fungicide. Gold nanorods were synthesized with an aspect ratio of 3.3 to match the 785 nm excitation wavelength used in the SERS studies. A thiolated cyclodextrin-based sensor molecule was synthesized, and its inclusion complex with carbendazim was formed and studied using SERS spectroscopy. SERS analysis of the inclusion complex at different concentrations in the presence of gold nanorods afforded good quality Raman spectra of carbendazim at micromolar concentrations. Quantitative analysis was preformed using partial least-squares (PLS), and a calibration plot for these data was generated. Results suggest that carbendazim concentrations as low as 50 microM can be accurately detected using the described SERS assay.

Surface-Enhanced Raman Scattering Based Ligase Detection Reaction

Genomics provides a comprehensive view of the complete genetic makeup of an organism. Individual sequence variations, as manifested by single nucleotide polymorphisms (SNPs), can provide insight into the basis for a large number of phenotypes and diseases including cancer. The ability rapidly screen for SNPs will have a profound impact on a number of applications, most notably personalized medicine. Here we demonstrate a new approach to SNP detection through the application of surface-enhanced Raman scattering (SERS) to the ligase detection reaction (LDR). The reaction uses two LDR primers, one of which contains a Raman enhancer and the other a reporter dye. In LDR, one of the primers is designed to interrogate the SNP. When the SNP being interrogated matches the discriminating primer sequence, the primers are ligated and the enhancer and dye are brought into close proximity enabling the dyes Raman signature to be detected. By detecting the Raman signature of the dye rather than its fluorescence emission, our technique avoids the problem of spectral overlap which limits number of reactions which can be carried out in parallel by existing systems. We demonstrate the LDR-SERS reaction for the detection of point mutations in the human K-ras oncogene. The reaction is implemented in an electrokinetically active microfluidic device that enables physical concentration of the reaction products for enhanced detection sensitivity and quantization. We report a limit of detection of 20 pM of target DNA with the anticipated specificity engendered by the LDR platform.

Multiplex single nucleotide polymorphism genotyping utilizing ligase detection reaction coupled surface enhanced Raman spectroscopy.

Single nucleotide polymorphisms (SNPs) are one of the key diagnostic markers for genetic disease, cancer progression, and pharmcogenomics. The ligase detection reaction (LDR) is an excellent method to identify SNPs, combining low detection limits and high specificity. We present the first multiplex LDR-surface enhanced Raman spectroscopy (SERS) SNP genotyping scheme. The platform has the advantage in that the diagnostic peaks of Raman are more distinct than fluorescence, and in theory, a clinically significant number of markers can be multiplexed in a single sample using different SERS reporters. Here we report LDR-SERS multiplex SNP genotyping of K-Ras oncogene alleles at 10 pM detection levels, optimization of DNA labeling as well as Raman conditions, and the linear correlation of diagnostic peak intensity to SNP target concentration in heterozygous samples. Genomic DNA from typed cells lines was obtained and scored for the K-Ras genotype. These advances are significant as we have further developed our new SNP genotyping platform and have demonstrated the ability to correlate genotype ratios directly to diagnostic Raman peak signal intensity.

PAA‐Derived Gold Nanorods for Cellular Targeting and Photothermal Therapy

A single-step LbL procedure to functionalize CTAB-capped GNRs via electrostatic self-assembly is reported. This approach allows for consistent biomolecule/GNR coupling using standard carboxyl-amine conjugation chemistry. The focus is on cancer-targeting biomolecule/GNR conjugates and selective photothermal destruction of cancer cells by GNR-mediated hyperthermia and NIR light. GNRs were conjugated to a single-chain antibody selective for colorectal carcinoma cells and used as probes to demonstrate photothermal therapy. Selective targeting and GNR uptake in antigen-expressing SW 1222 cells were observed using fluorescence microscopy. Selective photothermal therapy is demonstrated using SW 1222 cells, where >62% cell death was observed after cells are treated with targeted A33scFv-GNRs.

Entropically Driven Self-Assembly of Lysinibacillus sphaericus S-Layer Proteins Analyzed Under Various Environmental Conditions

S-Layer proteins are an example of bionanostructures that can be exploited in nanofabrication. In addition to their ordered structure, the ability to self-assembly is a key feature that makes them a promising technological tool. Here, in vitro self-assembly kinetics of SpbA was investigated, and found that it occurs at a rate that is dependent on temperature, its concentration, and the concentration of calcium ions and sodium chloride. The activation enthalpy (120.81 kJ . mol(-1)) and entropy (129.34 J . mol(-1) . K(-1)) obtained infers that the incorporation of monomers incurs in a net loss of hydrophobic surface. By understanding how the protein monomers drive the self-assembly at different conditions, the rational optimization of this process was feasible.

Nitric oxide-releasing polyacrylonitrile disperses biofilms formed by wound-relevant pathogenic bacteria

To test the antimicrobial and antibiofilm properties of a nitric oxide (NO)‐releasing polymer against wound‐relevant bacterial pathogens.

In vitro self-assembly of gold nanoparticle-coated poly(3-hydroxybutyrate) granules exhibiting plasmon-induced thermo-optical enhancements.

Polyhydroxyalkanoate (PHA) synthase attached to gold nanoparticles (AuNP) produce poly(3-hydroxybutyrate) (PHB) upon the addition of 3-hydroxybutyrate-CoA, and then coalesce to form micrometer-sized AuNP-coated PHB granules. These AuNP-coated PHB granules are potential theranostic agents that have enhanced imaging capabilities and are capable of heating upon near-infrared laser irradiation. The AuNP-coated PHB exhibited 11-fold enhancement in surface-enhanced Raman scattering over particles prior polymerization. Stained AuNP-coated PHB exhibited a 6-fold enhancement in fluorescence intensity as well as a 1.3-fold decrease in photobleaching rate compared to PHB granules alone. The granules were also shown to emit heat when illuminated at 808 nm with a 3.9-fold increase in heating rate compared to particles alone.

Integrated reactive ion etching to pattern cross-linked hydrophilic polymer structures for protein immobilization

Patterning of cross-linked hydrophilic polymer features using reactive ion etching (RIE) capable of covalently immobilizing proteins has been achieved. Projection photolithography was used to pattern photoresist to create micromolds. Vapor phase molecular self-assembly of polymerizable monolayer in molds allowed covalent binding of hydrogel on surface during free-radical polymerization. Excess hydrogel blanket film was consumed with oxygen RIE resulting into hydrogel pattern of 1μm size aligned to prefabricated silicon oxide structures. Proteins were finally coupled through their primary amine groups selectively to acid functionalized hydrogel features through stable amide linkages using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysulfosuccinimide.

New designs for portable Raman instrumentation in defense applications

The realization of global terrorism after the September 11 attacks led immediately to a need for rapid field analysis of materials. Colorimetric test kits existed, but they are very subjective to interpret and they require contact with the sample. A push for handheld spectrometers quickly led to FTIR systems with ATR sampling, handheld IMS systems, and handheld Raman spectrometers. No single technique solves all of the problems of field detection. We will discuss the development of Raman instrumentation and, in particular, cover the advantages and the problems that are inherent in Raman portability. Portable Raman instrumentation began with a limited number of accessories: a point-and-shoot and some sort of vial adaptor. Currently this has expanded to stand-off attachments for measurements at a distance, air sampling to look for toxic gasses or aerosols, Orbital Raster Scan (ORS) to spatially average over samples, SERS attachments for trace detection, and fiber optic probes.The realization of global terrorism after the September 11 attacks led immediately to a need for rapid field analysis of materials. Colorimetric test kits existed, but they are very subjective to interpret and they require contact with the sample. A push for handheld spectrometers quickly led to FTIR systems with ATR sampling, handheld IMS systems, and handheld Raman spectrometers. No single technique solves all of the problems of field detection. We will discuss the development of Raman instrumentation and, in particular, cover the advantages and the problems that are inherent in Raman portability. Portable Raman instrumentation began with a limited number of accessories: a point-and-shoot and some sort of vial adaptor. Currently this has expanded to stand-off attachments for measurements at a distance, air sampling to look for toxic gasses or aerosols, Orbital Raster Scan (ORS) to spatially average over samples, SERS attachments for trace detection, and fiber optic probes.