Guy D. Griffin

Antibody-Based Fiberoptics Biosensor for the Carcinogen Benzo(a)pyrene

A new antibody-based fiberoptics biosensor was used to detect the important carcinogen benzo(a)pyrene (BaP). The fiberoptics sensor utilizes anti-BaP antibodies covalently bound to its tip. A helium cadmium laser was used as the excitation source to induce fluorescence from BaP conjugated to the bound anti-BaP antibodies. The fiberoptics device can detect 1 femtomole of BaP in a 5-μL sample drop.

Intracellular integration of synthetic nanostructures with viable cells for controlled biochemical manipulation

We demonstrate the integration of vertically aligned carbon nanofibre (VACNF) elements with the intracellular domains of viable cells for controlled biochemical manipulation. Deterministically synthesized VACNFs were modified with either adsorbed or covalently-linked plasmid DNA and were subsequently inserted into cells. Post insertion viability of the cells was demonstrated by continued proliferation of the interfaced cells and long-term (}22

Antibody-based nanoprobe for measurement of a fluorescent analyte in a single cell

>> 22 day) expression of the introduced plasmid. Adsorbed plasmids were typically desorbed in the intracellular domain and segregated to progeny cells. Covalently bound plasmids remained tethered to nanofibres and were expressed in interfaced cells but were not partitioned into progeny, and gene expression ceased when the nanofibre was no longer retained. This provides a method for achieving a genetic modification that is non-inheritable and whose extent in time can be directly and precisely controlled. These results demonstrate the potential of VACNF arrays as an intracellular interface for monitoring and controlling subcellular and molecular phenomena within viable cells for applications including biosensors, in vivo diagnostics, and in vivo logic devices.

SURFACE-ENHANCED RAMAN SCATTERING (SERS) METHOD AND INSTRUMENTATION FOR GENOMICS AND BIOMEDICAL ANALYSIS

We report here the application of an antibody-based nanoprobe for in situ measurements of a single cell. The nanoprobe employs antibody-based receptors targeted to a fluorescent analyte, benzopyrene tetrol (BPT), a metabolite of the carcinogen benzo[a]pyrene (BaP) and of the BaP–DNA adduct. Detection of BPT is of great biomedical interest, since this species can serve as a biomarker for monitoring DNA damage due to BaP exposure and for possible precancer diagnosis. The measurements were performed on the rat liver epithelial clone 9 cell line, which was used as the model cell system. Before making measurements, the cells were treated with BPT. Nanoprobes were inserted into individual cells, incubated 5 min to allow antigen–antibody binding, and then removed for fluorescence detection. We determined a concentration of 9.6 ± 0.2 × 10−11 M for BPT in the individual cells investigated. The results demonstrate the possibility of in situ measurements inside a single cell using an antibody-based nanoprobe.

Plasmonic nanoprobes: from chemical sensing to medical diagnostics and therapy

The development of a surface-enhanced Raman scattering (SERS) method and instrument for use in biomedical and genomics analysis is described. The technology uses DNA gene probes based on SERS labels for gene detection and DNA mapping. The detection method uses nanostructured metallic substrates as SERS-active platforms. The surface-enhanced Raman gene (SERGen) probes can be used to detect DNA targets via hybridization to DNA sequences complementary to these probes. The probes do not require the use of radioactive labels and have great potential to provide both sensitivity and selectivity. Advanced instrumental systems designed for point-source spectral measurements and for multi-spectral imaging (MSI) are described. The MSI concept allows recoding the entire SERS spectrum for every pixel on the two-dimensional hybridization platform in the field of view with the use of a rapid-scanning solid-state device, such as the acousto-optic tunable filter (AOTF). The usefulness of the SERGen approach and its applications in biomedical diagnostics, high-throughput analysis and DNA mapping and sequencing are discussed. Copyright

Hyperspectral surface-enhanced Raman imaging of labeled silver nanoparticles in single cells

This article provides an overview of the development and applications of plasmonics-active nanoprobes in our laboratory for chemical sensing, medical diagnostics and therapy. Molecular Sentinel nanoprobes provide a unique tool for DNA/RNA biomarker detection both in a homogeneous solution or on a chip platform for medical diagnostics. The possibility of combining spectral selectivity and high sensitivity of the surface-enhanced Raman scattering (SERS) process with the inherent molecular specificity of nanoprobes provides an important multiplex diagnostic modality. Gold nanostars can provide an excellent multi-modality platform, combining two-photon luminescence with photothermal therapy as well as Raman imaging with photodynamic therapy. Several examples of optical detection using SERS and photonics-based treatments are presented to illustrate the usefulness and potential of the plasmonic nanoprobes for theranostics, which seamlessly combines diagnostics and therapy.

Surface-enhanced Raman scattering detection of chemical and biological agent simulants

We describe the development of an acousto-optic tunable filter (AOTF)-based hyperspectral surface-enhanced Raman imaging (HSERI) system equipped with an intensified charged coupled device and an avalanche photodiode. The AOTF device is a miniature rapid-scanning solid-state device that has no moving parts and can be rapidly tuned (microseconds) either sequentially or randomly, over a wide spectral range between 600 and 900nm [corresponding to a large relative wave number range (∼0–4500cm−1)], with respect to a 632.8nm excitation and can also acquire images at a fairly narrow band of ∼7cm−1. In this article we describe a confocal surface-enhanced Raman imaging (SERI) system developed in our laboratory that combines hyperspectral imaging capabilities with surface-enhanced Raman scattering (SERS) to identify cellular components with high spatial and temporal resolution. The HSERI system’s application to cellular imaging is demonstrated using SERS-labeled nanoparticles in cellular systems.

Sulfur mustard as a carcinogen: Application of relative potency analysis to the chemical warfare agents H, HD, and HT

Surface-enhanced Raman scattering spectra of chemical and biological agent simulants, such as dimethyl methylphonate, pinacolyl methylphosphonate, diethyl phosphoramidate, 2-chloroethyl ethylsulfide, bacillus globigii, erwinia herbicola, and bacillus thuringiensis were obtained from silver-oxide film-deposited substrates. Thin AgO films ranging in thickness from 50 to 250 nm were produced by chemical bath deposition onto glass slides. Further Raman intensity enhancements were noticed in UV irradiated surfaces due to photo-induced Ag nanocluster formation, which may provide a possible route to producing highly useful plasmonic sensors for the detection of chemical and biological agents upon visible-light illumination.

A Portable Fiberoptic Monitor for Fluorimetric Bioassays

A relative potency method for assessing potential human health effects from exposures to relatively untested chemicals is presented and documented. The need for such a method in evaluating the carcinogenic potential of the chemical warfare agent sulfur mustard (agent HD) from a limited data base is specifically addressed. The best-estimate potency factor for sulfur mustard relative to benzo[a]pyrene is 1.3, with an interquartile range of 0.6 to 2.9. The method is applied to (1) the estimated fence-boundary air concentrations of mustard during operation of a proposed agent incinerator at Aberdeen Proving Ground (APG), Maryland, and (2) the current approved general population exposure level of 1 X 10(-4) mg HD/m3 and the occupational exposure level of 3 X 10(-3) mg HD/m3. Maximum estimates of excess lifetime cancer risk for individuals at sites along the APG boundary range between 3 X 10(-8) and 1 X 10(-7). Lifetime cancer risk estimates less than or equal to 10(-6) are not now regulated by the U.S. Environmental Protection Agency or the Food and Drug Administration. Maximum estimates of excess lifetime cancer risk assuming daily exposure to the approved standards during the proposed 5 years of incinerator operation are on the order of 10(-5) for the general public and 10(-4) for the worker population. These values are considered upper limit estimates.

Nanosensor for in vivo measurement of the carcinogen benzo[a]pyrene in a single cell.

A simple and portable fiberoptic instrument for fluorimetric bioassays is described. The instrument is designed to be readily adaptable to commercially available biotesting wells on microplates. The microwells can also be used as interchangeable sensor heads. The utility of the fiberoptic biosensor is illustrated with a model enzyme-linked immunosorbent assay (ELISA) used to measure varying amounts of rabbit immunoglobulin G from 10 pg to 1 μg. The detection method utilizes an enzyme-amplified immunoassay of 4-methylumbelliferone phosphate as the enzyme substrate. The sensitivity of this assay is three orders of magnitude better than that obtained with a conventional ELISA absorption spectrometric assay using a commercially available reader.