Brian M. Cullum

Nanosensors and biochips: frontiers in biomolecular diagnostics ☆

Abstract In the past two decades, the biotechnology and medical fields have seen great advances in the development of novel technologies that open new horizons for identifying and quantifying biomolecules, and diagnosing diseases. This manuscript provides an overview of two of those important technologies, nanosensors and biochips. We describe various types of nanosensors and biochips that have been developed for biological and medical applications, along with significant advances achieved over the last several years in these technologies. Some applications of nanosensors, developed in our laboratory for single-cell analysis, and applications of biochips for biological sensing of pathogenic agents and medical diagnostics are described to illustrate the usefulness and potential of these technologies.

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

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.

The development of optical nanosensors for biological measurements

This article discusses and documents the basic concepts of, and developments in, the field of optical nanosensors and nanobiosensors. It describes the progression of this field of research from its birth up to the present, with emphasis on the techniques of sensor construction and their application to biological systems. After a brief overview of the techniques for fabricating nanometer-sized optical fibers, we describe the various types of transducer and bioreceptor molecule presently used for nanosensor and nanobiosensor fabrication.

Dual Layer and Multilayer Enhancements from Silver Film over Nanostructured Surface-Enhanced Raman Substrates

Novel dual layer and multilayer silver film over nanostructure (SFON) substrates have been developed that provide surface-enhanced Raman scattering (SERS) signal enhancements of greater than 1000% compared to conventional single layer SFON substrates. These substrates provide signal enhancement factors of 3.8 × 105 and greater for a variety of SERS active analytes. Substrate preparation is accomplished by vapor depositing a thick (∼100 nm) layer of silver on top of an underlying layer of alumina nanoparticles, followed by deposition of additional layers of silver with silver oxide layers between them. Unlike previous dual layer silver island based substrates that have been developed, these substrates do not rely on achieving an optimal morphology via deposition of silver. Instead, these substrates rely on the roughness being provided by the original under-layer, providing enhanced substrate homogeneity and more reproducible signals than either silver island substrates or colloidal substrates. In addition, the signal enhancement gives these substrates extended lifetimes compared to conventional single layer SFON substrates. Finally, this study also shows that geometric surface structure and surface roughness factors play little or no role in this enhancement process, allowing for this multilayer fabrication process to be applied to many different types of substrates achieving similar or even greater results.

Development of a compact, handheld Raman instrument with no moving parts for use in field analysis

This article describes a lightweight, self-contained, field portable Raman instrument that has been developed for rapid on-site determination of primary mixture components. The instrument consists of a helium neon (HeNe) laser for excitation, an acousto-optic tunable filter (AOTF) for wavelength discrimination, and an avalanche photodiode for detection. The AOTF is the primary component of this system and has been selected based on its spectral range (600–900 nm) along with its high resolution, ∼7.5 cm−1. Labview™ based software for controlling the AOTF frequency and the signal acquisition has also been developed. Several different samples were analyzed (both solids and liquids) using this instrument for the evaluation of parameters such as spectral resolution, sensitivity, and data acquisition speed for certain environmentally important compounds. The results from these measurements are compared with those using a conventional laboratory Raman system consisting of a grating-based spectrograph and a charg...

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

This work describes the fabrication and the application of an antibody-based fiber-optic nanosensor for in situ measurements of the carcinogen benzo[a]pyrene (BaP) in a single cell. This antibody-based spectroscopic nanosensor is miniaturized enabling the detection of fluorescent analytes in single cells. In addition to measuring fluorescent analytes in single cells, the nanosensor has the potential to be applied for both diagnostic and proteomics purposes. In this work, the human breast carcinoma cell line, MCF-7, was used as the model system to perform BaP measurements in single cells. A standard concentration curve for BaP was established and used to perform quantitative analyses of BaP in individual cells. From these analyses, it was estimated that the concentration of BaP in the individual cells investigated was approximately 3.61 x 10(-10) M. The results obtained demonstrate the application of antibody-based nanosensors for performing in situ measurements inside a single cell.

Development of nanosensors and bioprobes

We describe the development and application of nanosensors having bioreceptor probes for bioanalysis. The nanoprobes were fabricated with optical fibers pulled down to tips having distal end sizes of approximately 30–60 nm. The use of two different types of receptors was investigated. Fiberoptic nanoprobes were covalently bound either with bioreceptors, such as antibodies, or with other receptors, such as cyclodextrins that are selective for the size and chemical structure of the analyte molecules. Theoretical calculations were performed to model the binding of beta-cyclodextrin with pyrene and 5,6-benzoquinoline, and to illustrate the possibility of comparing experimental data with theoretical data. The antibody-based nanoprobe was used for in situ measurements of benzopyrene tetrol in single cells. The performance of the nanosensor is illustrated by intracellular measurements performed on a rat liver epithelial cell line (Clone 9) used as the model cell system. The usefulness and potential of these nanotechnology-based biosensors in biological research and applications are discussed.

Recent advances in plasmonic nanostructures for sensing: a review

Abstract. This review describes the recent advances in plasmonic nanostructures for various sensing applications. In particular, significant advances in surface-enhanced Raman, surface plasmon resonance, and metal-enhanced fluorescence-sensing methodologies associated with the introduction of plasmonic nanostructures, made over the past decade, are highlighted. Plasmonic properties of the various nanostructures employed for each sensing technique are also tabulated to provide a systematic overview of the state-of-the-art in each sensing field. This review is not intended to be a comprehensive compilation of the literature but rather a critical review of the recent significant advances in plasmonic nanostructures for each sensing regime.

Multilayer enhanced gold film over nanostructure surface-enhanced Raman substrates.

We have developed a novel class of gold multilayer, surface-enhanced Raman scattering (SERS) substrates that are capable of enhancing SERS signals by 15.3-fold over conventional gold film over nanostructure (GFON) SERS substrates, making them comparable in sensitivity to optimized silver film over nanostructure (SFON) substrates, while providing the long-term stability obtained from gold. They are fabricated by depositing 10 Å thick silver oxide islands on conventional GFON substrates, followed by deposition of a second continuous gold layer. The silver oxide layer acts as a dielectric spacer between the two continuous gold films and produces significantly enhanced SERS signals, as compared to optimized single layer substrates of the same geometry or comparable substrates prepared by deposition of silver islands that are not oxidized. In addition to the enhanced sensitivity of these multilayer substrates, they also exhibit long SERS active shelf-lives (i.e., months), with no measurable degradation in SERS enhancement, and relative standard deviations in SERS enhancement of less than 5.2% across the substrates surface.

Laser-induced fluorescence studies of polycyclic aromatic hydrocarbons (PAH) vapors at high temperatures.

In this work, we present the fluorescence spectra of anthracene and pyrene vapors at different elevated temperatures (from 150 to 650 degrees C) excited with the 337 nm line of a nitrogen laser. We describe the high temperature effects on the resulting spectral properties including spectral intensity, spectral bandwidth and spectral shift. We found that the PAH fluorescence spectral bandwidths become very broad as the temperature increases. The broadening is mainly due to thermal vibrational sequence congestion. We also have found that the fluorescence intensity of pyrene vapor increases with increasing temperature, which results from the increase of the pyrene vapor absorption cross section at 337 nm.