Rapid Nanophotonics Assay for Head and Neck Cancer Diagnosis.

Efficient and timely diagnosis of head and neck squamous cell carcinoma (HNSCC) is a critical challenge, particularly in low and middle income countries. These regions, which are expected to witness a drastic increase in HNSCC rates, are ill-prepared to handle the diagnostic burden due to limited resources, especially the low ratio of pathologists per population, resulting in delayed diagnosis and treatment. Here, we demonstrate the potential of an alternative diagnostic method as a low-cost, resource-efficient alternative to histopathological analysis. Our novel technology employs unique surface-enhanced Raman scattering (SERS) "nanorattles" targeting cytokeratin nucleic acid biomarkers specific for HNSCC. In this first study using SERS diagnostics for head and neck cancers, we tested the diagnostic accuracy of our assay using patient tissue samples. In a blinded trial, our technique demonstrated a sensitivity of 100% and specificity of 89%, supporting its use as a useful alternative to histopathological diagnosis. The implications of our method are vast and significant in the setting of global health. Our method can provide a rapid diagnosis, allowing for earlier treatment before the onset of distant metastases. In comparison to histopathology, which can take several months in remote limited-resources regions, our method provides a diagnosis within a few hours.

Author Correction: Rapid Nanophotonics Assay for Head and Neck Cancer Diagnosis.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Squamous Cell Carcinoma DNA Detection Using Ultrabright SERS Nanorattles and Magnetic Beads for Head and Neck Cancer Molecular Diagnostics.

A rise in head and neck cancers in low and middle countries over recent years has prompted the need for low-cost, resource-efficient diagnostic technologies. Standard diagnosis with histopathology is often not feasible due to the low number of trained pathologists in these regions, resulting in delayed diagnosis and treatment. This study presents an alternative diagnostic method to standard histopathology. We developed a surface enhanced raman scattering (SERS) based method to distinguish squamous cell carcinoma from other cell lines. Using a "sandwich" method employing ultrabright SERA nanorattles and magnetic beads, we directly targeted specific nucleic acid markers of squamous cells. Our method was able to detect the presence of squamous cells with high sensitivity and specificity, supporting its potential for use as a diagnostic tool in head and neck fine needle aspirations (FNA).

Deep UV nano-microstructuring of substrates for surface plasmon resonance imaging.

In this paper, we describe wafer-scale fabrication and characterization of plasmonic chips-containing different sizes and spacings of metallic micro- and nanoline structures-using deep UV lithography. Using a high dose (25 mJ cm( - 2)) and a proper lift-off process, feature sizes as small as 25 nm are obtained. Moreover, we study the dependence of surface plasmon resonance on the angle of incidence and wavelength for different micro- and nanoline size and spacing values, yielding localized to quasi-propagative plasmonic behaviors. Rigorous coupled wave analysis (RCWA) techniques are employed to numerically confirm these experimental observations. Finally, the refractive index of media around the SPRI sensor chips is varied, showing the angulo-spectral regions of higher sensitivity for each type of structure.

Synthesis and characterization of SERS gene probe for BRCA-1 (breast cancer).

A protocol for binding cresyl fast violet (CFV), a SERS-active dye (label) containing an aromatic amino group with a modified oligomer having a carboxy derivatized thymidine moiety using carbodiimide coupling has been achieved for the first time. Covalent coupling between CFV and the oligomer has been confirmed by mass spectral analysis of the labeled oligomer. The fluorescence, SERS and absorption characteristics of the labeled product have been evaluated. The chosen oligomer contains a BRCA-1 (breast cancer) sequence, and hence has the potential for being used as a gene probe to identify BRCA-1 gene. It has high potential for being used in polymerase chain reaction (PCR) amplification, as has been performed with labeled oligonucleotide for the HIV sequence.

Development of a fluorescence detection system using optical parametric oscillator (OPO) laser excitation for in vivo diagnosis.

In this work, the development and applications of a fluorescence detection system using optical parametric oscillator (OPO) laser excitation for in vivo disease diagnosis including oral carcinoma are described. The optical diagnosis system was based on an OPO laser for multi-wavelength excitation and time-resolved detection. The pulsed Nd-YAG-pumped OPO laser system (6 ns, 20 Hz) is compact and has a rapid, broad, and uniform tuning range. Time-gated detection of intensified charge-coupled device (ICCD) making use of external triggering was used to effectively eliminate the laser scattering and contribute to the highly sensitive in vivo measurements. Artificial tissue-simulating phantoms consisting of polystyrene microspheres and tissue fluorophores were tested to optimize the gating parameters. 51-ns gate width and 39-ns gate delays were determined to be the optimal parameters for sensitive detection. In vivo measurements with the optical diagnosis system were applied to esophagus, stomach, and small intestine using an endoscope in canine animal studies. The rapid tuning capability of the optical diagnosis system contributed greatly to the optimization of wavelength for the observation of porphyrin in the small intestine. When the small intestine was thoroughly washed with water, the emission band which corresponds to porphyrin disappeared. Based on this observation, it was concluded that the detected signal was yielded by porphyrin-containing bile secretion. Also, multispectral analyses using multiple excitations from 415 to 480 nm at 5 nm intervals confirmed the porphyrin detection in the small intestine. The optical diagnosis system was also applied to the detection of human xenograft of oral carcinoma in mice using 5-aminolevulinic acid (5-ALA) which is a photodynamic therapy (PDT) drug. Significant differences in protoporphyrin IX fluorescence intensity between normal and tumor tissue could be obtained 2 hours after the injection of 5-ALA into mice due to the preferential accumulation of 5-ALA in tumors. Results reported herein demonstrate potential capabilities of the LIF-OPO system for in vivo disease diagnosis.

Radiation dosimetry using three-dimensional optical random access memories.

The ability to determine particle type and energy plays an important role in the dosimetry of heavy charged particles (HCP) and neutrons. A new approach to radiation dosimetry is presented, which is shown to be capable of particle type and energy discrimination. This method is based on utilising radiation induced changes in the digital information stored on three-dimensional optical random access memories (3D ORAM). 3D ORAM is a small cube (a few mm3) composed of poly(methyl methacrylate) doped with a photochromic dye. and it was originally proposed as a memory device in high speed parallel computers. A Nd:YAG laser system is used to write and read binary information (bits) on the ORAM, which functions as a charged particle detector. Both the read and the write processes use two laser beams that simultaneously strike the material to cause a colour change at their intersection (similar to the darkening of light-sensitive sunglasses when exposed to sunlight.) The laser produces colour changes in the ORAM, which then reverts to the original colour ('bit-flips') at sites where energy is deposited from interaction with incident HCP or neutron-recoil protons. The feasibility of this approach was demonstrated both theoretically and experimentally. Calculations based on track structure theory (TST) predict that when HCP interact with the ORAM material, the local energy deposition is capable of inducing measurable 'bit-flips'. These predictions were recently confirmed experimentally using two types of ORAM systems, one based on spirobenzopyran and the other on anthracene, as the photochromic dyes.

Characterization of antibodies against benzo[a]pyrene with thermodynamic and kinetic constants.

Antibodies of a polyclonal antiserum against benzo[a]pyrene were characterized by determining thermodynamic and kinetic constants of the antigen-antibody reaction. Label-free binding assays with optical detection based on reflectometric interference spectroscopy were performed to determine these constants. Different evaluation methods for kinetic measurements were compared. Also, cross-reactivity against two other polycyclic aromatic hydrocarbons, chrysene and pyrene, was checked. The affinity constant between the antibodies and benzo[a]pyrene in homogeneous phase was determined to be K=(5.3+/-0.3)x10(7) M(-1) which was in the middle of the usual range of antibody affinities. The association rate constant for the reaction at the surface was determined to be (3.8+/-0.9)x10(5) M(-1) s(-1), the dissociation rate constant as (9.7+/-0.5)x10(-3) s(-1). Different evaluation methods applied to the kinetic measurements led to the same results. This antiserum would be suitable for the selective determination of benzo[a]pyrene in concentrated samples.

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.

Microarray sampling-platform fabrication using bubble-jet technology for a biochip system.

The fabrication of microarrays containing PCR-amplified genomic DNA extracts from mice tumors on a Zetaprobe membrane using a modified thermal ink-jet printer is described. A simple and cost-effective procedure for the fabrication of microarrays containing biological samples using a modified bubble-jet printing system is presented. Because of their mass-produced design, ink-jet printers are a much cheaper alternative to conventional spotting techniques. The usefulness of the biochip microarray platform is illustrated by the detection of human fragile histidine triad (FHIT), a tumor suppressor gene. Subcutaneous carcinomas were induced with MKN/FHIT and MKN/E4 cell lines in immunodeficient mice. Several weeks into their development, the tumors from both groups of mice were removed and subjected to DNA extraction by lysis of tissue samples. The extracted DNA samples were amplified by PCR (30 cycles) using the primers corresponding to nucleotides 2 to 18 of the FHIT sequence. The resulting solution was transferred to the individual reservoirs of a three-color cartridge from a conventional thermal ink-jet printer (HP 694C), and arrays were printed on to a Zetaprobe membrane. After spotting, these membranes were used in a hybridization assay, using fluorescent probes, and detected with a biochip.

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.

Application of an antibody biochip for p53 detection and cancer diagnosis.

Detection of the p53 tumor suppressor gene is important in early cancer diagnostics because alterations in the gene have been associated with carcinogenic manifestations in several tissue types in humans. We have developed an antibody-based detection instrument, the biochip, to detect the presence of the anti-p53 antibody in human serum. The design of this highly integrated detector system is based on miniaturized phototransistors having multiple optical sensing elements, amplifiers, discriminators, and logic circuitry on an IC board. The system utilizes laser excitation and fluorescence signals to detect complex formation between the p53 monoclonal antibody and the p53 antigen. Recognition antibodies are immobilized on a nylon membrane platform and incubated in solutions containing antigens labeled with Cy5, a fluorescent cyanine dye. Subsequently, this membrane is placed on the detection platform of the biochip and fluorescence signal is induced using a 632.8-nm He-Ne laser. Using this immuno-biochip, we have been able to detect binding of the p53 monoclonal antibody to the human p53 cancer protein in biological matrices. The performance of the integrated phototransistors and amplifier circuits of the biochip, previously evaluated through measurement of the signal output response for various concentrations of fluorescein-labeled molecules, have illustrated the linearity of the microchip necessary for quantitative analysis. The design of this biochip permits sensitive, selective and direct measurements of a variety of antigen-antibody formations at very low concentrations. Furthermore, the acquisitions of the qualitative and quantitative results are accomplished rapidly, in about 15 min. These features demonstrate the potential of this antibody-based biochip for simple, rapid and early biomedical diagnostics of cancer.

Detection of E. coli using a microfluidics-based antibody biochip detection system.

This work demonstrates the detection of E. coli using a 2-dimensional photosensor array biochip which is efficiently equipped with a microfluidics sample/reagent delivery system for on-chip monitoring of bioassays. The biochip features a 4 x 4 array of independently operating photodiodes that are integrated along with amplifiers, discriminators and logic circuitry on a single platform. The microfluidics system includes a single 0.4 mL reaction chamber which houses a sampling platform that selectively captures detection probes from a sample through the use of immobilized bioreceptors. The independently operating photodiodes allow simultaneous monitoring of multiple samples. In this study the sampling platform is a cellulosic membrane that is exposed to E. coli organisms and subsequently analyzed using a sandwich immunoassay involving a Cy5-labeled antibody probe. The combined effectiveness of the integrated circuit (IC) biochip and the immunoassay is evaluated for assays performed both by conventional laboratory means followed by detection with the IC biochip, and through the use of the microfluidics system for on-chip detection. Highlights of the studies show that the biochip has a linear dynamic range of three orders of magnitude observed for conventional assays, and can detect 20 E. coli organisms. Selective detection of E. coli in a complex medium, milk diluent, is also reported for both off-chip and on-chip assays.

Biomedical implications of protein folding and misfolding.

A review of protein-folding mechanisms is presented to indicate (i) the formation of correctly folded and misfolded forms, and (ii) the biomedical implications involved. Protein mechanisms may be classified into series and series-parallel mechanisms. The mechanism may involve one or more stable intermediate (partially folded, misfolded, with some structure) states. It is the parallel or the 'off-pathway' step(s) that lead to the aggregate (misfolded state). This aggregate state yields the amyloid deposits in human tissue that lead to diseases in humans that are resistant to treatment. Means by which the off-pathway step may be minimized and the correct folding pathway enhanced are presented. Particular emphasis is placed on the in vivo machinery (chaperones) that is in place in the cells, and how it functions to allow the proteins to fold in the correct and active form, thus minimizing the amyloid deposits in tissues. More effort needs to be placed to understand the function of these chaperones, and thus help facilitate the formation of active and stable proteins in the cells. Though more effort needs to be placed in the present treatment strategies, novel treatment procedures also need to be explored in order to develop effective strategies to treat these seemingly intractable diseases.

A kinetic analysis using fractals of cellular analyte-receptor binding and dissociation.

A fractal analysis is presented for cellular analyte-receptor binding and dissociation kinetics using a biosensor. Data taken from the literature may be modelled, in the case of binding, using a single-fractal analysis or a dual-fractal analysis. The dual-fractal analysis represents a change in the binding mechanism as the reaction progresses on the surface. The predictive relationship developed for the equilibrium constant, K (affinity which is equal to k(d)/k(1or2)), as a function of the analyte concentration is of particular value since it provides a means by which the affinity may be manipulated. This should be of assistance in cell-surface reactions, drug-candidate optimization and for the design of immunodiagnostic devices. Relationships are also presented for the binding and dissociation rate coefficients as a function of their corresponding fractal dimension, D(f) or the degree of heterogeneity that exists on the surface, and the analyte concentration in solution. When analyte-receptor binding or dissociation is involved, an increase in the heterogeneity on the surface (increase in D(f) or D(fd) as the case may be) leads to an increase in the binding and the dissociation rate coefficients. It is suggested that an increase in the degree of heterogeneity on the surface leads to an increase in the turbulence on the surface owing to the irregularities on the surface. This turbulence promotes mixing, minimizes diffusional limitations and leads subsequently to an increase in the binding and the dissociation rate coefficients. The binding and dissociation rate coefficients are rather sensitive to the degree of heterogeneity, D(f) and D(fd), respectively, that exists on the biosensor surface. The heterogeneity on the surface in general affects the binding and dissociation rate coefficients differently. In general, the analyte concentration in solution has a mild affect on the fractal dimension for binding or the fractal dimension for dissociation. This is indicated by the low values of the exponent in the predictive relationships developed.

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.

Antibody-based biosensor for breast cancer with ultrasonic regeneration.

We describe a novel method and instrumental setup for regenerating antibodies immobilized on a fiberoptic probe of an immunosensor using ultrasonic irradiation with broadband imaging transducers. The instrumental setup and irradiation conditions for antibody regeneration using ultrasound are described. The results of the measurements with antibody against breast cancer antigen illustrate the effectiveness and potential of the regenerable immunosensor. A 65% removal of the antigens bound to the Mab immobilized on the fiber surface is attained after ultrasound regeneration.

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+/-0.2x10(-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.

Three-dimensional optical random access memory materials for use as radiation dosimeters.

This article describes the development of the first three-dimensional optical random access memory (3D-ORAM) material and readout system for monitoring energetic neutrons. Two different photochromic dyes, 5'-chloro-6-nitro-1',3',3'-trimethylspiro-[2H-1-benzopyran-2,2'-in doline] (spirobenzopyran) and anthracene, have been investigated for use in these 3-D ORAM dosimeter materials. These dyes were immobilized in a poly(methyl methacrylate) support, and the resulting dosimeter materials were irradiated with neutrons from a Cf-252 source. Fluorescence measurements from the dosimeter show a dramatic decrease in the overall fluorescence intensity of the 3D-ORAM dosimeter exposed to the Cf-252, relative to a nonirradiated dosimeter. In addition, a two-photon excitation readout system has been developed for determining characteristics of the radiation that are necessary for estimating dose.