Biomedical subjects

Marc D Porter

Publications and source records attributed to Marc D Porter.

At least 19 recordsLinked to original sources

PubMed2006

Control of antigen mass transfer via capture substrate rotation: an absolute method for the determination of viral pathogen concentration and reduction of heterogeneous immunoassay incubation times.

Immunosorbent assays are commonly employed as diagnostic tests in human healthcare, veterinary medicine and bioterrorism prevention. These assays, however, often require long incubation times, limiting sample throughput. As an approach to overcome this weakness, this paper examines the use of rotating capture substrates to increase the flux of antigen to the surface, thereby reducing the incubation time. To assess the capability of this approach, porcine parvovirus (PPV) was selectively extracted from solution by systematically varying the rotation rate of a gold substrate modified with a layer of anti-PPV monoclonal antibodies. The captured PPV were then directly imaged and quantified by atomic force microscopy. The benefits of substrate rotation are demonstrated by comparing an assay performed under stagnant conditions to one carried out with substrate rotation at 800 rpm, both for 10 min incubations at 25 degrees C. The use of rotation lowered the limit of detection to 3.4x10(4)TCID50/mL (approximately 80 fM) from 3.2x10(5)TCID50/mL (approximately 800 fM) under stagnant conditions. Results are also presented that show this strategy can be used: (1) to determine antigen concentrations without standards and (2) to establish the numerical relationship between quantal concentration units (e.g., 50% tissue culture infective dose (TCID50)) and quantitative concentration units (e.g., viruses/mL) The potential to broadly apply this technique to heterogeneous immunoassays is also briefly discussed.

Antibodies, Monoclonal↗

PubMed2006

Labeled gold nanoparticles immobilized at smooth metallic substrates: systematic investigation of surface plasmon resonance and surface-enhanced Raman scattering.

This paper experimentally and theoretically investigates the influence of an underlying metallic substrate (i.e., gold and silver) on the surface plasmon resonance (SPR) of labeled gold nanoparticles and the concomitant impact on the surface-enhanced Raman scattering (SERS) signal from the labels. These experiments employ nanoparticles of varied sizes (30-100 nm) that are coated with a bifunctional Raman scatterer composed of (1) a disulfide for chemisorption to the nanoparticle surface, (2) a succinimidyl ester for formation of a covalent linkage to an amine-terminated self-assembled monolayer on the underlying substrate, and (3) an aryl nitro group with an intrinsically strong Raman active vibrational mode. This approach allows facile systematic assessments of how variations in nanoparticle size, substrate composition, and the gap between the nanoparticle and substrate affect the SPR of the bound particles. Both UV-vis transmission and reflection absorption (incident angle of 58 degrees ) spectroscopy are used to characterize the effect of each of these parameters on SPR. These results are then correlated with SERS enhancement factors (EFs) that were determined by accounting for particle surface concentrations, which were measured by atomic force microscopy, and the absolute number of labels, which were calculated on the basis of the surface area of each of the different-sized particles. All SERS spectra were collected at an incident angle of 58 degrees with respect to the surface normal. As expected, the SPR for particles in solution red-shifts with increasing particle size. More importantly, the SPR moves to even longer wavelengths as the size of immobilized particles increases and as the gap between the immobilized particle and substrate decreases. The red shift is also greater for a gold nanoparticle tethered to a gold substrate compared to a silver substrate. A theoretical model for the extinction of a particle above a flat substrate, corrected for surface scattering, radiation damping, and dynamic depolarization, is also briefly detailed. SPR results calculated with the model are consistent with the shifts observed in the SPR position for each of the manipulated experimental variables. The largest EFs are found for samples with an SPR maximum (lambda(max)) between the wavelengths for laser excitation (633 nm) and the Raman band for the symmetric nitro stretch of the particle coating (690 nm). As an example, an order of magnitude in the SERS enhancement factor is gained for a 60-nm particle immobilized 1.2 nm above a gold substrate (SPR lambda(max) = 657 nm) compared to that for a 30-nm particle (SPR lambda(max) = 596 nm).

Chemistry, Physical↗

PubMed2006

Single molecule adsorption at compositionally patterned self-assembled monolayers on gold: role of domain boundaries.

This paper examines the single-molecule adsorption of YOYO-I-labeled lambda-DNA at compositionally patterned self-assembled monolayers (SAMs). The interactions of fluorescently labeled lambda-DNA molecule with the patterned SAMs, which are comprised of different functional groups (i.e., amine-, alcohol-, and acid-terminated thiolates), were monitored at optically transparent gold films using total internal reflection fluorescence microscopy. The role of solution pH, lambda-DNA concentration, and domain size was investigated. In addition to delineation of the relative adsorption strength as a function of terminal group identity (NH2 > COOH > OH), the potential importance of structural defects was also revealed. The latter result, found both at the disordered boundaries between domains and at adlayers in which structural order was affected by the length of the alkyl chain, points to the subtle but preferential adsorption of the "sticky ends" of lambda-DNA. These experiments also detected an intriguing dependence of adsorption with respect to domain size.

Adsorption↗

PubMed2006

Fabrication of optically transparent carbon electrodes by the pyrolysis of photoresist films: approach to single-molecule spectroelectrochemistry.

This paper describes the preparation, physical and chemical characterization, and performance of carbon-based optically transparent electrodes (C-OTEs) fabricated by the pyrolysis of thin films of photoresist. The electrodes are prepared by spin coating dilute solutions of the positive photoresist AZ 4330 onto quartz substrates. Pyrolysis of these samples at 1000 degrees C in a reducing atmosphere yields optically transparent carbon films that have thicknesses ranging between 10 and 80 nm. Sheet resistance measurements, X-ray photoelectron spectroscopy, Raman spectroscopy, and atomic force microscopy were used to determine the physical and chemical properties of the films, and cyclic voltammetry and chronoabsorptometry were employed to delineate the electrochemical and conventional spectroelectrochemical performance of the C-OTEs. These findings showed that the transparency of this material improves as film thickness decreases, but at the expense of an increase in film resistance. At a wavelength of 500 nm, for example, 13- and 79-nm-thick films have transparencies of 47 and 10% and sheet resistances of 1100 and 210 Omega/ square, respectively. Importantly, adjusting the dilution factor allows the facile and reproducible variation of thickness and transparency. Preliminary results using these C-OTEs for single-molecule spectroelectrochemistry, which represents a new development in the merger of optical and electrochemical techniques, by probing the potential dependence of the adsorption of individual YOYO-I-labeled lambda-DNA are also presented.

Journal Article↗

PubMed2006

Ultrafast concentration and speciation of chromium(III) and (VI).

There is an increasing need to know the concentrations of chromium(III) and (VI) separately rather than only the total chromium content. A method is described for accomplishing this very quickly using only low-cost, portable equipment. Two small, resin-loaded extraction disks are placed one on top of the other in a plastic holder. Then a syringe containing the aqueous sample is attached to the holder and the sample is pushed through the disks. In a matter of seconds, all of the chromium(VI) is retained on the top anion-exchange disk and chromium(III) is extracted by the second cation-exchange disk. The concentrations on each disk are several hundredfold higher than they were in the original sample. The amounts of chromium(III) and (VI) extracted are measured directly on the surface of the respective disks by diffuse reflectance spectroscopy (DRS). Despite the low molar absorptivity of chromium(III) in aqueous solution, the concentration on the upper most layer on the extraction disk is high enough to permit the determination of chromium(III) in samples at the low mg/L range. Chromium(VI) can also be determined at low to sub-mg/L concentrations. A study of the cation-exchange disks was undertaken to compare the performance characteristics of disks containing sulfonated resins and those with iminodiacetate functionality. In addition, data are presented to show the effects of heating the iminodiacetate disks after the initial extraction. The disks were heated in hot water for 15-30 min to complete the slow complexation reaction on the surface.

Anion Exchange Resins↗

PubMed2005

Electrochemically modulated liquid chromatography and the Gibbs adsorption equation.

Electrochemically modulated liquid chromatography uses a conductive material like porous graphitic carbon (PGC) as a chromatographic stationary phase and a working electrode. This dual functionality enables manipulation of separations by changes in the potential applied (E(app)) to the packing. Thus, by monitoring the retention factor (k') with respect to E(app), a chromatographic tool for examination of electrosorption processes can be devised. This novel capability is developed herein by examining the retention of charged aromatic compounds at PGC. The Gibbs adsorption equation and related formulations (e.g., the Lippmann equation) are used to determine interfacial excesses (Gamma) of these solutes in different supporting electrolytes, changes in interfacial tension (dgamma), the charge on the electrode (q(M)), and the potential of zero charge (PZC). Values of the PZC were also determined from plots of ln k' versus E(app). In this case, the dependence of ln k' reveals a shift in the PZC to more cathodic values as the strength of specific adsorption by anions as the electrolyte concentration increases. Together, these results provide insights into the retention mechanism and, more generally, to adsorption at electrified carbon electrodes. Extensions of this strategy as a probe of electrified interfaces with respect to mobile-phase composition, temperature, and pressure are briefly described.

Adsorption↗

PubMed2005

Giant magnetoresistive sensors and superparamagnetic nanoparticles: a chip-scale detection strategy for immunosorbent assays.

Thin structures of alternating magnetic and nonmagnetic layers with a total thickness of a few hundred nanometers exhibit a phenomenon known as giant magnetoresistance. The resistance of microfabricated giant magnetoresistors (GMRs) is dependent on the strength of an external magnetic field. This paper examines magnetic labeling methodologies and surface derivatization approaches based on protein-protein binding that are aimed at forming a general set of protocols to move GMR concepts into the bioanalytical arena. As such, GMRs have been used to observe and quantify the immunological interaction between surface-bound mouse IgG and alpha-mouse IgG coated on superparamagnetic particles. Results show the response of a GMR network connected together as a set of two sense GMRs and two reference GMRs in a Wheatstone bridge as a means to compensate for temperature effects. The response can be readily correlated to the amount of the magnetically labeled alpha-mouse IgG that is captured by an immobilized layer of mouse IgG, the presence of which is confirmed with X-ray photoelectron spectroscopy and atomic force microscopy. These results, along with a detailed description of the experimental testing platform, are described in terms of sensitivity, detection limits, and potential for multiplexing.

Animals↗

PubMed2005

Low-level detection of viral pathogens by a surface-enhanced Raman scattering based immunoassay.

The need for rapid, highly sensitive, and versatile diagnostic tests for viral pathogens spans from human and veterinary medicine to bioterrorism prevention. As an approach to meet these demands, a diagnostic test employing monoclonal antibodies (mAbs) for the selective extraction of viral pathogens from a sample in a chip-scale, sandwich immunoassay format has been developed using surface-enhanced Raman scattering (SERS) as a readout method. The strengths of SERS-based detection include its inherent high sensitivity and facility for multiplexing. The capability of this approach is demonstrated by the capture of feline calicivirus (FCV) from cell culture media that is exposed to a gold substrate modified with a covalently immobilized layer of anti-FCV mAbs. The surface-bound FCVs are subsequently coupled with an extrinsic Raman label (ERL) for identification and quantification. The ERLs consist of 60-nm gold nanoparticles coated first with a layer of Raman reporter molecules and then a layer of mAbs. The Raman reporter molecule is strategically designed to chemisorb as a thiolate adlayer on the gold nanoparticle, to provide a strong and unique spectral signature, and to covalently link a layer of mAbs to the gold nanoparticle. The last feature provides a means to selectively tag substrate-bound FCV. This paper describes the development of the assay, which uses cell culture media as a sample matrix and has a linear dynamic range of 1 x 10(6)-2.5 x 10(8) viruses/mL and a limit of detection of 1 x 10(6) viruses/mL. These results reflect the findings from a detailed series of investigations on the effects of several experimental parameters (e.g., salt concentration, ERL binding buffer, and sample agitation), all of which were aimed at minimizing nonspecific binding and maximizing FCV binding efficiency. The performance of the assay is correlated with the number of captured FCV, determined by atomic force microscopy, as a means of method validation.

Buffers↗

PubMed2005

Assessment of supporting electrolyte contributions in electrochemically modulated liquid chromatography.

This paper reports the results of an investigation on the role of the supporting electrolyte in separations using electrochemically modulated liquid chromatography (EMLC) with a porous graphitic carbon stationary phase. With respect to the identity of the supporting electrolyte, the elution strength of the electrolyte anion increased as F- < OH- < BF4- < ClO4- < PF6- for injections of negatively charged aromatic molecules, whereas a 10-fold increase in electrolyte concentration induced a 60% change in retention for the same solutes. Furthermore, both the concentration and composition of the supporting electrolyte affected retention in a manner that varied with the charge of the analyte and applied potential. This behavior is explained using Gouy-Chapman diffuse double layer theory, coupled with comparisons of this theory with closely related models for ion-pair chromatography. Insights into the retention mechanism reveal that an ion-exchange mechanism controls the retention of negatively charged solutes at applied potentials removed from the potential of zero charge (PZC). At potentials close to the PZC, the electrostatic model is less effective with the predominant retention mechanism likely involving hydrophobic interactions with the carbonaceous stationary phase. The combined effects of these findings are demonstrated by using a temporal gradient in supporting electrolyte concentration to optimize an EMLC separation.

Chromatography, Liquid↗

PubMed2005

Single DNA molecules as probes of chromatographic surfaces.

YOYO-I-labeled lambda-DNA was employed as a nanoprobe for different functionalized surfaces to elucidate adsorption in chromatography. While the negatively charged backbone is not adsorbed, the 12-base unpaired ends of this DNA provide exposed purine and pyrimidine groups for adsorption. Self-assembled monolayers (SAMs) formed on gold substrate provide a wide range of choices of surface with well-defined and well-organized functional groups. Patterns of amino-terminated, carboxylic acid-terminated, and hydroxyl-terminated SAMs are generated by lithography. Patterns of metal oxides are generated spontaneously after deposition of metals. By recording the real-time dynamic motion of DNA molecules at the SAMs/aqueous interface, one can study the various parameters governing the retentivity of an analyte during chromatographic separation. Even subtle differences among adsorptive forces can be revealed.

Adsorption↗

PubMed2004

Electrochemically modulated liquid chromatographic separations of inorganic anions.

Inorganic anion retention on a porous graphitic carbon (PGC) stationary phase is investigated by electrochemically modulated liquid chromatography (EMLC). Through various combinations of the potential applied (Eapp) to the PGC packing and/or changes in the composition (sodium salts of tetrafluoroborate, sulfate, and fluoride) and concentration (10, 25, and 50 mM) of an aqueous mobile phase, conditions for the separation of two different inorganic anion mixtures (iodate, bromide, nitrite, and nitrate or iodate, bromate, and chlorate) are developed. Results show that retention was affected by both variables, with the analyte retention factor, k', changing in a few cases by as much as a factor of ca. six. Moreover, plots of In k' are linearly dependent on both Eapp and In [SE], where [SE] is the supporting electrolyte concentration. Based on these findings, insights into the retention mechanism are briefly discussed by drawing on the theory for ion exchange chromatography.

Anions↗

PubMed2004

High-speed electrochemically modulated liquid chromatography.

The performance advantages of carrying out electrochemically modulated liquid chromatography (EMLC) at elevated temperatures and mobile-phase flow rates are investigated. EMLC has the unique ability to manipulate analyte retention and enhance separation efficiencies through changes in the potential applied to a conductive stationary phase. Operation of high-performance liquid chromatography systems at elevated column temperatures also provides pathways to improve chromatographic performance by enhancing analyte diffusivity and facilitating the use of higher mobile-phase flow rates than conventionally attainable. The results show that performing EMLC separations at elevated temperatures (e.g., 100 degrees C) reduces the analysis time of a mixture of aromatic sulfonates in a mixed mobile phase by more than a factor of 20. Moreover, use of higher operating temperatures enables the separation of this mixture with an entirely aqueous mobile phase in less than 2 min.

Chromatography, High Pressure Liquid↗

PubMed2004

Nanoparticle coding: size-based assays using atomic force microscopy.

Described herein is a novel strategy for the construction and interrogation of an assay platform based on (1) the size encoding of labeled nanoparticles; (2) the high imaging resolution of atomic force microscopy; and (3) evaporatively driven self-assembly of dense nanoparticle layers. This strategy employs two different sized nanoparticles that couple in the presence of a target analyte. In this example, one set of particles is a few hundred nanometers in size and acts as a capture substrate, while a second set of smaller particles serve as the analyte label. Thus, by forming an evaporatively assembled layer from a mixture of the two particle dispersions, the imaged size of the smaller particles when bound to the larger capture particles identifies the presence of the analyte. This letter demonstrates the feasibility of our bar-code strategy by concept tests using the binding specificity of biotin-modified silica nanoparticles (300-nm diameter) with streptavidin-labeled gold nanoparticles (10-nm diameter). The potential to extensively multiplex this assay strategy is briefly discussed.

Journal Article↗

PubMed2004

Multiplexed colorimetric solid-phase extraction: determination of silver(I), nickel(II), and sample Ph.

This paper reports the design and ground-based testing of a multiplexed colorimetric solid-phase extraction (MC-SPE) platform for the rapid determination of multiple water quality parameters in a simple set of operational steps. Colorimetric solid-phase extraction (C-SPE) is an analytical platform that combines impregnated colorimetric reagents on a solid-phase extraction membrane and diffuse reflectance spectroscopy to quantify trace analytes in water. In extending C-SPE to MC-SPE, a filter holder that incorporates discrete analysis channels and a jig that facilitates the concurrent operation of multiple syringes have been designed, enabling the simultaneous determination of three different measures of water quality. Separate, single-parameter membranes, placed in a readout cartridge create unique, parameter-specific addresses at the exit of each channel. Following sample exposure, the diffuse reflectance spectrum of each address is collected serially and the Kubelka-Munk function is used to quantify each water quality parameter via calibration curves. Performance evaluations of the MC-SPE platform were conducted using sample pH, silver(I), and nickel(II). Determinations of silver(I) (0.05-0.5 ppm) and nickel(II) (1.8-5.0 ppm) follow established C-SPE methods on reversed-phase extraction membranes using 5-(p-dimethylaminobenzylidene)rhodanine and dimethylglyoxime, respectively, as colorimetric reagents. Sample pH (2.5-5.0) is measured using an anion-exchange membrane impregnated with fluorescein. These determinations require approximately 120 s to complete using a total sample volume of 3.0 mL. The extension of MC-SPE to the determination of a greater number of analytes and its potential application to space and earth-bound monitoring needs are briefly discussed.

Journal Article↗

PubMed2004

Novel biosensor chip for simultaneous detection of DNA-carcinogen adducts with low-temperature fluorescence.

A monoclonal antibody (MAb)-gold biosensor chip with low-temperature laser-induced fluorescence detection for analysis of DNA-carcinogen adducts is described. Optimization of the detection limit, dynamic range, and biosensing applicability of the MAb-gold biosensor chip was achieved by: (1) using dithiobis(succinimidyl propionate (DSP)) as a protein linker and (2) employing recombinant protein A to provide oriented immobilization of the MAbs. The use of DSP, which has a short methylene chain length, led to faster protein binding kinetics and higher protein surface density than a longer dithiobis(succinimidyl undecanoate) (DSU) linker. The incorporation of recombinant protein A increased the distance between the oriented MAb-bound analytes and the gold surface. The increased distance minimized fluorescence quenching, resulting in about a 10-fold increase in the fluorescence signal in comparison with a chip without protein A. The improved chip architecture was used to demonstrate that biosensing of two structurally similar benzo[a]pyrene (BP)-derived DNA adducts, BP-6-N7Gua and BP-diolepoxide-10-N2dG, bound to two specific MAbs immobilized from a mixture at the same address on the chip, is feasible. These mutagenic adducts are formed by one-electron oxidation and monooxygenation pathways, and are depurinating and stable DNA adducts, respectively. It is shown that the DNA adducts can be easily identified at the same address using time-resolved, low-temperature laser-based fluorescence spectroscopy. The current limit of detection is in the low femtomole range. These results indicate that a single biosensor chip consisting of a Au/DSP/protein A/MAb nano-assembly, with analyte-specific MAbs and low-temperature fluorescence detection should be suitable for simultaneous detection and quantitation of the above adducts, as well as the luminescent antigens for which selective MAbs exist.

Benzo(a)pyrene↗

PubMed2003

Femtomolar detection of prostate-specific antigen: an immunoassay based on surface-enhanced Raman scattering and immunogold labels.

A novel reagent for low-level detection in immunoadsorbent assays is described. The reagent consists of gold nanoparticles modified to integrate bioselective species (e.g., antibodies) with molecular labels for the generation of intense, biolyte-selective surface-enhanced Raman scattering (SERS) responses in immunoassays and other bioanalytical applications. The reagent is constructed by coating gold nanoparticles (30 nm) with a monolayer of an intrinsically strong Raman scatterer. These monolayer-level labels are bifunctional by design and contain disulfides for chemisorption to the nanoparticle surface and succinimides for coupling to the bioselective species. There are two important elements in this label design; it both minimizes the separation between label and particle surface and maximizes the number of labels on each particle. This approach to labeling also exploits several other advantages of SERS-based labels: narrow spectral bandwidth, resistance to photobleaching and quenching, and long-wavelength excitation of multiple labels with a single excitation source. The strengths of this strategy are demonstrated in the detection of free prostate-specific antigen (PSA) using a sandwich assay format based on monoclonal antibodies. Detection limits of approximately 1 pg/mL in human serum and approximately 4 pg/mL in bovine serum albumin have been achieved with a spectrometer readout time of 60 s. The extension of the method to multianalyte assays (e.g., the simultaneous determination of the many complexed forms of PSA) is discussed.

Animals↗

PubMed2003

Rapid determination of ions by combined solid-phase extraction--diffuse reflectance spectroscopy.

We introduce colorimetric solid-phase extraction (C-SPE) for the rapid determination of selected ions. This new technique links the exhaustive concentration of an analyte by SPE onto a membrane disk surface for quantitative measurement with a hand-held diffuse reflectance spectrometer. The concentration/measurement procedure is complete in approximately 1 min and can be performed almost anywhere. This method has been used to monitor iodine and iodide in spacecraft water in the 0.1-5.0 ppm range and silver(I) in the range of 5.0-1000 microg/l. Applications to the trace analysis of copper(II), nickel(II), iron(III) and chromium(VI) are described. Studies on the mechanism of extraction showed that impregnation of the disk with a surfactant as well as a complexing reagent results in uptake of additional water, which markedly improves the extraction efficiency.

Cations↗

PubMed2002

Thin films of block copolymer blends for enhanced performance of acoustic wave-based chemical sensors.

The performance of quartz crystal oscillator-based volatile organic compound (VOC) sensors has been enhanced by using coatings made from poly(styrene-block-ethylene-co-butylene-block-styrene) block copolymers blended with resins and homopolymers. Enhanced performance is characterized by a wider operational temperature range (-10 to +50 degrees C) over which the sensors displayed, concurrently, an analyte sensitivity of >0.2 Hz/ppm toluene, minimal energy loss (resistance <120 ohms), and response times of <20 min (time required to reach 90% of full response). Atomic force microscopy images are consistent with a process in which the additive associates with the polystyrene portions of the microphase-separated block copolymer. This association reinforces the rigidity of the polystyrene network while allowing the rapid uptake of VOCs by the softer polyethylene/butylene phase.

Air Pollutants↗