Thayne L. Edwards

Multiplexed microneedle-based biosensor array for characterization of metabolic acidosis.

The development of a microneedle-based biosensor array for multiplexed in situ detection of exercise-induced metabolic acidosis, tumor microenvironment, and other variations in tissue chemistry is described. Simultaneous and selective amperometric detection of pH, glucose, and lactate over a range of physiologically relevant concentrations in complex media is demonstrated. Furthermore, materials modified with a cell-resistant (Lipidure(®)) coating were shown to inhibit macrophage adhesion; no signs of coating delamination were noted over a 48-h period.

Integrated carbon fiber electrodes within hollow polymer microneedles for transdermal electrochemical sensing.

In this study, carbon fiber electrodes were incorporated within a hollow microneedle array, which was fabricated using a digital micromirror device-based stereolithography instrument. Cell proliferation on the acrylate-based polymer used in microneedle fabrication was examined with human dermal fibroblasts and neonatal human epidermal keratinocytes. Studies involving full-thickness cadaveric porcine skin and trypan blue dye demonstrated that the hollow microneedles remained intact after puncturing the outermost layer of cadaveric porcine skin. The carbon fibers underwent chemical modification in order to enable detection of hydrogen peroxide and ascorbic acid; electrochemical measurements were demonstrated using integrated electrode-hollow microneedle devices.

Lithographically defined 3D nanoporous nonenzymatic glucose sensors.

Nonenzymatic glucose oxidation is demonstrated on highly faceted palladium nanowflower-modified porous carbon electrodes fabricated by interference lithography. Varying electrodeposition parameters were used to control the final shape and morphology of the deposited nanoparticles on the 3D porous carbon which showed a 12 times increase in the electrochemically active surface area over analogous planar electrodes. Extremely fast amperometric glucose responses (achieving 95% of the steady state limiting current in less than 5s) with a linear range from 1 to 10mM and a detection limit of 10 μM were demonstrated. The unusual surface properties of the pyrolyzed photoresist films produced strongly adhered palladium crystal structures that were stable for hundreds of cycles towards glucose oxidation without noticeable current decay.

Orthogonal Cell-Based Biosensing: Fluorescent, Electrochemical, and Colorimetric Detection with Silica-Immobilized Cellular Communities Integrated with an ITO–Glass/Plastic Laminate Cartridge

This is the first report of a living cell-based environmental sensing device capable of generating orthogonal fluorescent, electrochemical, and colorimetric signals in response to a single target analyte in complex media. Orthogonality is enabled by use of cellular communities that are engineered to provide distinct signals in response to the model analyte. Coupling these three signal transduction methods provides additional and/or complementary data regarding the sample which may reduce the impact of interferants and increase confidence in the sensors output. Long-term stability of the cells was addressed via 3D entrapment within a nanostructured matrix derived from glycerated silicate, which allows the device to be sealed and stored under dry, ambient conditions for months with significant retention in cellular activity and viability (40% viability after 60 days). Furthermore, the first co-entrapment of eukaryotic and bacterial cells in a silica matrix is reported, demonstrating multianalyte biodetection by mixing disparate cell lines at intimate proximities which remain viable and responsive. These advances in cell-based biosensing open intriguing opportunities for integrating living cells with nanomaterials and macroscale systems.

A parallel microfluidic channel fixture fabricated using laser ablated plastic laminates for electrochemical and chemiluminescent biodetection of DNA

Herein is described the fabrication and use of a plastic multilayer 3-channel microfluidic fixture. Multilayer devices were produced by laser machining of plastic polymethylmethacrylate and polyethyleneterapthalate laminates by ablation. The fixture consisted of an array of nine individually addressable gold or gold/ITO working electrodes, and a resistive platinum heating element. Laser machining of both the fluidic pathways in the plastic laminates, and the stencil masks used for thermal evaporation to form electrode regions on the plastic laminates, enabled rapid and inexpensive implementation of design changes. Electrochemiluminescence reactions in the fixture were achieved and monitored through ITO electrodes. Electroaddressable aryl diazonium chemistry was employed to selectively pattern gold electrodes for electrochemical multianalyte DNA detection from double stranded DNA (dsDNA) samples. Electrochemical detection of dsDNA was achieved by melting of dsDNA molecules in solution with the integrated heater, allowing detection of DNA sequences specific to breast and colorectal cancers with a non-specific binding control. Following detection, the array surface could be renewed via high temperature (95u2009°C) stripping using the integrated heating element. This versatile and simple method for prototyping devices shows potential for further development of highly integrated, multi-functional bioanalytical devices.

4D-4 Love Wave Acoustic Array Biosensor Platform for Autonomous Detection

The rapid autonomous detection of pathogenic microorganisms and bioagents by field deployable platforms is critical to human health and safety. To achieve a high level of sensitivity for fluidic detection applications, we have developed a 330 MHz Love wave acoustic biosensor on 36deg YX lithium tantalate (LTO). Each die has four delay-line detection channels, permitting simultaneous measurement of multiple analytes or for parallel detection of single analyte containing samples. Crucial to our biosensor was the development of a transducer that excites the shear horizontal (SH) mode, through optimization of the transducer, minimizing propagation losses and reducing undesirable modes. Detection was achieved by comparing the reference phase of an input signal to the phase shift from the biosensor using an integrated electronic multi- readout system connected to a laptop computer or PDA. The Love wave acoustic arrays were centered at 330 MHz, shifting to 325-328 MHz after application of the silicon dioxide waveguides. The insertion loss was -6 dB with an out-of-band rejection of 35 dB. The amplitude and phase ripple were 2.5 dB p-p and 2-3deg p-p, respectively. Time-domain gating confirmed propagation of the SH mode while showing suppression of the triple transit. Antigen capture and mass detection experiments demonstrate a sensitivity of 7.19plusmn0.74deg mm2/ng with a detection limit of 6.7plusmn0.40 pg/mm2 for each channel.

Rapid Detection of Human Immunodeficiency Virus Types 1 and 2 by Use of an Improved Piezoelectric Biosensor

ABSTRACT Disasters can create situations in which blood donations can save lives. However, in emergency situations and when resources are depleted, on-site blood donations require the rapid and accurate detection of blood-borne pathogens, including human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). Techniques such as PCR and antibody capture by an enzyme-linked immunosorbent assay (ELISA) for HIV-1 and HIV-2 are precise but time-consuming and require sophisticated equipment that is not compatible with emergency point-of-care requirements. We describe here a prototype biosensor based on piezoelectric materials functionalized with specific antibodies against HIV-1 and HIV-2. We show the rapid and accurate detection of HIV-1 and HIV-2 in both simple and complex solutions, including human serum, and in the presence of a cross-confounding virus. We report detection limits of 12 50% tissue culture infective doses (TCID50s) for HIV-1 and 87 TCID50s for HIV-2. The accuracy, precision of measurements, and operation of the prototype biosensor compared favorably to those for nucleic acid amplification. We conclude that the biosensor has significant promise as a successful point-of-care diagnostic device for use in emergency field applications requiring rapid and reliable testing for blood-borne pathogens.

Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.

Reactive Ion Etching of Gold‐Nanoparticle‐Modified Pyrolyzed Photoresist Films

The high surface-area-to-volume ratio of nanometer-sized particles results in many unique properties. In many instances metal nanoparticles exhibit a higher degree of catalytic activity when compared to their bulk materials. As a result, nanoparticle-modified electrodes have been extensively studied for the preparation of highly active electrode surfaces. In addition to providing catalytic sites, nanoparticles can be used to increase conductivity, and provide a suitable surface for the immobilization of various ligands and biomolecules as well as facilitating the direct electron transfer for a number of redox-active enzymes.While there are many techniques used to adhere metal nanoparticles to electrode surfaces electrodeposition is one of the most common and facile methods due to simplicity and ease of preparation while the final nanoparticle size and surface density can be controlled by varying the deposition time, potential, and metal ion concentration in solution. Upon longer deposition times, however, the nucleation and growth of deposited particles becomes difficult to control with the result being larger, polydisperseparticles.Hereinwe report that theunique surface properties of pyrolyzed photoresist films (PPF) leads to a smaller and higher density of gold nanoparticles (AuNP) when compared to similar electrodeposition conditions onto other common electrode materials. Further, reactive ion etching of

A system of parallel and selective microchannels for biosensor sample delivery and containment

This paper presents an integrated microfluidic system for selectively interrogating parallel biosensors at programmed time intervals. Specifically, the microfluidic system is used for delivering a volume of sample from a single source to a surface-based arrayed biosensor. In this case the biosensors were an array of electrochemical electrodes modified with sample specific capture probes. In addition, the sample was required to be captured, stored and removed for additional laboratory analysis. This was accomplished by a plastic laminate stack in which each thin laminate was patterned by CO2 laser ablation to form microchannels and two novel valves. The first valve was a normally closed type opened by heat via an electrically resistive wire. The second valve was a check type integrated into a removable storage chamber. This setup allows for remote and leave-behind sensing applications and also containment of sensed sample for further laboratory analysis.