Darren W. Branch

Long-term stability of grafted polyethylene glycol surfaces for use with microstamped substrates in neuronal cell culture

Crucial to long-term stability of neuronal micropatterns is functional retention of the underlying substratum while exposed to cell culture conditions. We report on the ability of covalently bound PEG films in long-term cell culture to continually retard protein adhesion and cell growth. PDMS microstamps were used to create poly-d-lysine (PDL) substrates permissive to cell attachment and growth, and polyethylene glycol (PEG) substrates were used to minimize protein and cell adhesion. Film thickness was measured using null ellipsometry and atomic force microscopy (AFM). Organosilane film structure was examined using Fourier transform infrared (FT-IR) spectroscopy. Long-term film stability in cell culture conditions was tested by immersion in 0.1 M sodium phosphate buffer pH 7.4 for up to one month. Null ellipsometry and water contact measurements indicated that organosilane films were stable up to one month, whereas the PEG film thickness declined rapidly after day 25. Hippocampal cells plated at 200 cells/mm2 on uniform PEG substrates gave a steady increase in biofilm thickness on PEG films throughout the culture, possibly from proteins of neuronal origin. We found that all the layers in the cross-linking procedure were stable in cell culture conditions, with the exception of PEG, which degraded after day 25.

Microstamp patterns of biomolecules for high-resolution neuronal networks

A microstamping technique has been developed for high-resolution patterning of proteins on glass substrates for the localisation of neurons and their axons and dendrites. The patterning process uses a microfabricated polydimethylsiloxane stamp with micrometer length features to transfer multiple types of biomolecules to silanederivatised substrates, using glutaraldehyde as a homobifunctional linker. To test the efficacy of the procedure, substrates are compared in which poly-d-lysine (PDL) was physisorbed and patterned by photoresist with those stamped with PDL. Fluorescein isothiocyanate labelled poly-I-lysine was used to verify the presence and uniformity of the patterns on the glass substrates. As a biological assay, B104 neuroblastoma cells were plated on stamped and physisorbed glass coverslips. Pattern compliance was determined as the percentage of cells on the pattern 8h after plating. Results indicate that the stamping and photoresist patterning procedure are equivalent. Substrates stamped with PDL had an average pattern compliance of 52.6±4.4%, compared to 54.6±8.1% for physisorbed substrates. Measures of background avoidance were also equivalent. As the procedure permits successive stamping of multiple proteins, each with its own micropattern, it should be very useful for defining complex substrates to assist in cell patterning and other cell guidance studies.

Long-term maintenance of patterns of hippocampal pyramidal cells on substrates of polyethylene glycol and microstamped polylysine

For neurons to attach and remain in precise micropatterns for weeks in culture, background molecules that remain nonpermissive for extended culture durations need to be identified. Nonpermissive background molecules of either polyethylene glycol (PEG) or the amino acid serine (C/sub 3/H/sub 7/NO/sub 3/) were evaluated. The foreground regions were microstamped with 3-, 5-, or 10-/spl mu/m lines of poly-D-lysine (PDL), which promotes neural attachment and growth. After 29 days in culture the foreground compliance, or the fraction of all live somata which rested on the desired PDL surface, averaged 86% for serine and 90% for PEG, with only a small decline. The background compliance, or the fraction of square areas in the pattern background which were free of neurite extension, declined from highs of 40% and 55% (midculture) to 5.5% and 12% (29 days) for serine and PEG, respectively. Images of the cultures suggest that PEG is significantly more effective as a nonpermissive substrate. The authors conclude that these materials, especially PEG, are adequate for the maintenance of long-term patterned cultures of neurons. They believe that this is the first report of high-quality long-term patterning of cultured neurons.

Microcontact Printing for Precise Control of Nerve Cell Growth in Culture

Microcontact printing, facilitated by silane linker chemistry and high-relief stamps, creates precise patterns of proteins, which in turn control growth of hippocampal neurons in culture. This additive, multi-mask technique permits several different molecules to be patterned on the same substrate. The covalent linker technology permits relatively long-term (two-week) compliance of neurons to the stamped pattern against a polyethylene glycol background. When polylysine was stamped adjacent to a laminin/polylysine mixture, neural somata and dendrites preferred the polylysine while axons prefer the mixture or the border between the two.

Detection of bioagents using a shear horizontal surface acoustic wave biosensor

Viruses are of high medical and biodefense concern and their detection at concentrations well below the threshold necessary to cause health hazards continues to be a challenge with respect to sensitivity, specificity, and selectivity. Ideally, assays for accurate and real time detection of viral agents would not necessitate any pre-processing of the analyte, which would make them applicable for example to bodily fluids (blood, sputum) and man-made as well as naturally occurring bodies of water (pools, rivers). We describe herein a robust biosensor that combines the sensitivity of surface acoustic waves (SAW) generated at a frequency of 325MHz with the specificity provided by antibodies for the detection of viral agents. A lithium tantalate-based SAW transducer with silicon dioxide waveguide sensor platform featuring three test and one reference delay lines was used to adsorb antibodies directed against either Coxsackie virus B4 or the category A bioagent Sin Nombre virus (SNV), a member of the genus Hantavirus, family Bunyaviridae, negative-stranded RNA viruses. Rapid detection (within seconds) of increasing concentrations of viral particles was linear over a range of order of magnitude for both viruses, although the sensor was approximately 5 x 10(5)-fold more sensitive for the detection of SNV. For both pathogens, the sensors selectivity for its target was not compromised by the presence of confounding Herpes Simplex virus type 1. The biosensor was able to detect SNV at doses lower than the load of virus typically found in a human patient suffering from hantavirus cardiopulmonary syndrome (HCPS). Further, in a proof-of-principle real world application, the SAW biosensor was capable to selectively detect SNV agents in complex solutions, such as naturally occurring bodies of water (river, sewage effluent) without analyte pre-processing. This is the first study that reports on the detection of viral agents using an antibody-based SAW biosensor that has the potential to be used as a hand-held and self-contained device for rapid viral detection in the field.

Removal of Nonspecifically Bound Proteins on Microarrays Using Surface Acoustic Waves

Nonspecific binding of proteins is an ongoing problem that dramatically reduces the sensitivity and selectivity of biosensors. We demonstrate that ultrasonic waves generated by surface acoustic wave (SAW) devices remove nonspecifically bound proteins from the sensing and nonsensing regions of the microarrays. We demonstrate our approach for controllably and nondestructively cleaning the microarray interface. In this work, SAWs were generated using 128 YX lithium niobate, chosen for its high coupling coefficient and efficient power transfer to mechanical motion. These waves propagating along the surface were coupled into specifically bound and nonspecifically bound proteins on a patterned surface of 40 mum feature size. Fluorescence intensity was used to quantify cleaning efficacy of the microarrays. Our results have shown that excess protein layers and aggregates are removed leaving highly uniform films as evidenced by fluorescence intensity profiles. Selected antigen-receptor interactions remained bound during the acoustic cleaning process when subjected to 11.25 mW of power and retained their efficacy for subsequent antigen capture. Results demonstrate near-complete fluorescence signal recovery for both the sensing and nonsensing regions of the microarrays. Of significance is that our approach can be integrated into existing array technologies where sensing and nonsensing regions are extensively fouled. We believe that this technology will be pivotal in the development and advancement of microsensors and their biological applications.

Microcontact printing: A versatile technique for the study of synaptogenic molecules

During synaptogenesis information exchanged locally between synaptic partners results in precise alignment of morphological and molecular specializations. For example, agrin derived from motoneurons induces localized postsynaptic differentiation at the neuromuscular synapse. Similar information molecules are thought to act at other synapses; however, techniques for directly evaluating synaptogenic activities of such molecules are lacking. Here we use agrin-induced differentiation as a model system to validate a novel approach for characterizing synaptogenic molecules. Proteins are patterned with micron scale resolution on glass coverslips by covalent microcontact printing and these substrates are used for cell culture. Postsynaptic molecules accumulate specifically at sites of contact between muscle cells and patterned agrin: a response which is quantifiable. Our results demonstrate that microcontact printing is applicable to the analysis of cellular response to locally immobilized information molecules.

Nonspecific binding removal from protein microarrays using thickness shear mode resonators

Nonspecific binding is a universal problem that reduces bioassay sensitivity and specificity. We demonstrate that ultrasonic waves, generated by 5-MHz quartz crystal resonators, accelerate nonspecifically bound protein desorption from sensing and nonsensing areas of micropatterned protein arrays, controllably and nondestructively cleaning the micropatterns. Nonsensing area fluorescent intensity values dropped by more than 85% and sensing area fluorescent intensity dropped 77% due to nonspecific binding removal at an input power of 14 W. After patterning, antibody films were many layers thick with nonspecifically bound protein, and aggregates obscured patterns. Quartz crystal resonators removed excess antibody layers and aggregates leaving highly uniform films, as evidenced by smaller spatial variations in fluorescent intensity and atomic force microscope surface roughness values. Fluorescent intensity values obtained after 14-W QCR operation were more repeatable and uniform.

Fully integrated switchable filter banks

Fully integrated switchable filter have been successfully demonstrated using a ra CMOS SOI process in conjunction with an a (AlN) microresonator process. Single pole-mul were developed in the CMOS SOI process th multi-project wafer runs while the filters were aluminum nitride based microresonators. Each concurrent design cycles and was demonstrated to integration. After design improvements to bo full monolithic integration was implem microresonator filters with the CMOS switc compatibility of the two technologies. A four ch switchable bank of ∼7MHz bandwidth filters demonstrated exhibiting approximately 8 dB of 60dB of stop band rejection.

Rapid Detection of Ebola Virus with a Reagent-Free, Point-of-Care Biosensor

Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. This preliminary study demonstrates SAW biosensor detection of the Ebola virus in a concentration-dependent manner. The detection limit with this methodology is below the average level of viremia detected on the first day of symptoms by PCR. We observe a log-linear sensor response for highly fragmented Ebola viral particles, with a detection limit corresponding to 1.9 × 104 PFU/mL prior to virus inactivation. We predict greatly improved sensitivity for intact, infectious Ebola virus. This point-of-care methodology has the potential to detect Ebola viremia prior to symptom onset, greatly enabling infection control and rapid treatment. This biosensor platform is powered by disposable AA batteries and can be rapidly adapted to detect other emerging diseases in austere conditions.