Onnop Srivannavit

Individually addressable parallel peptide synthesis on microchips

Miniaturized, spatially addressable microchips of peptides and peptidomimetics are powerful tools for high-throughput biomedical and pharmaceutical research and the advancement of proteomics. Here we report an efficient and flexible method for the parallel synthesis of peptides on individually addressable microchips, using digital photolithography and photogenerated acid in the deprotection step. We demonstrate that we are able to synthesize thousands of peptides in a 1 cm2 area on a microchip using 20 natural amino acids as well as synthetic amino acid analogs, with high stepwise yields and short reaction-cycle times. Epitope screening experiments using a p53 antibody (PAb240) produced clearly defined binding patterns. The peptidomimetic sequences on the microchip show specific antibody binding and provide insights into the molecular details responsible for specificity of epitope binding. Our approach requires just a conventional synthesizer and a computer-controllable optical module, thereby allowing potential development of peptide microchips for various pharmaceutical and proteomic applications in routine research laboratories.

μParaflo™ Biochip for Nucleic Acid and Protein Analysis

We describe in this chapter the use of oligonucleotide or peptide microarrays (arrays) based on microfluidic chips. Specifically, three major applications are presented: (1) microRNA/small RNA detection using a microRNA detection chip, (2) protein binding and function analysis using epitope, kinase substrate, or phosphopeptide chips, and (3) protein-binding analysis using oligonucleotide chips. These diverse categories of customizable arrays are based on the same biochip platform featuring a significant amount of flexibility in the sequence design to suit a wide range of research needs. The protocols of the array applications play a critical role in obtaining high quality and reliable results. Given the comprehensive and complex nature of the array experiments, the details presented in this chapter is intended merely as a useful information source of reference or a starting point for many researchers who are interested in genome- or proteome-scale studies of proteins and nucleic acids and their interactions.

Chronic In Vivo Evaluation of PEDOT/CNT for Stable Neural Recordings

Objective: Subcellular-sized chronically implanted recording electrodes have demonstrated significant improvement in single unit (SU) yield over larger recording probes. Additional work expands on this initial success by combining the subcellular fiber-like lattice structures with the design space versatility of silicon microfabrication to further improve the signal-to-noise ratio, density of electrodes, and stability of recorded units over months to years. However, ultrasmall microelectrodes present very high impedance, which must be lowered for SU recordings. While poly(3,4-ethylenedioxythiophene) (PEDOT) doped with polystyrene sulfonate (PSS) coating have demonstrated great success in acute to early-chronic studies for lowering the electrode impedance, concern exists over long-term stability. Here, we demonstrate a new blend of PEDOT doped with carboxyl functionalized multiwalled carbon nanotubes (CNTs), which shows dramatic improvement over the traditional PEDOT/PSS formula. Methods: Lattice style subcellular electrode arrays were fabricated using previously established method. PEDOT was polymerized with carboxylic acid functionalized carbon nanotubes onto high-impedance (8.0 ± 0.1 MΩ: M ± S.E.) 250-μm2 gold recording sites. Results: PEDOT/CNT-coated subcellular electrodes demonstrated significant improvement in chronic spike recording stability over four months compared to PEDOT/PSS recording sites. Conclusion: These results demonstrate great promise for subcellular-sized recording and stimulation electrodes and long-term stability. Significance: This project uses leading-edge biomaterials to develop chronic neural probes that are small (subcellular) with excellent electrical properties for stable long-term recordings. High-density ultrasmall electrodes combined with advanced electrode surface modification are likely to make significant contributions to the development of long-term (permanent), high quality, and selective neural interfaces.

A Three-Dimensional 64-Site Folded Electrode Array Using Planar Fabrication

Neuroscience and neuroprosthetic devices are increasingly in need of more compact less invasive 3-D electrode arrays for interfacing with neural tissue. To meet these needs, a folding 64-site 3-D array architecture has been developed. The microstructure, in which four probes and two platforms are fabricated as a single planar unit, results in a low-profile (<; 350-μm) narrow-platform (0.604-mm2 silicon footprint) implant for cortical use. Signals are routed from 177-μm2 iridium sites through polysilicon lines to the probe back end and then across 4-μm-thick parylene-encased electroplated-gold folding lead transfers to the associated platform. Three levels of interconnect with a 10-μm minimum pitch are utilized for the 32 leads that traverse the platforms. After rapid microassembly, micromachined latches are used to fasten the folded device. Two flexible parylene cables with gold leads at a 20- μm pitch are monolithically integrated with the probes to minimize tethering and avoid any need for lead bonding within the array, and these cables carry the neural signals to a remote circuit module or percutaneous connector. With thin (~15-μm) boron-doped shanks at a ~ 200-μm pitch, the array displaces only 1.7% of the 0.64-mm2 instrumented tissue area, assuming a 100-μm recording range. Neural signals were recorded in vivo from the guinea pig auditory cortex.

A 3-D 160-Site Microelectrode Array for Cochlear Nucleus Mapping

A 3-D application-specific microelectrode array has been developed for physiological studies in guinea pig cochlear nucleus (CN). The batch-fabricated silicon probes contain integrated parylene cables and use a boron etch-stop to define 15μm-thick shanks and limit tissue displacement. Targeting the ventral (three probes) and dorsal (two probes) subnuclei, the custom four-shank 32-site probes are combined in a slotted block platform having a 1.18-mm2 footprint. The device has permitted, for the first time, high-density 3-D in vivo studies of ventral CN to dorsal CN connections, stimulating with 1000 μm2 sites in one subnucleus while recording with 177 μm 2 sites in the other. Through these experiments, it has demonstrated the efficacy of bimodal silicon arrays to better understand the central nervous system at the circuit level. The 160 electrode sites also provide a high-density neural interface, which is an essential aspect of auditory prosthesis prototypes.

Cytophobic surface modification of microfluidic arrays for in situ parallel peptide synthesis and cell adhesion assays.

A combination of PEG‐based surface passivation techniques and spatially addressable SPPS (solid‐phase peptide synthesis) was used to demonstrate a highly specific cell‐peptide adhesion assay on a microfluidic platform. The surface of a silicon‐glass microchip was modified to form a mixed self‐assembled monolayer that presented PEG moieties interspersed with reactive amino terminals. The PEG provided biomolecular inertness and the reactive amino groups were used for consequent peptide synthesis. The cytophobicity of the surface was characterized by on‐chip fluorescent binding assays and was found to be resistant to nonspecific attachment of cells and proteins. An integrated system for parallel peptide synthesis on this reactive amino surface was developed using photogenerated acid chemistry and digital microlithography. A constant synthesis efficiency of >98% was observed for up to 7mer peptides. To demonstrate specific cell adhesion on these synthetic peptide arrays, variations of a 7mer cell binding peptide that binds to murine B lymphoma cells were synthesized. Sequence‐specific binding was observed on incubation with fluorescently labeled, intact murine B lymphoma cells, and key residues for binding were identified by deletional analysis.

Light-directed simultaneous synthesis of oligopeptides on microarray substrate using a photogenerated acid.

Photogenerated acid (PGA) was used as the acid to remove the protection group from amino acids or peptide oligomers. Comparative study of the deprotection using a PGA, trisarylsulfonium antimonyhexafluoride (SSb), and trifluoroacetic acid (TFA) was performed on glass microscope slides. The results showed that PGA can replace TFA in the deprotection step of oligopeptide synthesis with comparable efficiencies. Acids needed for the deprotection step were generated in situ by light activiation of the precursor molecule on the microwell substrate. A maskless laser light illumination system was used to activate the precursor. The accuracy of the amino acid sequence of the synthesized oligopeptide and the location of the synthesis was illustrated by the specific recognition binding of two different models: lead(II) ion‐peptide biosensor for lead(II) and human protein p53 (residue 20–25)‐mouse MAb DO1. After parallel synthesis of the target peptide models and their analogues based on the predetermined pattern, specific binding treatment, and fluorescence labeling, the fluorescence emission images of the oligopeptide microarray showed fluorescence intensity as a result of specific binding at the correct locations of the array. The stepwise synthesis efficiencies of pentapeptide synthesis on the microwell substrate range are ∼96–100% and do not decrease with respect to the chain length of the peptide.

A conjugated polymer–peptide hybrid system for prostate-specific antigen (PSA) detection

We developed fast and readily applicable microarray chips to detect PSA by designing a novel conjugated polymer (energy donor) and combining it with on-chip peptide synthesis. The selective cleavage of a probing peptide labelled with a dye or a quencher (energy acceptor) produced a fluorescence sensory signal via fluorescent energy resonance transfer (FRET).

Target concentration dependence of DNA melting temperature on oligonucleotide microarrays

The design of microarrays is currently based on studies focusing on DNA hybridization reaction in bulk solution. However, the presence of a surface to which the probe strand is attached can make the solution‐based approximations invalid, resulting in sub‐optimum hybridization conditions. To determine the effect of surfaces on DNA duplex formation, the authors studied the dependence of DNA melting temperature (Tm) on target concentration. An automated system was developed to capture the melting profiles of a 25‐mer perfect‐match probe–target pair initially hybridized at 23°C. Target concentrations ranged from 0.0165 to 15 nM with different probe amounts (0.03–0.82 pmol on a surface area of 1018 Å2), a constant probe density (5 × 1012 molecules/cm2) and spacer length (15 dT). The authors found that Tm for duplexes anchored to a surface is lower than in‐solution, and this difference increases with increasing target concentration. In a representative set, a target concentration increase from 0.5 to 15 nM with 0.82 pmol of probe on the surface resulted in a Tm decrease of 6°C when compared with a 4°C increase in solution. At very low target concentrations, a multi‐melting process was observed in low temperature domains of the curves. This was attributed to the presence of truncated or mismatch probes.

A Compact Chemical-Resistant Microvalve Array Using Parylene Membrane and Pneumatic Actuation

A pneumatic microvalve array was designed and fabricated using silicon/glass bulk micromachining and a new parylene bonding technique. The valve membrane is made of parylene, thus has very compact size (300 µm x 300 µm) and excellent chemical resistance. The operation of valves was characterized to reveal the effects of several parameters such as actuation and inlet pressure. The valve demonstrates a flow rate as high as 0.33ml/min in open state with 15.5psi inlet pressure, and very low leaking rate. With the proposed novel control logic, the microfabricated valve array device is expected to be very suitable for fluidic manipulation in integrated lab-on-a-chip systems in which aggressive chemicals are involved and high throughputs are required.