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We report an approach for spatially selective assembly of an enzyme onto selected patterns of microfabricated chips. Our approach is based on electrodeposition of the aminopolysaccharide chitosan onto selected electrode patterns and covalent conjugation of a target enzyme to chitosan upon biochemical activation of a genetically fused "pro-tag." We report assembly of S-adenosylhomocysteine nucleosidase (Pfs) fused with a C-terminal pentatyrosine pro-tag. Pfs is a member of the bacterial autoinducer-2 biosynthesis pathway, catalyzing the irreversible cleavage of S-adenosylhomocysteine. The assembled Pfs retains its catalytic activity and structure, as demonstrated by retained antibody recognition. Assembly is controlled by the electrode area, resulting in reproducible rates of catalytic conversion for a given area, and thus allowing for area-based manipulation of catalysis and small molecule biosynthesis. Our approach enables optimization of small molecule biosynthesis in 1-step as well as multistep enzymatic reactions, including entire metabolic pathways, and we envision a wide variety of potential applications.

An alternative route to protein assembly at surfaces based on using the unique capabilities of biological materials for the spatially selective assembly of proteins is described. Specifically, the stimuli-responsive properties of aminopolysaccharide chitosan are combined with the molecular-recognition capabilities of biotin-streptavidin binding. Biotinylated chitosan retains its stimuli-responsive properties and is capable of electrodepositing at specific electrode addresses. Once deposited, it is capable of binding streptavidin, which can mediate the subsequent assembly of biotinylated proteins. Spatially selective protein assembly using biotinylated Protein A and fluorescently-labeled antibodies is demonstrated.

We report a biofunctionalization strategy for the assembly of catalytically active enzymes within a completely packaged bioMEMS device, through the programmed generation of electrical signals at spatially and temporally defined sites. The enzyme of a bacterial metabolic pathway, S-adenosylhomocysteine nucleosidase (Pfs), is genetically fused with a pentatyrosine "pro-tag" at its C-terminus. Signal responsive assembly is based on covalent conjugation of Pfs to the aminopolysaccharide, chitosan, upon biochemical activation of the pro-tag, followed by electrodeposition of the enzyme-chitosan conjugate onto readily addressable sites in microfluidic channels. Compared to traditional physical entrapment and surface immobilization approaches in microfluidic environments, our signal-guided electrochemical assembly is unique in that the enzymes are assembled under mild aqueous conditions with spatial and temporal programmability and orientational control. Significantly, the chitosan-mediated enzyme assembly can be reversed, making the bioMEMS reusable for repeated assembly and catalytic activity. Additionally, the assembled enzymes retain catalytic activity over multiple days, demonstrating enhanced enzyme stability. We envision that this assembly strategy can be applied to rebuild metabolic pathways in microfluidic environments for antimicrobial drug discovery.

We report a versatile approach for covalent surface-assembly of proteins onto selected electrode patterns of pre-fabricated devices. Our approach is based on electro-assembly of the aminopolysaccharide chitosan scaffold as a stable thin film onto patterned conductive surfaces of the device, which is followed by covalent assembly of the target protein onto the scaffold surface upon enzymatic activation of the protein's "pro-tag." For our demonstration, the model target protein is green fluorescent protein (GFP) genetically fused with a pentatyrosine pro-tag at its C-terminus, which assembles onto both two-dimensional chips and within fully packaged microfluidic devices in situ and under flow. Our surface-assembly approach enables spatial selectivity and orientational control under mild experimental conditions. We believe that our integrated approach harnessing genetic manipulation, in situ enzymatic activation, and electro-assembly makes it advantageous for a wide variety of bioMEMS and biosensing applications that require facile "biofunctionalization" of microfabricated devices., ((c) 2007 Wiley Periodicals, Inc.)

Microelectronic devices employ electrons for signaling whereas the nervous system signals using ions and chemicals. Bridging these signaling differences would benefit applications that range from biosensing to neuroprosthetics. Here, we report the use of localized electrical signals to perform an operation common to chemical signaling in the nervous system. Specifically, we employ electrical signals to restrain vesicles reversibly. We perform this operation using the stimuli-responsive aminopolysaccharide chitosan that is able to electrodeposit onto cathode surfaces in response to localized electrical stimuli. We show that surfactant-vesicles and liposomes can be co-deposited with chitosan and are entrapped (i.e., restrained) within the deposited film's matrix. Vesicle co-deposition could be controlled spatially and temporally using microfabricated wafers with independent electrode addresses. Finally, we show that vesicles restrained within the deposited chitosan matrix can be mobilized under mildly acidic conditions (pH <6.5) that resolubilize chitosan. Potentially, the ability to restrain and mobilize chemical signals that are segregated within vesicles may allow microfluidic systems to access the rich diversity offered by chemical signaling.

While microcantilevers offer exciting opportunities for mechano-detection, they often suffer from limitations in either sensitivity or selectivity. To address these limitations, we electrodeposited a chitosan film onto a cantilever surface and mechano-transduced detection events through the chitosan network. Our first demonstration was the detection of nucleic acid hybridization. In this instance, we electrodeposited the chitosan film onto the cantilever, biofunctionalized the film with oligonucleotide probe, and detected target DNA hybridization by cantilever bending in solution (static mode) or resonant frequency shifts in air (dynamic mode). In both detection modes, we observed a two-order of magnitude increase in sensitivity compared to values reported in literature for DNA immobilized on self-assembled monolayers. In our second demonstration, we coupled electrochemical and mechanical modes to selectively detect the neurotransmitter dopamine. A chitosan-coated cantilever was biased to electrochemically oxidize dopamine solution. Dopamine's oxidation products react with the chitosan film and create a tensile stress of approximately 1.7 MPa, causing substantial cantilever bending. A control experiment was performed with ascorbic acid solution. It was shown that the electrochemical oxidation of ascorbic acid does not lead to reactions with chitosan and does not change cantilever bending. These results suggest that chitosan can confer increased sensitivity and selectivity to microcantilever sensors.

We report facile in situ biomolecule assembly at readily addressable sites in microfluidic channels after complete fabrication and packaging of the microfluidic device. Aminopolysaccharide chitosan's pH responsive and chemically reactive properties allow electric signal-guided biomolecule assembly onto conductive inorganic surfaces from the aqueous environment, preserving the activity of the biomolecules. A transparent and nonpermanently packaged device allows consistently leak-free sealing, simple in situ and ex situ examination of the assembly procedures, fluidic input/outputs for transport of aqueous solutions, and electrical ports to guide the assembly onto the patterned gold electrode sites within the channel. Both in situ fluorescence and ex situ profilometer results confirm chitosan-mediated in situ biomolecule assembly, demonstrating a simple approach to direct the assembly of biological components into a completely fabricated device. We believe that this strategy holds significant potential as a simple and generic biomolecule assembly approach for future applications in complex biomolecular or biosensing analyses as well as in sophisticated microfluidic networks as anticipated for future lab-on-a-chip devices.

The patterning of nanoparticles represents a significant obstacle in the assembly of nanoscale materials and devices. In this report, cysteine residues were genetically engineered onto the virion surface of tobacco mosaic virus (TMV), providing attachment sites for fluorescent markers. To pattern these viruses, labeled virions were partially disassembled to expose 5' end RNA sequences and hybridized to virus-specific probe DNA linked to electrodeposited chitosan. Electron microscopy and RNAase treatments confirmed the patterned assembly of the virus templates onto the chitosan surface. These findings demonstrate that TMV nanotemplates can be dimensionally assembled via nucleic acid hybridization.

We report a new approach for microfluidic optical bioanalysis that is based on the electrically driven assembly of bio-components on a transparent sidewall and the optical detection of the assembled components using planar waveguides. This allows localized electrical signals for bio-assembly and optical signals for bio-detection that can easily be applied in MEMS systems. We demonstrate a BioMEMS design incorporating this scheme and its output signal when using fluorescent detection.

A nucleic acid hybridization assay was assembled onto a robust and readily addressable silicon-based chip using polysaccharide chitosan as a scaffold for the covalent coupling of probe DNA to the chip's surface. Chitosan is a unique polymer, ideally suited for this application because its net charge and solubility are pH dependent. Specifically in this work, gold-patterned electrodes were created using standard photolithographic techniques, chitosan was electrodeposited in a spatially resolved manner onto the polarized electrodes, probe DNA was covalently assembled onto the chitosan, and both DNA:DNA and DNA:mRNA hybridization detection schemes were evaluated. Hybridization of target nucleic acid was quantifiable, reproducible, and robust; the surface was regenerated and rehybridized up to eight times without loss of signal. Finally, transcriptional upregulation of the Escherichia coli chaperone, DnaK, which is an indicator of cellular stress, was observed using the hybridization chip sandwich assay. Thus, this method enables rapid and facile monitoring of gene expression in a format that is reusable and requires minimal reagent quantities.