Mukilteo, WA, United States
Mukilteo, WA, United States
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Chamberlain J.W.,University of Washington | Maurer K.,CombiMatrix | Cooper J.,CombiMatrix | Cooper J.,CustomArray, Inc. | And 3 more authors.
Biosensors and Bioelectronics | Year: 2012

Carbohydrate-mediated host-pathogen interactions are essential to bacterial and viral pathogenesis, and represent an attractive target for the development of antiadhesives to prevent infection. We present a versatile microelectrode array-based platform to investigate carbohydrate-mediated protein and bacterial binding, with the objective of developing a generalizable method for screening inhibitors of host-microbe interactions. Microelectrode arrays are well suited for interrogating biological binding events, including proteins and whole-cells, and are amenable to electrochemical derivitization, facilitating rapid deposition of biomolecules. In this study, we achieve microelectrode functionalization with carbohydrates via controlled polymerization of pyrrole to individual microelectrodes, followed by physisorption of neoglycoconjugates to the polypyrrole-coated electrodes. Bioactivity of the immobilized carbohydrates was confirmed with carbohydrate-binding proteins (lectins) detected by both fluorescent and electrochemical means. The platform's ability to analyze whole-cell binding was demonstrated using strains of Escherichia coli and Salmonella enterica, and the dose-dependent inhibition of S. enterica by a soluble carbohydrate antiadhesive. © 2012 Elsevier B.V.

Hu L.,Washington University in St. Louis | Stuart M.,Washington University in St. Louis | Tian J.,Washington University in St. Louis | Maurer K.,CombiMatrix | And 2 more authors.
Journal of the American Chemical Society | Year: 2010

Site-selective Pd(0)-catalyzed reactions have been developed to functionalize a microelectrode array. Heck, Suzuki, and allylation reactions have all been accomplished. The reactions are compatible with both 1K and 12K arrays and work best when a nonsugar porous reaction layer is used. Suzuki reactions are faster than the Heck reactions and thus require more careful control of the reactions in order to maintain confinement. The allylation reaction requires a different confining agent than the Heck and Suzuki reactions but can be accomplished nicely with quinone as an oxidant for Pd(0). © 2010 American Chemical Society.

Amstutz U.,University of Bern | Amstutz U.,University of British Columbia | Andrey-Zurcher G.,University of Bern | Suciu D.,CustomArray, Inc. | And 3 more authors.
Clinical Chemistry | Year: 2011

BACKGROUND: Molecular genetic testing is commonly used to confirm clinical diagnoses of inherited urea cycle disorders (UCDs); however, conventional mutation screenings encompassing only the coding regions of genes may not detect disease-causing mutations occurring in regulatory elements and introns. Microarray-based target enrichment and next-generation sequencing now allow more-comprehensive genetic screening. We applied this approach to UCDs and combined it with the use of DNA bar codes for more cost-effective, parallel analyses of multiple samples. METHODS: We used sectored 2240-feature mediumdensity oligonucleotide arrays to capture and enrich a 199-kb genomic target encompassing the complete genomic regions of 3 urea cycle genes, OTC (ornithine carbamoyltransferase), CPS1 (carbamoyl-phosphate synthetase 1, mitochondrial), and NAGS (Nacetylglutamate synthase). We used the Genome Sequencer FLX System (454 Life Sciences) to jointly analyze 4 samples individually tagged with a 6-bpDNAbar code and compared the results with those for an individually sequenced sample. RESULTS: Using a low tiling density of only 1 probe per 91 bp, we obtained strong enrichment of the targeted loci to achieve ≥ 90% coverage with up to 64% of the sequences covered at a sequencing dept ≥ 10-fold.We observed a very homogeneous sequence representation of the bar-coded samples, which yielded a >30% increase in the sequence data generated per sample, compared with an individually processed sample. Heterozygous and homozygous disease-associated mutations were correctly detected in all samples. CONCLUSIONS: The use of DNA bar codes and the use of sectored oligonucleotide arrays for target enrichment enable parallel, large-scale analysis of complete genomic regions for multiple genes of a disease pathway and for multiple samples simultaneously. This approach thus may provide an efficient tool for comprehensive diagnostic screening of mutations. © 2010 American Association for Clinical Chemitry.

Tanabe T.,Washington University in St. Louis | Bi B.,Washington University in St. Louis | Hu L.,Washington University in St. Louis | Maurer K.,CustomArray, Inc. | Moeller K.D.,Washington University in St. Louis
Langmuir | Year: 2012

A new amino acid derived fluorescent linker for attaching molecules to the surface of a microelectrode array has been developed. Molecules to be monitored on an array are attached to the C-terminus of the linker, the N-terminus is then used to attach the linker to the array, and the side chain is used to synthesize a fluorescent tag. The fluorescent group is made with a one-step oxidative cycloaddition reaction starting from a hydroxyindole group. The linker is compatible with site-selective Cu(I)-chemistry on the array, it allows for quality control assessment of the array itself, and it is compatible with the electrochemical impedance experiments used to monitor binding events on the surface of the array. © 2012 American Chemical Society.

Bi B.,Washington University in St. Louis | Maurer K.,CustomArray, Inc. | Moeller K.D.,Washington University in St. Louis
Journal of the American Chemical Society | Year: 2010

A "safety-catch" linker strategy has been used to site-selectively cleave and characterize molecules from a microelectrode array. The linkers are attached to the array by means of an ester and contain either a protected amine or protected alcohol nucleophile that can be released using acid generated at the microelectrodes. © 2010 American Chemical Society.

Agency: Department of Defense | Branch: Army | Program: SBIR | Phase: Phase II | Award Amount: 728.16K | Year: 2012

CustomArray produces a field-deployable electrochemical microarray platform ideally suited for the multiplexed detection of pathogenic organisms. In this Phase II proposal, we will continue our Phase I effort to build an automated arthropod-borne virus detection system. The Phase I assay work-flow included the following three steps: (1) Extract RNA from Mosquitoes; (2) Amplify and label viral RNA loci; and (3) Detect amplification product on microarray. The results of the assay were analyzed, using software we created, to make a pathogen call. We will use this prototype detection assay as a launching pad for developing the instrumentation necessary to automate the assay and create a truly field-deployable system. In this proposal, we outline our strategy for this automation. We will (1) automate the sample prep system to extract and purify the RNA, requiring only an input of mosquitoes; (2) create an amplification system which will enable multiplexed amplification of all medically relevant arboviruses; and (3) automate the hybridization and electrochemical detection for hands free processing. We envision integrating the automated sample prep/amplification system into one disposable cartridge which will insert into a device for processing and have an output of hybridization ready material appropriate interrogation using the developed hybridizer/reader.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 198.72K | Year: 2011

This proposal seeks to develop a cancer diagnostic based on microRNA (miRNA) expression patterns found in human serum. Mounting evidence is showing that patterns of miRNA expression and the appearance of these signals in circulating extra-cellular compartments are hallmarks of proliferating tumor cells. Our preliminary results have shown a clear ability to distinguish sera derived from cancer patients for 5 different types of cancer. In a data set involving 246 patient-derived serum samples, we were able todistinguish cancer derived sera from normal controls with a sensitivity of 76% and a specificity of 84%. These data, though promising, also indicated to us that a more sensitive assay is necessary in order to resolve these distinct patterns of miRNA expression. To this end, we propose the development of a multiplexed padlock probe assay, integrated with microarray detection that will simultaneously detect most of the known human miRNAs. Our unique customizable DNA microarray synthesis platform allows us toboth produce the padlock probes and to detect signal from the amplified mixture. Finally, using this assay, we will investigate the extracellular compartment or structure that protects the miRNA molecules that are secreted by proliferating tumor cells. Inphase II, we will apply this assay to look for patterns of miRNA and other non-coding RNA (ncRNA) expression from normal and cancer patient serum samples, repeating our previous work with this novel assay.

Agency: Department of Defense | Branch: Army | Program: SBIR | Phase: Phase I | Award Amount: 66.32K | Year: 2011

CustomArray produces a field-deployable electrochemical microarray platform that is ideally suited to the multiplexed detection of pathogenic organisms. In this proposal, we will build a complete detection process that will include: Probe design, sample prep, multiplexed PCR amplification, hybridization, detection and data analysis. We have developed a bioinformatic pipeline called GenoTyper that is able to design primers and probes for the simultaneous amplification, detection, interrogation and typing of families of related, rapidly evolving sequence families. We have successfully used this system in the past to detect and type many viral families including, among many others: influenza, alpha-virus, and 16S. In this proposal we will use this bioinformatic system to create an updated pan-flavivirus and pan-alphavirus detection system. This is a proven detection system that relies on primer design that generates broad amplification followed by hybridization probe design and data analysis that is able to interrogate and accurately type the amplified material. Because we can use up to 2240 probes per assay, we are able to offer a resolution and redundancy that is unprecedented in a compact, field-deployable device. We will use our expertise and know-how in sample prep to create a simple method for harvesting viral DNA from arthropod samples. We will incorporate a novel multiplex PCR amplification system that will allow multiple independent PCR amplification reactions to take place in a single-injection device. Hybridization will be performed on our customizable semiconductor-based microarray platform. Hybridization detection will be performed using our electrochemical detection system. The detection device itself is a hand-held product that requires a USB connection, and uses that connection as its sole power source. It can read an entire array in 20 sec. This platform is the only electrochemical detection system of its kind that is as sensitive as fluorescence detection. No other system exists that has such potential to revolutionize the breadth, accuracy and robustness of field-deployable pathogen detection.

There is disclosed an electrode array architecture employing continuous and discontinuous circumferential electrodes. There is further disclosed a process for the neutralization of acid generated at anode(s) by base generated at cathode(s) circumferentially located to each other so as to confine a region of pH change. The cathodes can be displayed as concentric rings (continuous) or as counter electrodes in a cross pattern (discontinuous). In this way reagents, such as acid, generated in a center electrode are countered (neutralized) by reagents, such as base, generated at the corners or at the outer ring.

There is disclosed an electrochemical deblocking solution for use on an electrode microarray. There is further disclosed a method for electrochemical synthesis on an electrode array using the electrochemical deblocking solution. The solution and method are for removing acid-labile protecting groups for synthesis of oligonucleotides, peptides, small molecules, or polymers on a microarray of electrodes while substantially improving isolation of deblocking to active electrodes. The method comprises applying a voltage or a current to at least one electrode of an array of electrodes. The array of electrodes is covered by the electrochemical deblocking solution.

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