High-throughput epitope binning of therapeutic monoclonal antibodies: why you need to bin the fridge

ScienceDirect  DOI: 10.1016/j.drudis.2014.05.011

B. Brooks, A. Miles, Y. Abdiche
Wasatch Microfluidics, LLC, 825 North 300 West, Suite C325, Salt Lake City, UT 84103, USA. ben@brooks.nu.
Rinat Laboratories, Pfizer, South San Francisco, CA 94080, USA

Analytical tools are evolving to meet the need for the higher-throughput characterization of therapeutic monoclonal antibodies. An antibody’s epitope is arguably its most important property because it underpins its functional activity but, because epitope selection is innate, it remains an empirical process. Here, we focus on the emergence of label-free biosensors with throughput capabilities orders of magnitude higher than the previous state-of-the-art, which can facilitate large assays such as epitope binning so that they can be incorporated alongside functional activity screens, enabling the rapid identification of leads that exhibit unique and functional epitopes. In addition to streamlining the drug development process by saving time and cost, the information from epitope binning assays could provide the basis for intellectual property protection.

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High-throughput epitope binning of therapeutic monoclonal antibodies: why you need to bin the fridge

ScienceDirect  DOI: 10.1016/j.drudis.2014.05.011

B. Brooks, A. Miles, Y. Abdiche
Wasatch Microfluidics, LLC, 825 North 300 West, Suite C325, Salt Lake City, UT 84103, USA. ben@brooks.nu.
Rinat Laboratories, Pfizer, South San Francisco, CA 94080, USA

Analytical tools are evolving to meet the need for the higher-throughput characterization of therapeutic monoclonal antibodies. An antibody’s epitope is arguably its most important property because it underpins its functional activity but, because epitope selection is innate, it remains an empirical process. Here, we focus on the emergence of label-free biosensors with throughput capabilities orders of magnitude higher than the previous state-of-the-art, which can facilitate large assays such as epitope binning so that they can be incorporated alongside functional activity screens, enabling the rapid identification of leads that exhibit unique and functional epitopes. In addition to streamlining the drug development process by saving time and cost, the information from epitope binning assays could provide the basis for intellectual property protection.

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The Importance of Epitope Binning for Biological Drug Discovery

NCBI PMC  Curr Drug Discov Technol. Jun 2014; 11(2): 109-112

B. Brooks
Wasatch Microfluidics, LLC, 825 North 300 West, Suite C325, Salt Lake City, UT 84103, USA. ben@brooks.nu.

The pharmaceutical industry is experiencing comeback sales growth due in large part to the industry’s R&D efforts that center on biologics drug development. To facilitate that effort, tools are being developed for more effective biologic drug development. At the forefront of this effort is epitope characterization, in particular epitope binning, primarily due to the role an epitope plays in drug function. Here we detail the financial advantages of epitope binning including (1) increased R&D productivity due to increased work in process, (2) reduced number of “dead-end”candidates, and (3) increased ability to re-engineer antibodies based on the epitope. With the arrival of high throughput biosensors, this manuscript serves as a call to push epitope binning earlier in the biological drug discovery process.

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High-Throughput Epitope Binning Assays on Label-Free Array-Based Biosensors Can Yield Exquisite Epitope Discrimination That Facilitates the Selection of Monoclonal Antibodies with Functional Activity

PLoS One 9(3): e92541, Mar. 2014

Y. Abdiche, A. Miles, J. Eckman, D. Foletti, T.J. Van Blarcom, Y.A. Yeung, J. Pons, A. Rajpal
Wasatch Microfluidics, LLC, 825 North 300 West, Suite C325, Salt Lake City, UT 84103, USA. ben@brooks.nu.

Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs). In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb’s epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi) integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI) detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic mAbs, depending upon the sample availability, and the number and diversity of the interactions being studied.

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Sensitivity of protein array deposition using continuous flow printing for fluorescent microarray applications - biomed 2013

Biomed Sci Instrum. 2013;49:117-23.

Romanov V1, Miles A, Gale B, Eckman J, Brooks B.
Wasatch Microfluidics, LLC, 825 North 300 West, Suite C325, Salt Lake City, UT 84103, USA. ben@brooks.nu.

The promise of antibody and protein microarrays to revolutionize disease diagnostics has failed to live up to the hype primarily due to the problems associated with the printing of the antibodies and/or proteins onto the detection surface. The current standard in printing proteins is pin printing. An alternative to the pin printer is the continuous-flow microspotter (CFM), a protein printer that uses microfluidic flow to print down the proteins. The advantages of the CFM include consistent spot morphology, spot-to-spot uniformity and enhanced surface concentration. Further, the CFM is effective at capturing proteins and antibodies from either dilute or complex (e.g. blood or tissue) samples. In this study, the sensitivity of CFM printing Cy3 and Cy5 fluorescently labeled proteins was determined. Values were obtained at low concentrations tens of ng/mL with low coefficients of variation. Thus, the CFM can effectively print and quantify proteins and antibodies from low concentration and complex buffered samples.

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Continuous scaling 3d micro flow printing for improved spot morphology in protein microarrays - biomed 2013.

Biomed Sci Instrum. 2013;49:25-31.

Romanov V, Gale B, Eckman J, Miles A, Brooks B.
Wasatch Microfluidics, LLC, 825 North 300 West, Suite C325, Salt Lake City, UT 84103, USA. ben@brooks.nu.

The protein microarray platform while innovative still poses a number of challenges which can only be met through creative and sophisticated system design. Pin printing while allowing for flexibility as to the type of medium printed does not offer the kind of spot reproducibility that a very sensitive application may require. The Continuous Flow Microspotter (CFM) was designed to not only allow for flexibility and reproducibility but to also achieve solution stability through flow scaling. This study uses the emerging CFM for printing protein and antibodies three dimensionally for general protein microarray applications. Consistent spot morphology, a continual and persistent problem in traditional pin printed microarrays, was compared under variable printed flow rates. The final assessment was performed using a rudimentary shear model. Force effects discussion and statistical data was used to demonstrate the versatility of the system.

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Imaging surface plasmon resonance for multiplex microassay sensing of mycotoxins

Anal Bioanal Chem. 2011 Jul;400(9):3005-11

Dorokhin D, Haasnoot W, Franssen MC, Zuilhof H, Nielen MW.
Laboratory of Organic Chemistry, Wageningen University, The Netherlands.

A prototype imaging surface plasmon resonance-based multiplex microimmunoassay for mycotoxins is described. A microarray of mycotoxin-protein conjugates was fabricated using a continuous flow microspotter device. A competitive inhibition immunoassay format was developed for the simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEN), using a single sensor chip. Initial in-house validation showed limits of detection of 21 and 17 ng/mL for DON and 16 and 10 ng/mL for ZEN in extracts, which corresponds to 84 and 68 μg/kg for DON and 64 and 40 μg/kg for ZEN in maize and wheat samples, respectively. Finally, the results were critically compared with data obtained from liquid chromatography-mass spectrometry confirmatory analysis method and found to be in good agreement. The described multiplex immunoassay for the rapid screening of several mycotoxins meets European Union regulatory limits and represents a robust platform for mycotoxin analysis in food and feed samples.

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Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling

Nature. 2011 Jul 4;476(7360):293-7.

de Lau W, Barker N, Low TY, Koo BK, Li VS, Teunissen H, Kujala P, Haegebarth A, Peters PJ, van de Wetering M, Stange DE, van Es JE, Guardavaccaro D, Schasfoort RB, Mohri Y, Nishimori K, Mohammed S, Heck AJ, Clevers H.
Hubrecht Institute and University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands.

The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues.

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Identification of Autoantibody Profiles by Monitoring Autoantibody Biomarkers in Rheumatoid Arthritis with Microarray Surface Plasmon Resonance Imaging

Arthritis & Rheumatism, Volume 63, November 2011 Abstract Supplement

Van Beers1,  Joyce J.B.C., Segbers-Lokate1,  Angelique M.C., Egberts1,  Wilma T.M. Vree, Schasfoor2,  Richard B.M., Pruijn1,  Ger J.M.

1Radboud University Nijmegen, Nijmegen, Netherlands
2University of Twente and IBIS Technologies BV, Enschede, Netherlands

Autoantibodies against citrullinated proteins (ACPA) are specifically found in approximately 75% of rheumatoid arthritis (RA) patients. Citrullination is the post-translational conversion of peptidylarginine into peptidylcitrulline, which is catalyzed by peptidylarginine deiminase (PAD) in a calcium-dependent manner. Several citrullinated antigens have been identified in the inflamed joints of RA patients. These include fibrinogen, alpha-enolase, vimentin and collagen type II. Accumulating evidence suggests a role of citrullinated proteins and ACPA in the pathophysiology of RA. The results of many studies indicate that the ACPA response is highly heterogeneous with diverse patterns of reactivity to distinct citrullinated epitopes. This study aimed to identify clinically meaningful ACPA profiles in RA patients using a microarray containing different citrullinated peptides in combination with surface plasmon resonance imaging (iSPR).

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Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Arthritis Res Ther. 2010;12(6):R219.

van Beers JJ1, Raijmakers R, Alexander LE, Stammen-Vogelzangs J, Lokate AM, Heck AJ, Schasfoort RB, Pruijn GJ.
Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences, Institute for Molecules and Materials, Radboud University, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands.

Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.

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High-throughput screening of drug-lipid membrane interactions via counter-propagating second harmonic generation imaging

Anal Chem. 2011 Aug 1;83(15):5979-88.

Nguyen TT, Conboy JC.
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States.

Here we report the use of counter-propagating second harmonic generation (SHG) to image the interactions between the local anesthetic tetracaine and a multicomponent planar supported lipid bilayer array in a label-free manner. The lipid bilayer arrays, prepared using a 3D continuous flow microspotter, allow the effects of lipid phase and cholesterol content on tetracaine binding to be examined simultaneously. SHG images show that tetracaine has a higher binding affinity to liquid-crystalline phase lipids than to solid-gel phase lipids. The presence of 28 mol % cholesterol decreased the binding affinity of tetracaine to bilayers composed of the mixed chain lipid, 1-steroyl-2-oleoyl-sn-glycero-3-phophocholine (SOPC), and the saturated lipids 1,2-dimyristoyl-sn-glycero-3-phophocholine (DMPC) and 1,2-dipamitoyl-sn-glycero-3-phophocholine (DPPC) while having no effect on diunsaturated 1,2-dioleoyl-sn-glycero-3-phophocholine (DOPC). The maximum surface excess of tetracaine increases with the degree of unsaturation of the phospholipids and decreases with cholesterol in the lipid bilayers. The paper demonstrates that SHG imaging is a sensitive technique that can directly image and quantitatively measure the association of a drug to a multicomponent lipid bilayer array, providing a high-throughput means to assess drug-membrane interactions.

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Using micropatterned lipid bilayer arrays to measure the effect of membrane composition on merocyanine 540 binding

Biochim Biophys Acta. 2011 Jun;1808(6):1611-7.

Smith KA, Conboy JC.
Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.

The lipophilic dye merocyanine 540 (MC540) was used to model small molecule-membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 binding in DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule-membrane interactions in a high-throughput manner.

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In situ microarray fabrication and analysis using a microfluidic flow cell array integrated with surface plasmon resonance microscopy

Anal Chem. 2009 Jun 1;81(11):4296-301.

Liu J, Eddings MA, Miles AR, Bukasov R, Gale BK, Shumaker-Parry JS.
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.

Surface Plasmon Resonance Microscopy (SPRM) is a promising label-free analytical tool for the real-time study of biomolecule interactions in a microarray format. However, flow cell design and microarray fabrication have hindered throughput and limited applications of SPRM. Here we report the integration of a microfluidic flow cell array (MFCA) with SPRM enabling in situ microarray fabrication and multichannel analysis of biomolecule probe-target interactions. We demonstrate the use of the MFCA for delivery of sample solutions with continuous flow in 24 channels in parallel for rapid microarray creation and binding analysis while using SPRM for real-time monitoring of these processes. Label-free measurement of antibody-antibody interactions demonstrates the capabilities of the integrated MFCA-SPRM system and establishes the first steps of the development of a high-throughput, label-free immunogenicity assay. After in situ probe antibody immobilization, target antibody binding was monitored in real time in 24 channels simultaneously. The limit of detection for this particular antibody pair is 80 ng/mL which is approximately 6 times lower than the industry recommended immunogenicity assay detection limit. The integrated MFCA-SPRM system is a powerful and versatile combination for a range of array-based analyses, including biomarker screening and drug discovery.

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The importance of epitope binning for biological drug discovery

Curr Drug Discov Technol. 2014;11(2):109-12.

Brooks BD.
Wasatch Microfluidics, LLC, 825 North 300 West, Suite C325, Salt Lake City, UT 84103, USA. ben@brooks.nu.

The pharmaceutical industry is experiencing comeback sales growth due in large part to the industry’s R&D efforts that center on biologics drug development. To facilitate that effort, tools are being developed for more effective biologic drug development. At the forefront of this effort is epitope characterization, in particular epitope binning, primarily due to the role an epitope plays in drug function. Here we detail the financial advantages of epitope binning including (1) increased R&D productivity due to increased work in process, (2) reduced number of “dead-end”candidates, and (3) increasedability to reengineer antibodies based on the epitope. With the arrival of high throughput biosensors, this manuscript serves as a call to push epitope binning earlier in the biological drug discovery process.

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In situ microarray fabrication and analysis using a microfluidic flow cell array integrated with surface plasmon resonance microscopy

Anal Chem. 2009 Jun 1;81(11):4296-301. doi: 10.1021/ac900181f.

Liu J1, Eddings MA, Miles AR, Bukasov R, Gale BK, Shumaker-Parry JS.
1Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.

Surface Plasmon Resonance Microscopy (SPRM) is a promising label-free analytical tool for the real-time study of biomolecule interactions in a microarray format. However, flow cell design and microarray fabrication have hindered throughput and limited applications of SPRM. Here we report the integration of a microfluidic flow cell array (MFCA) with SPRM enabling in situ microarray fabrication and multichannel analysis of biomolecule probe-target interactions. We demonstrate the use of the MFCA for delivery of sample solutions with continuous flow in 24 channels in parallel for rapid microarray creation and binding analysis while using SPRM for real-time monitoring of these processes. Label-free measurement of antibody-antibody interactions demonstrates the capabilities of the integrated MFCA-SPRM system and establishes the first steps of the development of a high-throughput, label-free immunogenicity assay. After in situ probe antibody immobilization, target antibody binding was monitored in real time in 24 channels simultaneously. The limit of detection for this particular antibody pair is 80 ng/mL which is approximately 6 times lower than the industry recommended immunogenicity assay detection limit. The integrated MFCA-SPRM system is a powerful and versatile combination for a range of array-based analyses, including biomarker screening and drug discovery.

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Detergent screening of a G-protein-coupled receptor using serial and array biosensor technologies

Anal Biochem. 2009 Mar 1;386(1):98-104. doi: 10.1016/j.ab.2008.12.011. Epub 2008 Dec 24.

Rich RL1, Miles AR, Gale BK, Myszka DG.
1Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah, Salt Lake City, 84132, USA.

We describe the benefits and limitations of two biosensor approaches for screening solubilization conditions for G-protein-coupled receptors (GPCRs). Assays designed for a serial processing instrument (Biacore 2000/3000/T100) and an array platform (Biacore Flexchip) were used to examine how effectively 96 different detergents solubilized the chemokine receptor CCR5 while maintaining its binding activity for a conformationally sensitive Fab (2D7). Using the serial processing instrument, we were able to analyze three samples in each 30-min binding cycle, thereby requiring approximately 24h to screen an entire 96-well plate of conditions. In-line capturing allowed us to normalize the 2D7 binding responses for different receptor capture levels. In contrast, with the array system, we could characterize the effects of all 96 detergents simultaneously, completing the assay in less than 1h. But the current array technology requires that we capture the GPCR preparations off-line, making it more challenging to normalize for receptor capture levels. Also, the array platform is less sensitive than the serial platforms, thereby limiting the size of the analyte to larger molecules (>5000Da). Overall, the two approaches proved to be highly complementary; both assays identified identical detergents that produced active solubilized CCR5 as well as those detergents that either were ineffective solubilizers or inactivated the receptor.

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Spot and hop: internal referencing for surface plasmon resonance imaging using a three-dimensional microfluidic flow cell array

Anal Biochem. 2009 Feb 15;385(2):309-13. doi: 10.1016/j.ab.2008.11.014. Epub 2008 Nov 20.

Eddings MA1, Eckman JW, Arana CA, Papalia GA, Connolly JE, Gale BK, Myszka DG.
1Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA.

We have developed a novel referencing technique for surface plasmon resonance imaging systems referred to as “spot and hop.” The technique enables internal referencing for individual flow cells in a parallel processing microfluidic network. Internal referencing provides the ability to correct for nonspecific binding and instrument drift, significantly improving data quality at each region of interest. The performance of a 48-flow-cell device was demonstrated through a series of studies, including “rise and fall” time, ligand preconcentration, ligand immobilization, analyte binding, and regeneration tests. Interfacing parallel processing fluidics with imaging systems will significantly expand the throughput and applications of array-based optical biosensors while retaining high data quality.

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Micropatterned fluid lipid bilayer arrays created using a continuous flow microspotter

Anal Chem. 2008 Nov 1;80(21):7980-7. doi: 10.1021/ac800860u. Epub 2008 Oct 8.

Smith KA1, Gale BK, Conboy JC.
1Department of Chemistry, University of Utah, 315 South 1400 East RM. 2020, Salt Lake City, Utah 84112, USA.

We have developed a new method for creating micropatterned lipid bilayer arrays (MLBAs) using a 3D microfluidic system. An array of fluid lipid membranes was patterned onto a glass substrate using a Continuous Flow Microspotter. Fluorescence microscopy experiments were used to verify the formation of a bilayer structure on the glass substrate. Fluorescence recovery after photobleaching experiments demonstrated the bilayers’ fluidity was maintained while being individually corralled on the substrate. The reproducibility of bilayer formation within an array was demonstrated by the linear response of membrane fluorescence versus mol % rhodamine functionalized lipids incorporated into the vesicles prior to fusion to the surface. The highly customizable nature of the MLBAs was demonstrated utilizing three different fluorescently labeled lipids to generate a multiple component lipid array. Finally, the cholera toxin B/ganglioside GM 1, antidinitrophenyl (DNP) antibody/DNP, and NeutrAvidin/biotin protein-ligand systems were used to model multiple protein-ligand binding on the MLBAs. The multicomponent patterned bilayers were functionalized with GM 1, DNP, and biotin lipids, and binding curves was generated by recording surface fluorescence versus increasing concentration of membrane bound ligands.

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Parallel determination of phenotypic cytotoxicity with a micropattern of mutant cell lines

Biomed Microdevices. 2009 Apr;11(2):443-52. doi: 10.1007/s10544-008-9250-z.

Sincic RS1, Chang-Yen DA, Eddings M, Barrows LR, Gale BK.
1Department of Biomedical Engineering, University of Utah, 50 S. Central Campus Dr., Rm. 2480 MEB, Salt Lake City, UT 84112-9202, USA. sincic@gmail.com

This work presents a novel tool, the Continuous Flow Microspotter (CFM) and its use in patterning cellular microarrays of multiple cell types into the bottom of a tissue culture well. The CFM uses a system of isolated microfluidic channels to make an array of localized microspots of adhesion dependent cells in the bottom of a conventional tissue culture well. With this device we have created micropatterns of multiple cell lines in a single tissue culture well and used this system to conduct simultaneous cytotoxicity tests and recover dose survival curves in a parallel study. This mechanism of parallel testing allows the researcher to employ the use of positive and negative controls, as well as compare the chemical response of phenotypes in a tightly controlled microenvironment. For the experiments presented in this paper we have fabricated a CFM with a set of ten microchannels (five inlet channels and five outlet channels) to pattern a row of five microspots consisting of four cellular microspots and one empty spot for background measurements. Micropatterns containing a set of four different Chinese hamster ovarian cell (CHO) mutant phenotypes were deposited into the bottom of commercially available tissue culture wells then interrogated with mitomycin C, a chemotherapeutic agent. This study shows statistically significant (P < 0.05) hypersensitivity of the UV20 CHO mutant to a DNA interstrand cross-linking agent (mitomycin C). Because the CFM is also capable of depositing proteins and other biomolecules to the individual microspots of the array we foresee capabilities of the 48 microspot CFM to multiplex 48 cell types with 48 chemical reagents all within the confines of a 60 mm(2) area.

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Optimal conditions for protein array deposition using continuous flow

Anal Chem. 2008 Nov 15;80(22):8561-7. doi: 10.1021/ac8014609. Epub 2008 Oct 22.

Natarajan S1, Hatch A, Myszka DG, Gale BK.
1Department of Chemical Engineering, University of Utah, Salt Lake City, Utah 84112, USA.

Optimal conditions for depositing protein microarrays using a continuous-flow microfluidic device, the continuous-flow microspotter (CFM), have been determined using a design of experiments approach. The amount of protein deposited on the surface depends on the rates of convective and diffusive transport to the surface and binding at the surface. These rates depend on parameters such as the flow rate, time, and capture mechanism at the surface. The process parameters were optimized, and uniform protein spots were obtained at a protein concentration of 10 microg/mL and even at 0.4 microg/mL. A 150-fold dilution in protein concentration in the sample solution decreased surface concentration by a factor of only 16. If the capture mechanism of the protein on the substrate is nonspecific, optimal deposition is obtained at higher flow rates for short periods of time. If the capture mechanism is specific, such as biotin-avidin, deposition is optimal at medium flow rates with little advantage beyond 30 min. The CFM can be used to deposit protein arrays with good spot morphology, spot-to-spot uniformity and enhanced surface concentration. The CFM was used to deposit an array of various antibodies, and their interactions with an antigen were studied using surface plasmon resonance (SPR). Affinity values were obtained at low antibody concentrations (5 microg/mL) with low coefficients of variation. Thus, the CFM can be used to effectively capture proteins and antibodies from dilute samples while depositing multiple spots, thereby increasing the quality of spots in protein microarrays and especially improving screening throughput of SPR.

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Improved continuous-flow print head for micro-array deposition

Anal Biochem. 2008 Nov 1;382(1):55-9. doi: 10.1016/j.ab.2008.07.031. Epub 2008 Aug 3.

Eddings MA1, Miles AR, Eckman JW, Kim J, Rich RL, Gale BK, Myszka DG.
1Department of Bioengineering, University of Utah, Salt Lake City, UT 84132, USA.

Limitations in depositing ligands using conventional micro-array pin spotting have hindered the application of surface plasmon resonance imaging (SPRi) technology. To address these challenges we introduce a modification to our continuous-flow micro-spotting technology that improves ligand deposition. Using Flexchip protein A/G and neutravidin capturing surfaces, we demonstrate that our new microfluidic spotter requires 1000 times less concentrated antibodies and biotinylated ligands than is required for pin spotting. By varying the deposition flow rate, we show that the design of our tip overlay flow cell is efficient at delivering sample to the substrate surface. Finally, contact time studies show that it is possible to capture antibodies and biotinylated ligands at concentrations of less than 0.1 ug/ml and 100 pM, respectively. These improvements in spotting technology will help to expand the applications of SPRi systems in areas such as antibody screening, carbohydrate arrays, and biomarker detection.

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Large-area, high-aspect-ratio SU-8 molds for the fabrication of PDMS microfluidic devices

Journal of Micromechanics and Microengineering Volume 18 Number 4

S Natarajan1,2 D A Chang-Yen2,3 and B K Gale2,3

1Department of Chemical Engineering, University of Utah, USA
2Center of Excellence for Biomedical Microfluidics, University of Utah, USA
3Department of Mechanical Engineering, University of Utah, USA

A relatively low-cost fabrication method using soft lithography and molding for large-area, high-aspect-ratio microfluidic devices, which have traditionally been difficult to fabricate, has been developed and is presented in this work. The fabrication process includes novel but simple modifications of conventional microfabrication steps and can be performed in any standard microfabrication facility. Specifically, the fabrication and testing of a microfluidic device for continuous flow deposition of bio-molecules in an array format are presented. The array layout requires high-aspect-ratio elastomeric channels that are 350 µm tall, extend more than 10 cm across the substrate and are separated by as little as 20 µm. The mold from which these channels were fabricated consisted of high-quality, 335 µm tall SU-8 structures with a high-negative aspect ratio of 17 on a 150 mm silicon wafer and was produced using spin coating and UV-lithography. Several unique processing steps are introduced into the lithographic patterning to eliminate many of the problems experienced when fabricating tall, high-aspect-ratio SU-8 structures. In particular, techniques are used to ensure uniform molds, both in height and quality, that are fully developed even in the deep negative-aspect-ratio areas, have no leftover films at the top of the structures caused by overexposure and no bowing or angled sidewalls from diffraction of the applied UV light. Successful microfluidic device creation was demonstrated using these molds by casting, curing and bonding a polydimethylsiloxane (PDMS) elastomer. A unique microfluidic device, requiring these stringent geometries, for continuous flow printing of a linear array of 16 protein and antibody spots has been demonstrated and validated by using surface plasmon resonance imaging of printed arrays.

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Continuous-flow microfluidic printing of proteins for array-based applications including surface plasmon resonance imaging

Anal Biochem. 2008 Feb 1;373(1):141-6. Epub 2007 Aug 19.

Natarajan S1, Katsamba PS, Miles A, Eckman J, Papalia GA, Rich RL, Gale BK, Myszka DG.
1Department of Chemical Engineering, University of Utah, Salt Lake City, UT 84132, USA.

Arraying proteins is often more challenging than creating oligonucleotide arrays. Protein concentration and purity can severely limit the capacity of spots created by traditional pin and ink jet printing techniques. To improve protein printing methods, we have developed a three-dimensional microfluidic system to deposit protein samples within discrete spots (250-microm squares) on a target surface. Our current technology produces a 48-spot array within a 0.5 x 1 cm target area. A chief advantage of this method is that samples may be introduced in continuous flow, which makes it possible to expose each spot to a larger volume of sample than would be possible with standard printing methods. Using Biacore Flexchip (Biacore AB) surface plasmon resonance array-based biosensor as a chip reader, we demonstrate that the microfluidic printer is capable of spotting proteins that are dilute (10,000-fold molar excess). We also show that the spots created by the microfluidic printer are more uniform and have better-defined borders than what can be achieved with pin printing. The ability to readily print proteins using continuous flow will help expand the application of protein arrays.

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Direct Adsorption and Detection of Proteins, Including Ferritin, onto Microlens Array Patterned Bioarrays

J. Am. Chem. Soc., 2007, 129 (30), pp 9252–9253

Feng Zhang, Richard J. Gates,Vincent S. Smentkowski, Sriram Natarajan,Bruce K. Gale,Richard K. Watt, Matthew C. Asplund,and Matthew R. Linford
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602
GE Global Research, 1 Research Circle, Niskayuna, New York
Departments of Chemical Engineering and Mechanical Engineering, University of Utah, Salt Lake City, Utah 84112

Protein arrays continue to increase in importance as tools for analysis of biological samples. This paper describes a new method for preparing bioarrays that is compatible with high throughput manufacturing. First, a native oxide terminated silicon substrate is coated with a monolayer of a polyethylene-glycol-containing silane. The coated substrate is then placed beneath a microlens array (MA) and the array is irradiated with a brief (4 ns) pulse of 532 nm laser light. The MA focuses the laser light onto the substrate, causing monolayer removal. The microlenses in the array employed in this work are square packed and have a spacing of 100 μm, that is, there are 10000 microlenses/cm2 in the optic and therefore 10000 spots/cm2 on surfaces patterned with this array. The patterned substrate is then immersed in a dilute (10-5 M) solution of a protein. Time-of-flight secondary ion mass spectrometry shows that all of the proteins studied, including avidin, BSA, ferritin, lysozyme, myoglobin, protein A, and streptavidin, adsorb selectively into the spots in the array. The stability of these adsorbed proteins is shown. The retention of activity of avidin after adsorption is demonstrated. Protein localization on the arrays is demonstrated using a microfluidic spotter. Additionally, it is shown that this method can be used to confirm the location of the metal (iron) in ferritin.

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A Novel PDMS Microfluidic Spotter for Fabrication of Protein Chips and Microarrays

Microelectromechanical Systems, Journal of (Volume:15, Issue: 5 )

Chang-Yen, D.A.
Utah State Center of Excellence for Biomed. Microfluidics, Utah Univ., Salt Lake City, UT

A novel polydimethyl siloxane (PDMS) microfluidic spotter system has been developed for the patterning of surface microarrays that require individually addressing each spot area and high probe density. Microfluidic channels are used to address each spot region, and large spot arrays can be addressed in parallel. Fluorescence intensity measurement of dye-spotted samples compared to control and pipetted drops demonstrated a minimum of three-fold increase in dye surface density compared to pin-spotted dyes. Surface plasmon resonance (SPR) measurement of protein-spotted samples as compared to pin-spotted samples demonstrated an 86-fold increase in protein surface concentration. The spotting system has been applied successfully to protein microarrays for SPR applications, in both a 12-spot linear and 48-spot two-dimensional (2-D) array format. This novel spotter system can be applied to the production of high-throughput arrays in the fields of genomics, proteomics, immunoassays, and fluorescence or luminescence assays.

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