Multifunctional Protein Microarrays for Cultivationof Cells and Immunodetection of Secreted CellularProducts

Caroline N. Jones,† Ji Youn Lee,† James Zhu,† Gulnaz Stybayeva,†,‡ Erlan Ramanculov,‡

Mark A. Zern,§ and Alexander Revzin*,†

Department of Biomedical Engineering and Department of Medicine, Transplant Research Institute, University ofCalifornia, Davis, California 95616, and National Center for Biotechnology, Astana, Republic of Kazakhstan

The microarray format is being used extensively forcombinatorial screening of cellular interactions with pro-teins, small molecules, or biomaterials. The utility ofmicroarray-based cell cultivation approaches may beenhanced further by incorporating biosensing elementsalongside the cell-adhesive ligands to enable local detec-tion of secreted cellular products. The concept of com-bining cells and sensing elements in the same microarrayis demonstrated in the present paper with hepatocytesserving as a model cellular system. Robotic microarrayingwas employed to print arrays of 300-µm-diameter collagen(I) spots alongside the antibody (Ab) spots specific to liverproteins: albumin and r1-antitrypsin (r1-AT). Proteinmicroarrays were printed onto poly(ethylene glycol) hy-drogel-coated glass slides, thus eliminating nonspecificadsorption of cells or proteins. When incubated withprinted microarrays, hepatocytes became localized oncollagen (I) domains but did not attach on Ab spots orelsewhere on hydrogel-coated glass substrates. Liver-specific proteins secreted by hepatocytes were capturedon Ab domains in the immediate vicinity of the cells,detected with a sandwich immunofluorescent assay andquantified using a microarray scanner. Importantly, he-patic albumin and r1-AT production detected in themicroarray was comparable to enzyme-linked immun-osorbent assay measurements of these proteins. In thefuture, the juxtaposition of sensing Ab regions with cellarrays will be particularly useful for the detection of localappearance or loss of phenotype of cells interacting withthe printed components of the cellular microenvironment.

Micropatterning and surface engineering techniques1–3 haveemerged as important tools for dissecting the effects of cell-surface, cell-cell, or cell-solution interactions on cellular

behavior.4–8 In particular, there has been a strong recent effortto adapt DNA microarray technologies9 to combinatorial screeningof cell-surface interactions.1–3,10–14 The microarray format has abroad appeal to multiple areas of cell biology/biotechnology dueto the possibility of performing multiple experiments in parallel,while utilizing a small number of cells and small amounts ofreagents. Given the difficulties associated with stem cell cultivationand the poor understanding of the signals required for stem celllineage selection, the microarray format has been used for high-throughput screening of the effects of extracellular matrix pro-teins,1 growth factors,12 small molecules,2 and biomaterials3 onthe differentiation of adult and embryonic stem cells. In otherstudies, arrays of antibodies have been employed in immunology/diagnostics for parallel capture and characterization of leukocytesubsets, thus determining disease signatures based on the types/numbers of surface-bound cells.15,16 Microarrays of RNA interfer-ence molecules have been employed for the assessment of geneknockdown and loss of function in a highly parallel fashion.13,17

Use of microarrays for cell capture/cultivation requires comple-mentary technologies for detecting location-specific differencesin cell function occurring within the cell microarray. For the mostpart, the differences in cell function within the microarray havebeen assessed with reporter genes or by intracellular immunof-luorescent staining of biomolecules (e.g., proteins, mRNA) using

* To whom correspondence should be addressed. Phone: 530-752-2383. Fax:530-754-5739. E-mail: arevzin@ucdavis.edu.

† Department of Biomedical Engineering, University of California, Davis.‡ National Center for Biotechnology.§ Department of Medicine, Transplant Research Institute, University of

California, Davis.(1) Flaim, C. J.; Chien, S.; Bhatia, S. N. Nat. Methods 2005, 3, 119.(2) Ding, S.; Schultz, P. G. Nat. Biotechnol. 2004, 22, 833–849.(3) Anderson, D. G.; Levenberg, S.; Langer, R. Nat. Biotechnol. 2004, 22, 863–

866.

(4) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X.; Ingber, D. E. Annu.Rev. Biomed. Eng. 2001, 3, 335–373.

(5) Folch, A.; Toner, M. Annu. Rev. Biomed. Eng. 2000, 2, 227.(6) Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I. C.;

Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696–698.(7) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science

1997, 276, 1425–1428.(8) Jeon, N. L.; Baskaran, H.; Detringer, S. K. W.; Whitesides, G. M.; Van de

Water, L.; Toner, M. Nat. Biotechnol. 2002, 20, 826–830.(9) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270,

467–470.(10) Revzin, A.; Rajagopalan, P.; Tilles, A. W.; Berthiaume, F.; Yarmush, M. L.;

Toner, M. Langmuir 2004, 20, 2999–3005.(11) Soen, Y.; Chen, D. S.; Kraft, D. L.; Davis, M. M.; Brown, P. O. PLOS Biol.

2003, 1, 429–438.(12) Soen, Y.; Mori, A.; Palmer, T. D.; Brown, P. O. Mol. Syst. Biol. 2006, 2,

1–14.(13) Wheeler, D B.; Sabatini, D. M. Nat. Methods 2004, 1, 127–132.(14) Ziauddin, J.; Sabatini, D. M. Nature 2001, 411, 107–110.(15) Belov, L.; de la Vega, O.; dos Remedios, C. G.; Mulligan, S. P.; Christo-

pherson, R. I. Cancer Res. 2001, 61, 4483–4489.(16) Zhu, H.; Macal, M.; George, M. D.; Dandekar, S.; Revzin, A. Anal. Chim.

Acta 2008, 608, 186–196.(17) Wu, R. Z.; Bailey, S. N.; Sabatini, D. M. Trends Cell Biol. 2002, 12, 485–

488.

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10.1021/ac8007626 CCC: $40.75 2008 American Chemical Society 6351Analytical Chemistry, Vol. 80, No. 16, August 15, 2008Published on Web 07/22/2008

microscopy-based screening.1,13,17 Recently, we employed lasercapture microdissection in tandem with reverse-transcriptase PCRto retrieve cells from desired locations within the microarray fordownstream gene expression studies.18 However, this approachdid not account for translational differences or post-translationalmodifications of proteins and, therefore, offered an incompletepicture of cellular phenotype. Given that intracellular protein-staining approaches used commonly in screening of cell microar-rays provide only qualitative information about the presence orabsence of specific proteins, the goal of the present study was toquantify proteins secreted in the cell microarray.

In addition to its use in cell microenvironment screeningexperiments, the microarray format has been used extensively tocreate biosensors for parallel detection of analytes.19–24 Specifi-cally, printed arrays of capture antibodies (Abs) have been usedas a base of multiplexed immunoassays for detection of proteinsor bacteria using a sandwich Ab format.19,21,25 Ab microarray-based immunoassays are analogous in design and sensitivity toenzyme-linked immunosorbent assays (ELISA) but offer a signifi-cant advantage of multiplexed analyte detection in a small volume.Recently, Chen et al. proposed to extend the microarray immu-noassay concept to detection of cytokines secreted by surface-bound T-lymphocytes.26 In order to colocalize cells and biorec-ognition molecules, microarrays with individual spots containinga mixture of anticytokine capture Abs and cell-adhesive peptideswere printed. This method allowed detection of cytokines secretedby T-cells bound on the spot, but was not suitable for quantifyinglevels of protein production.

In the present paper, sensing arrays of Ab spots werejuxtaposed with arrays of hepatic cells to enable local andmultiplexed detection of cell function within the microarray(Figure 1A). Robotic microarraying was used to print arrays of300-µm-diameter spots of cell-adhesive collagen (I) molecules nextto spots of Abs against liver-specific proteins, albumin and R1-antitrypsin (R1-AT). The microarrays were printed onto glassslides coated with a poly(ethylene glycol) (PEG) hydrogel layeremployed to eliminate nonspecific protein or cell adhesion. Whenincubated with protein microarrays, hepatocytes became localizedexclusively on collagen (I) domains and did not attach on Ab spotsor elsewhere on the hydrogel-coated glass slides. The liver-specificproteins were then detected on adjacent Ab spots using sandwichimmunoassay and fluorescence intensity was quantified with amicroarray scanner. The microarray immunoassay had a limit ofdetection of ∼6 ng/mL for R1-AT and ∼13 ng/mL for human orrat albumin. Importantly, levels of both secreted proteins detectedwith Ab microarrays were comparable to ELISA. In the future,the ability to juxtapose sensing Ab regions with printed compo-nents of cellular microenvironment will be utilized to detect localappearance or loss of cell function within the cell microarray.

MATERIALS AND METHODSChemicals and Materials. Glass slides (75 × 25 mm2) were

obtained from VWR (West Chester, PA). (3-Acryloxypropyl)-trichlorosilane was purchased from Gelest, Inc. (Morrisville, PA).Sulfuric acid, hydrogen peroxide, ethanol, collagen from rat tail(type I), poly(ethylene glycol) diacrylate (PEG), and 2-hydroxy-2-methylpropiophenone (photoinitiator) were obtained from Sigma-Aldrich (St. Louis, MO). Concentrated phosphate-buffered saline(10× PBS) was purchased from Lonza (Walkersville, MD).Minimal essential medium (MEM), sodium pyruvate, nonessentialamino acids, fetal bovine serum (FBS), and streptavidin, AlexaFluor 546 conjugate were purchased from Invitrogen (Carlsbad,CA). The 384-well polypropylene microarray plates were obtainedfrom Genetix (New Milton, NH). Goat antihuman cross-adsorbedalbumin antibody, goat antihuman albumin antibody-HRP con-jugate, reference serum, and goat IgG ELISA quantitation kit wereobtained from Bethyl Laboratories (Montgomery, TX). Gaskets

(18) Lee, J. Y.; Jones, C. N.; Zern, M. A.; Revzin, A. Anal. Chem. 2006, 78,8305–8312.

(19) Delehanty, J. B.; Ligler, F. S. Anal. Chem. 2002, 74, 5681–5687.(20) Taitt, C. R.; Anderson, G. P.; Lingerfelt, B. M.; Feldstein, M. J.; Ligler, F. S.

Anal. Chem. 2002, 74, 6114–6120.(21) Li, Y.; Reichert, W. M. Langmuir 2003, 19, 1557–1566.(22) Li, Y.; Nath, N.; Reichert, W. M. Anal. Chem. 2003, 75, 5274.(23) Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloshchuk, S.; Chupeeva, V.;

Mirzabekov, A. Anal. Biochem. 2000, 278, 123–131.(24) Bailey, R. C.; Kwong, G. A.; Radu, C. G.; Witte, O. N.; Heath, J. R. J. Am.

Chem. Soc. 2007, 129, 1959–1967.(25) Wang, C. C. J. Proteome Res. 2002, 1, 337–343.(26) Chen, D. S.; Soen, Y.; Stuge, T. B.; Lee, P. P.; Weber, J. S.; Brown, P. O.;

Davis, M. M. PLOS Med. 2005, 2, 1018–1030.

Figure 1. Merging cells and immunosensing elements in the same microarray. (A) Schematic demonstrating the concept of merging cell andantibody microarrays for local detection of secreted proteins. Binding of hallmark liver proteins (albumin and R1-AT) secreted by the hepatocyteswas detected on the adjacent sensing Ab spots using a sandwich immunofluorescent assay and was quantified using a microarray scanner. (B)A map of the 12 × 20 protein microarray composed of hepatocyte-adhesive collagen (I) islands juxtaposed with antialbumin (liver protein),anti-R1-AT (liver protein), antimouse IgG (negative control), and biotin (positive control). Microarrays were printed onto PEG hydrogel-coatedglass slides and were fitted with silicone gaskets to define three reaction chambers (100 µL each) on a standard glass slide.

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were purchased from Grace Bio-Laboratories (Bend, OR). For-malin was purchased from Fisher (Pittsburgh, PA). Biotin, EZbiotin quantitation kit, and Slide-A-Lyzer mini dialysis units werepurchased from Pierce Biotechnology (Rockford, IL).

Preparation of Glass Substrates. Glass slides were cleanedby immersion in “piranha” solution consisting of 3:1 ratio ofaqueous solutions of 50% v/v sulfuric acid and 30% w/v hydrogenperoxide for 30 min (caution: this mixture reacts violently withorganic materials and must be handled with extreme care). The glassslides were thoroughly rinsed with deionized water, dried undernitrogen, and kept in Class 10000 air prior to use. For silanemodification, the glass slides were exposed to oxygen plasma for5 min at 300 W (YES3, Yield Engineering Systems, Livermore,CA) and then placed for 10 min in a 2 mM solution of (3-acrylopropyl)trichlorosilane diluted in anhydrous toluene. Thereaction was performed in a glovebox under a nitrogen blanketto avoid exposure to atmospheric moisture. After silanization, theslides were rinsed with fresh toluene, dried under nitrogen, andcured at 100 °C for 4 h. Silanized glass slides were then coatedwith PEG hydrogel according to the procedures reported earlier.27,28

PEG gel-coated glass slides were dehydrated by lyophilization for24 h and were stored in a desiccator prior to microarray printing.

Printing Microarrays of Cell Adhesive Ligands and Anti-bodies. All proteins employed for printing were dissolved in 1×PBS with 0.005% Tween-20 at 0.2 mg/mL concentration. Proteinmicroarrays were contact-printed under ambient conditions onPEG-gel modified 75 × 25 mm2 glass slides using the GMS 417pin-and-ring robotic microarrayer (Affymetrix, Santa Clara, CA).A robotic microarrayer collected protein (0.2 mg/mL in printingbuffer) from a 384-well plate, dispensing 1-2 nL of solution ontothe hydrogel-coated glass slide and forming circular spots ∼300µm in diameter with 375-µm center-to-center spacing. A typicalmicroarray used in our experiments contained one row of ∼300-µm-diameter collagen (I) spots (12 × 1) with adjacent perpendicu-lar rows of antialbumin, anti-R1-AT, biotinylated IgG (positivecontrol), and antimouse IgG (negative control) Ab spots forminga 12 × 20 array (Figure 1B).

Protein arrays were kept in a refrigerator before use and werefunctional for at least three weeks. The quality of printed arraywas assessed using an Agilent G2565BA laser microarray scanner(Agilent, Santa Clara, CA) and Zeiss LSM 5 Pascal confocalmicroscope (Carl Zeiss, Thornwood, NJ).

Stability of Printed Antibody Arrays. In order to evaluateretention of printed Ab molecules, hydrogel-coated glass slidescontaining Ab microarrays were incubated in cell culture mediafor one or three days. After the desired incubation time, glassslides were removed from culture media and were exposed to a100-µL volume of protein extraction buffer consisting of 4 Mguanidine hydrochloride (pH 7.2) supplemented with 0.2% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS),10 mM EDTA, 0.05 M Tris, and protease inhibitor cocktail (Sigma-Aldrich). The Ab microarrays were incubated with the guanidineextraction solution for 30 min, after which the solution withextracted proteins was buffer-exchanged with 6 M urea containing0.05 M Tris (pH 7.4) using Slide-A-Lyzer Mini Dialysis Units(Pierce Biotechnology) with 3500 MWCO for 1 h. Concentration

of antibody after dialysis was determined using the goat IgGELISA Quantitation Kit (Bethyl Laboratories).

Creating Cellular Microarrays. Human hepatoma (HepG2)cells and primary rat hepatocytes were used in cell micropattern-ing and liver protein detection studies. HepG2 cells were main-tained in MEM supplemented with 10% FBS, 200 units/mLpenicillin, 200 µg/mL streptomycin, 1 mM sodium pyruvate, and1 mM nonessential amino acids at 37 °C in a humidified 5% CO2

atmosphere. Cells were cultured until ∼90% confluence and thenpassaged. Primary rat hepatocytes were isolated from adult femaleLewis rats (Charles River Laboratories, Boston, MA) weighing125-200 g, using a two-step collagenase perfusion procedure asdescribed previously.29 Typically, 100-200 million hepatocyteswere obtained with viability of >90% as determined by trypan blueexclusion. Primary hepatocytes were maintained in DMEMsupplemented with epidermal growth factor, glucagon, hydrocor-tisone sodium succinate, recombinant human insulin, 200 units/mL penicillin, 200 µg/mL streptomycin, and 10% FBS.

For cell seeding experiments, a glass slide containing printedarrays of collagen (I) and Ab spots was placed into a P100 dishand was outfitted with a silicone rubber gasket (see Figure 1B)to reduce cell culture volume to 100 µL. Each glass slide containedthree regions for cell cultivation and protein detection. Hepatocyteswere seeded to form cellular arrays using protocols describedearlier.10 In brief, glass slides containing printed collagen (I) andAb spots were first exposed to 100 µL of HepG2 cells or ratprimary hepatocytes suspended in culture medium at a concentra-tion of 1 × 106 cells/mL. After 1 h of incubation at 37 °C, hepaticcells became localized on collagen (I) domains but did not attachon Ab regions or the surrounding PEG hydrogel matrix. Hepaticcells were incubated in culture media for periods of time rangingfrom 16 h to 3 days before fixation and immunofluorescentstaining.

Detecting Albumin and r1-AT Production with Ab Mi-croarrays. Prior to detection of secreted liver proteins, calibrationcurves were constructed to correlate fluorescence signals with con-centrations of albumin and R1-AT. To construct calibration curves,proteins were printed onto hydrogel-coated glass slides at con-centrations ranging from 0 to 100 ng/mL. Immunostaining ofprinted protein arrays with biotinylated antialbumin and anti-R1-AT Abs was followed by SA-Alexa-546 labeling, revealing protein-concentration-dependent changes in fluorescence intensity. Inorder to create a quantitative readout of fluorescence signalemanating from the array, the laser microarray scanner wasemployed to scan the glass slides at a spot pixel resolution of 5µm. The fluorescence intensity of each array element wasdetermined using GenePix Pro 6.0 data analysis software (Mo-lecular Devices, Downingtown, PA).

In order to detect cellular products with Ab microarrays,hepatic cells were cultivated on 300-µm-diameter collagen (I)domains in the immediate vicinity of antialbumin and anti-R1-ATAb spots. To detect binding of hepatic proteins, substrates withcell/Ab microarrays were removed at the desired time points andfixed by exposure to 3% formalin in 1× PBS for 20 min. The slideswere then incubated with 1 µg/mL (in 1× PBS) of biotinylatedantibody at 37 °C for 2 h followed by incubation in 10 µg/mL

(27) Revzin, A.; Tompkins, R. G.; Toner, M. Langmuir 2003, 19, 9855–9862.(28) Sekine, K.; Revzin, A.; Tompkins, R. G.; Toner, M. J. Immunol. Methods

2006, 313, 96–109.(29) Dunn, J. C.Y.; Yarmush, M. L.; Koebe, H. G.; Tompkins, R. G. FASEB J.

1989, 3, 174–179.

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streptavidin, Alexa Fluor 546 conjugate for 1 h. Cells were washedbetween each staining step with warm 1× PBS. All incubationswere performed at room temperature if not stated otherwise.

Immunostained arrays were visualized and imaged using aconfocal microscope and quantified using laser scanner asdescribed before. The hepatic synthesis of albumin and R1-ATdetected with Ab microarrays was compared with ELISA analysisof these proteins. For ELISA experiments, 75 × 25 mm2 hydrogel-coated glass slides containing collagen (I) arrays were diced usinga scribing diamond pen and placed into 6-well plates. A glass piecein each well of the plate contained a 12 × 12 array of collagen (I)spots and was immersed in 3 mL of cell culture media. Hepato-cytes were seeded and cultivated on microarrays as describedabove. The cell culture medium was collected periodically andanalyzed for albumin and R1-AT using standard ELISA protocols.29

RESULTS AND DISCUSSIONIn this study, arrays of hepatic cells were juxtaposed with

sensing Ab spots in order to detect production of liver-specificproteins (Figure 1B). When seeded onto printed microarrays,hepatic cells attached preferentially to collagen (I) regions,becoming localized next to antialbumin and anti-R1-AT Ab spots.Immunofluorescent staining of the Ab domains followed byimaging with a laser microarray scanner allowed the quantificationof secreted liver-specific proteins. In the future, colocalizing cellsand multiple biorecognition elements in the same microarray mayenable monitoring of location-specific differences in cell phenotypeexpression.

Creating Microarrays of Cells and Antibodies. In order toensure rapid incorporation and uniform distribution of biomol-ecules, PEG hydrogel-coated glass slides were dehydrated bylyophilization prior to printing of microarrays. Rehydration of thehydrogel matrix imprinted with protein microarrays for 30 minin cell culture media (MEM without phenol red) was sufficientto create a nonfouling PEG gel matrix around protein spots.Printed Ab molecules remained entrapped in the hydrogel layerover the course of cell cultivation and protein detection experi-ment, as discussed in the latter section of this paper.

When seeded on glass substrates imprinted with proteinmicroarrays, hepatocytes attached on collagen (I) regions formingcircular cell clusters ∼300 µm in diameter but did not adhere on

Ab domains or the surrounding PEG hydrogel matrix. Figure 2A,showing primary rat hepatocytes residing next to antialbumin(red) and anti-R1-AT (green) domains, highlights the possibilityof juxtaposing cells with multiple immunosensing domains. Thecolocalization of cells and sensing elements allowed the detectionof secreted cellular products in the immediate vicinity of the cells.Figure 2B,C shows bright-field and fluorescence images obtainedafter 16-h incubation of hepatic (HepG2) cell clusters in themicroarrays containing negative control (mouse IgG) spots onthe left and antialbumin Ab spots on the right of the cell clusters.The immunofluorescent staining of microarrays performed after16-h incubation revealed a strong fluorescence signal fromantialbumin domains and no signal from the negative control IgGdomain (Figure 2C). These results demonstrate proof-of-conceptdetection of specific proteins secreted by the neighboring cells.Characterization of the immunosensing microarrays described inthe latter sections of this paper allowed us to quantify levels ofthe secreted hepatic proteins.

Stability of Printed Antibody Arrays. The ability to detectbinding of cellular products within the microarray is stronglydependent on the retention and availability of the sensing Abmolecules over the course of the experiment. In order tocharacterize stability of biomolecules, arrays of antialbumin andanti-R1-AT spots were printed onto PEG hydrogel-coated glasssubstrates and incubated in cell culture media at 37 °C for timepoints ranging from 1 to 3 days. The substrates with printedmicroarrays were then exposed to guanidine-based buffer in orderto extract Ab molecules immobilized in the hydrogel matrix.Quantification of goat IgG proteins with ELISA was used tocharacterize time-based retention of printed Ab molecules. As seenfrom the results presented in Figure 3, surface density of Abmolecules decreased by 17.1 ± 4.0% between the initial (sampledimmediately after printing) and one-day time points. However,further incubation in cell culture media (up to 3 days) did notresult in additional loss of the Abs from the hydrogel matrix. Theseresults are encouraging and point to long-term stability of theimmunosensing Ab molecules immobilized in the PEG hydrogelmatrix after robotic printing. The initial decrease in Ab surface

Figure 2. Placing hepatic cell clusters next to sensing Ab spots. (A) Primary rat hepatocytes attached on 300-µm-diameter collagen (I) domainsin the immediate vicinity of antialbumin (Cy3, red) and anti-R1-AT (FITC, green) spots. Notice limited nonspecific cell adhesion on IgG Ab spotsand the surrounding, PEG hydrogel-coated glass surface. (B, C) Bright-field and fluorescence images of 300-µm-diameter clusters of humanhepatoma (HepG2) cells residing on collagen (I) spots with antimouse IgG regions on the left and antialbumin domains on the right of the cells.After 16-h incubation, cell/Ab microarrays were immunostained with biotinylated antialbumin followed by incubation with streptavidin-Alexa-546(red). (C) The fluorescence signal emanating from antialbumin Ab spots points to secretion and binding of this liver-specific protein. Negativecontrol spots to the left of the cells remain nonfluorescent.

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density is likely due to desorption of loosely bound moleculesoccurring shortly after immersion of microarrays into cell culturemedium.

Detection of Hepatic Albumin and r1-AT with Ab Mi-croarrays. To quantify production of liver-specific proteins,calibration curves correlating analyte concentration and thefluorescence intensity were created. In these experiments, humanalbumin, rat albumin, or human-R1-AT were printed onto hydrogel-coated glass substrates at solution concentrations ranging from3 to 100 ng/mL. Printed arrays of proteins were then immun-ostained by incubation with biotinylated Abs, followed by labelingwith streptavidin, Alexa Fluor-546 conjugate. Figure 4A showsconcentration-dependent modulation in fluorescence intensity ofthe printed R1-AT spots. Calibration curves for albumin and R1-AT shown in Figure 4B,C were then constructed by scanningfluorescently labeled protein arrays and plotting analyte concentra-tion as a function of fluorescence intensity. The higher sensitivityof microarray-based R1-AT biosensor compared to albumin sensor(seen in Figure 4B) is feasibly due to differences in association/dissociation kinetics of the antigen-antibody interaction sincesimilar trends were observed with traditional ELISA (data notshown). Calibration curves were fitted with a logarithmic best-fitcurve with R -value >0.98. Importantly, the detection range ofimmunosensing Ab spots was well within the levels of proteinproduction by hepatocytes, thus allowing us to use the calibrationcurves in Figure 4 to quantify production of albumin and R1-ATin a manner analogous to ELISA.

In order to quantify production of liver-specific proteins withinthe hepatocyte array, HepG2 cells (human) were cultivated oncollagen (I) domains in the vicinity of antialbumin, anti-R1-AT,and antimouse IgG (negative control) Ab spots (Figure 5A). After16-h incubation, cellular/Ab microarrays were fixed, immunof-luorescently labeled, and imaged using a laser microarray scanner.Figure 5A shows a representative region of the cell/Ab microarraywith a line scan across the sensing spots corresponding tofluorescence intensity profile presented in Figure 5B. As seen fromthe signal profile (Figure 5B), there was a clear difference inalbumin and R1-AT signals pointing to higher rate of productionof R1-AT by the HepG2 cells. This fact was corroborated by ELISA

experiments described later in this section. It should also be notedthat mouse IgG Ab spots used as an on-chip negative control hada low background signal leading to a high signal-to-noise ratio(S/N ratio of 7.2 for the example shown in Figure 5A,B) of ourimmunosensing Ab arrays. Using calibration curves discussedabove, the fluorescence intensity of the albumin biosensor wasconverted to synthesis rate of 4.1 ± 0.2 pg cell-1 day-1 while thesignal of R1-AT biosensor corresponded to protein production of11.7 ± 0.4 pg cell-1 day-1. ELISA analysis, performed to validateprotein levels detected with Ab microarrays, revealed similartrends of lower albumin production (4.5 ± 0.1 pg cell-1 day-1)compared to R1-AT (8.4 ± 0.5 pg cell-1 day-1). The levels ofalbumin production detected with Ab microarrays and ELISA werecomparable (no significant difference, p-value <0.005) whilemicroarray-based immunosensors yielded somewhat higher (∼30%)levels of R1-AT compared with ELISA. The discrepancy betweenAb microarray and ELISA measurements of R1-AT production maybe explained by experiment-to-experiment variability in hepaticphenotype expression of HepG2 cells. Overall, detection ofsecreted liver proteins bound on Ab microarrays closely paralleled

Figure 3. Quantifying retention of antialbumin and anti-R1-AT Abarrays on PEG hydrogel-coated glass slides. In these experiments,incubation of Ab microarrays for a desired period of time in cell culturemedia was followed by extraction of the remaining Ab molecules fromthe hydrogel matrix. Our data point to initial desorption of Abs between0 (immediately after printing) and 1 day time points, followed bystabilization of Ab spots.

Figure 4. Construction of calibration curves for detection of liver-specific proteins. (A) Different concentrations R1-AT printed onto PEGhydrogel-covered glass slides and detected by secondary labelingwith anti-R1-AT-biotin/streptavidin-Alexa 546. (B, C) Calibrationcurves correlating R1-AT and albumin concentrations to fluorescenceintensity were constructed by scanning fluorescence images shownin (A). Limits of detection of 6.3 ng/mL for R1-AT and 12.5 ng/mL foralbumin observed in our microarray immunoassays were comparableto standard ELISA.

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the levels of the same proteins measured in the cell culture mediaby ELISA. However, there are notable differences betweenstandard ELISA and our strategy for detection of cellular products.The microarray immunoassay described here is placed in situ atthe site of the cells; therefore, in cell culture experiments requiringfrequent changes of media (e.g., primary cell or stem cellcultures), immunosensing spots will be exposed to analyteconcentrations varying from high (just before media exchange)to low (immediately after changing media). In order to avoid thiscomplication, the data presented in Figure 5C were based on ashort-term (16 h) cell culture experiment that did not involvemedia exchange. Correlating in situ immunosensors to ELISA ina complex, multiday experiment would require coupling rate ofcellular protein production with association/dissociation kineticsof the antigen-antibody complexes to account for dissociationoccurring at low analyte concentration. With this in mind, cell/Ab microarrays described here may be more suitable for detectingrelative protein expression occurring in different locations of thesame cell culture substrate.

Interestingly, we did not observe gradients in concentrationof surface-bound proteins. This fact may be attributed to arelatively large hepatic population (12 cell clusters, ∼80 cells/cluster) robustly synthesizing proteins over relatively long incuba-

tion times ranging from hours to days. It is likely that reductionin the number of cells will lead to weaker signals with proteinconcentration decreasing as a function of distance from the cells.

The Ab microarrays were also employed to detect changes inliver phenotype expression over time. In these experiments,primary rat hepatocytes were cultivated on 300-µm-diametercollagen (I) spots in juxtaposition with sensing antialbumin Abdomains. As seen from Figure 5D, the rate of albumin synthesisincreased significantly (p-value <0.005) between one and threedays in culture. Given that primary rat hepatocytes have limitedproliferative capacity, the upregulation in albumin productiondetected with immunosensing Ab spots was likely due to posti-solation recovery and rescue of liver function in these cells. Similartrends of short-term phenotype recovery of primary hepatocytesfollowed by dedifferentiation and loss of liver function wereobserved previously.10

The data presented in Figure 5 point to the possibility ofintegrating sensing Ab spots into cellular microarrays to quantifyproduction of tissue-specific proteins. Our experiments focusedon proof-of-concept detection of two hallmark liver proteins,albumin and R1-AT; however, robotic microarraying technologyaffords almost unlimited opportunities for expanding the panel ofprinted Abs. Placement of multianalyte immunosensors with built-

Figure 5. Quantifying liver-specific protein production in microarrayed hepatocytes. (A) Immunofluorescent staining of a mixed array of HepG2cell clusters (300-µm diameter) and sensing Ab spots performed after 16-h incubation shows strong fluorescence signals from anti-R1-AT andantialbumin Ab spots and no signal from mouse IgG spot serving as a negative control (-). White trace shows a line scan from a microarrayscanner. (B) Fluorescence intensity profiles of Ab spots, corresponding to line scan in (A), was converted to protein concentration using calibrationcurves. (C) Comparing Ab microarray-based detection of liver proteins secreted by HepG2 cells with traditional ELISA. Levels of albumin detectedby the two methods were comparable while R1-AT production was overestimated by ∼30% in our immunosensing scheme. Experiments wererun in triplicate for each experimental method. (D) Ab microarray-based detection of time-dependent enhancement of albumin production inprimary rat hepatocytes. Given that these cells do not proliferate in vitro, the significant increase (p < 0.005) in albumin production from day 1to day 3 may be attributed to enhanced liver phenotype expression. Protein was produced by 12 clusters (300-µm diameter each) of primaryhepatocytes and detected on 20 antialbumin Ab spots.

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in negative/positive controls and analyte reference points at thesite of the cells allows the detection of multiple cellular productsin a small volume. Importantly, a number of detectable analytesin our microarray-based approach can be increased while main-taining a small (microliter) volume of the cell culture system. Incontrast, detection using traditional ELISA approaches requiresa relatively large volume of ∼100 µL for each analyte. There arealternatives to traditional ELISA (e.g., bead-based flow-throughassays) that allow for multianalyte, high-throughput detection tobe performed in small volumes;30,31 however, the ability to connectsecreted cellular products to the local microenvironment ofadherent cells is not afforded by these methodologies. The Abmicroarrays for detection of soluble proteins may be coupled withlaser-mediated retrieval of cells from specific locations on thesurface for downstream gene expression analysis.18 The combina-tion of these two approaches would provide transcriptional andtranslational information about phenotype expression in cellularmicropatterns or microarrays. In the future, the microarray-basedsensing system described here may be further improved byreplacing a sandwich immunoassay with aptamer beacons32,33 toallow for real-time detection of analyte binding, and obviating theneed for secondary Abs or fluorescence reporter molecules.

CONCLUSIONSThe present paper demonstrates merging of cellular and

immunoassay arrays for on-chip detection of secreted cellularproducts. Printing arrays of cell-adhesive domains alongside Ab

regions selective for liver-derived proteins, albumin and R1-AT,allowed the juxtaposition of hepatocytes and tissue-specific im-munosensors. Expression of liver-specific function of the hepato-cytes was then detected on the adjacent antialbumin and anti-R1-AT Ab spots using a sandwich immunofluorescent assay and wasquantified with a microarray scanner. Using the same microarrayfor both cell cultivation and biosensing enabled us to perform acomplex immunoassay for simultaneous detection of two analyteswith built-in redundancy and positive/negative controls at the siteof a small (∼800 cells) group of hepatocytes. In the future,integrating biosensing elements with screens of cell-microen-vironment interactions may allow for the monitoring of theappearance or loss of cell phenotype in a location-specific andmultiplexed fashion and, thus, will be particularly useful for stemcell differentiation/lineage selection studies.

ACKNOWLEDGMENTWe thank Prof. Soichiro Yamada’s laboratory in the Depart-

ment of Biomedical Engineering at UC Davis for assistance withconfocal microscopy. We thank Prof. Hari Reddi for usefuldiscussions and suggestions regarding testing of the stability ofprinted Ab arrays. Scanning of Ab microarrays was performed atExpression Analysis Facility, Genome Center at UC Davis.Financial support for this work was provided by NIH (DK073901).J.Z. was supported through an NIH Training Grant (EB003827).G.S. was supported through a Biotechnology Fellowship fromNational Center for Biotechnology, Republic of Kazakhstan.

Received for review April 16, 2008. Accepted June 9,2008.

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(30) Nolan, J. P.; Sklar, L. A. Trends Biotechnol. 2002, 20, 9–12.(31) Pregibon, D. C.; Toner, M.; Doyle, P. S. Science 2007, 22, 5122–5128.(32) Cho, E. J.; Collett, J. R.; Szafranska, A. E.; Ellington, A. D. Anal. Chim.

Acta 2006, 564, 82–90.(33) Hamaguichi, N.; Ellington, A. D.; Stanton, M. Anal. Biochem. 2001, 294,

126–131.

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