Published: April 14, 2011

r 2011 American Chemical Society 6116 dx.doi.org/10.1021/la1050833 | Langmuir 2011, 27, 6116–6123

ARTICLE

pubs.acs.org/Langmuir

Simple One-Step Process for Immobilization of Biomolecules onPolymer Substrates Based on Surface-Attached Polymer NetworksMartin Rendl,† Andreas B€onisch,† Andreas Mader,†,‡ Kerstin Schuh,† Oswald Prucker,†

Thomas Brandstetter,*,† and J€urgen R€uhe†

†Chemistry and Physics of Interfaces, Department of Microsystems Engineering (IMTEK), University of Freiburg,Georges-K€ohler-Allee 103, D-79110 Freiburg, Germany‡Institute of Pathology, Freiburg University Medical Center, Breisacher Strasse 115a, D-79106 Freiburg, Germany

bS Supporting Information

1. INTRODUCTION

Microarrays are versatile tools in biomedical research, as theypermit highly parallel analytics of various samples such as blood,urine, saliva, or tissues.1 However, despite considerable effort, thistechnology still suffers from rather complex and cost-intensivemanufacturing processes, tedious handling, and poor reproducibi-lity2,3 which still prevents a wider acceptance in today’s clinicalroutine diagnostics. The development of a suitable strategy, whichallows the simple and efficient immobilization of probemolecules tothe chip surfaces, plays a key role in the successful design andimplementation of a microarray.2 High surface concentration ofprobe molecules and retention of their biological activity, highsensitivity, and signal to background ratio as well as low unspecificbinding are crucial parameters of such a process. To permitquantitative or semiquantitative bioassays and to allow for low intra-and interchip variance, control over the amount of probe moleculesper area, their orientation, and reliable and robust immobilizationare decisive requirements. To meet these requirements, a widespectrum of different immobilization strategies has been developedand published over the years.

Commonly, biomolecules such as DNA strands or proteins areimmobilized on substrates through binding to a monolayer forthe functionalization of surfaces. To achieve efficient coupling,

several different chemistries have been developed. Most tech-nologies are based on surface modification of the substrate withactive groups reacting with more or less specific parts of thebiomolecule itself or conjugated linker groups such as biotiny-lated or amino-modified DNA or proteins.3,4 The strategies forthe immobilization of biomolecules can be divided into threecategories, in which the surface attachment of the biomolecules isbased on either covalent, physical, or affinity based bindinginteractions.5 Strategies which use covalent binding are oftenbased on amine chemistry, where, for example, aminosilanes orlysine moieties are used as anchoring groups for biomolecules.Probably the most commonly used binding mechanisms rely onsuccinimidyl esters (NHS), epoxy, aldehyde, or carbodiimidecontaining compounds, which covalently bind to amine groupsof the biomolecule.2 Covalent binding strategies yielding or-iented immobilization of proteins are based on maleimides anddisulfide derivates binding to cysteines of disulfide bridges in thehinge region of antibodies. Amino groups or hydrazines bindingto carbohydrate residues of the Fc portion of antibodies provide

Received: December 23, 2010Revised: March 16, 2011

ABSTRACT: For the miniaturization of biological assays, espe-cially for the fabrication of microarrays, immobilization of biomo-lecules at the surfaces of the chips is the decisive factor.Accordingly, a variety of binding techniques have been developedover the years to immobilize DNA or proteins onto such sub-strates. Most of them require rather complex fabrication processesand sophisticated surface chemistry. Here, a comparatively simpleimmobilization technique is presented, which is based on the localgeneration of small spots of surface attached polymer networks.Immobilization is achieved in a one-step procedure: probe mol-ecules are mixed with a photoactive copolymer in aqueous buffer,spotted onto a solid support, and cross-linked as well as bound tothe substrate during brief flood exposure to UV light. Thedescribed procedure permits spatially confined surface functionalization and allows reliable binding of biological species toconventional substrates such as glass microscope slides as well as various types of plastic substrates with comparable performance.The latter also permits immobilization on structured, thermoformed substrates resulting in an all-plastic biochip platform, which issimple and cheap and seems to be promising for a variety of microdiagnostic applications.

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an alternative covalent, oriented binding mechanism.6 Physicaladsorption of biomolecules onto substrates via intermolecularforces, ionic bonds, and hydrophobic and polar forces mainlyoccurs with porous materials such as polyaniline modifiedpolypropylene membranes or poly-L-lysine coated substrates.Bioaffine attachment of biomolecules often relies on bio-tin�avidin interactions. This system is known to form one ofthe strongest noncovalent bonds.5,6

All of these techniques are limited by the surface area availablefor binding. Due to the inherent limitation to a monomolecular(and thus two-dimensional) layer, only a limited amount ofbiologically active species can be immobilized per spot of themicroarray. The maximum surface density of the immobilizedmolecules is further limited, as the density of capture moleculescannot be increased too strongly without running into the dangerof losing their specific binding capabilities.7 For higher concen-trations, intermolecular distances are a crucial factor as theyinfluence the nativity of the bound species and the accessibilityfor target molecules as too dense binding results in strong sterichindrance. Additionally, the efficiency of immobilization isdrastically reduced for higher concentrations of probe molecules,as the space available for binding is limited.8,9

To increase the surface density of biological species onmicroarray substrates, novel immobilization strategies based onpolymer hydrogels have been developed.7,10�23 The establishedmethods can be divided into laminar coatings of the entiresubstrate10,15,16,19,20,22�24 and local modifications of the surfaceby means of dispensing,25�28 contact printing,29 or microstruc-turing.17,30�32 An increased binding capacity of polymer com-pared to planar spots was reported, coming along with improvedactivity of the immobilized biological species.17,23 This wasrelated to an increased intermolecular distance providing re-duced steric hindrance and hence better accessibility, as well asbetter retention of nativity.7,11,33 The latter is a crucial factorespecially for immunoassay applications.

However, most approaches rely on rather complex surfacechemistries, highly sophisticated fabrication processes, and complex,time-consuming handling procedures. In the following, a simpleone-step immobilization process for the fabrication ofmicroarrays ispresented. It is based on the photogeneration of surface attachedpolymer networks with simultaneously covalent anchoring ofoligonucleotides and proteins. We demonstrate that the processgenerally does not require a complex surface pretreatment and canbe used on standard plastic and glass surfaces as well as on morecomplex three-dimensional (3D) structured surfaces such as micro-fluidic channels or microwells. Furthermore, we study the compat-ibility of such functionalized surfaces with biological standardprocedures such as polymerase chain reaction (PCR)34,35 anddirect as well as sandwich immunoassays. The performance of thisimmobilization technique for typical microarray applications wasdetermined for DNA assays, genotyping human papilloma virus(HPV) and protein assays detecting autoimmune factors TPO andJo-1.

2. MATERIALS AND METHODS

2.1. Materials. Substrates. Injection molded microscopy slides(75 � 25 � 1 mm3) fabricated from polycarbonate (PC-08), polystyr-ene (PS-04), cyclic olefin copolymer (TOPAS-02), polypropylene (PP-02), and cyclic olefin polymer (COP-01) were obtained from micro-fluidic ChipShop GmbH, Jena, Germany. Bulk commercially availabletransparent foils of acrylic glass with a thickness of 1 mm were laser-cut

to microscope slide dimensions, and the protective adhesive foil wasremoved. All plastic substrates were ultrasonically cleaned as previouslydescribed by Neumann et al.36 Glass slides Nexterion Glass B (SchottAG, Germany) and epoxysilane modified glass substrates (NexterionSlide E, Schott AG, Germany) were used as obtained.

Oligonucleotides/DNA.Three specific oligonucleotide probes againstHPV genotypes 6 and 16 as well as control oligonucleotides (couplingcontrol and detection control) were chosen exemplarily. The samesequences have been used in previous work for microarray basedgenotyping of human papilloma virus.34,35 Oligonucleotides as well asHPV specific primers were synthesized by TIB MOLBIOL GmbH,Berlin, Germany. The sequences of all used oligonucleotides are listed inthe Supporting Information.

Proteins. For immunoassay tests, a sandwich immunoassay specificfor thyroid peroxidase (TPO, 101 kDa) and Jo-1 (58 kDa) wasperformed. These factors were chosen as clinically relevant parameters forthe indication of certain autoimmune diseases.37�40Human sera containingantibodies against these parameters were used as analytes. Cy5-conjugatedsecondary anti-human IgG antibody (109-175-098, Jackson Immunore-search, West Grove, PA) was used for labeling of bound species. Allparameters were obtained from Diarect AG (Freiburg, Germany). Cy5-conjugated anti-mouse secondary antibodies (ab6563, abcam, U.K.) wereused in coupling controls for protein microarrays. For direct protein assays,antibodies against human IgG (109-005-003, Jackson Immunoresearch,West Grove, PA) were used as capture antibodies and human IgG (009-000-003, Jackson Immunoresearch, West Grove, PA) was used as analyte.The latter was labeled with DY647 fluorophores using a Fluoro Spin 647labeling kit (empbiotechGmbH,Berlin,Germany).The labeling procedurewas carried out according to the recommendations of the manufacturer.Likewise, the degree of labeling and the resulting effective concentration oflabeled protein were determined.2.2. Synthesis and Characterization of the Photoactive

Copolymer. The copolymer was synthesized using a standard freeradical polymerization process. In a typical run, N,N-dimethylacrylamide(DMAA, 92.5 mol %), methacryloyloxybenzophenone (MaBP, 5 mol %),8

and Na-4-styrenesulfonate (SSNa, 2.5 mol %) were dissolved undernitrogen in methanol with a concentration of 2 mol/L, and 0.1 mol % R,R0-azoisobutyronitrile (AIBN) was added. After five freeze and thaw cycles,the solution was placed into a preheated water bath and kept at 60 �C for20 h. After completion of the polymerization reaction, the polymer wasprecipitated by dropwise addition of the solution to a large excess ofdiethylether, filtered off, and purified by reprecipitation from methanol.After freeze-drying, the copolymer was obtained as a white powder in atypical yield of 50% and Mw of around 300 000 g/mol.12 The synthesizedand freeze-dried copolymer was stored at 4 �C protected from light. Adetailed chemical characterization of the copolymer including NMR, UV/vis, and FTIR spectra can be found in the Supporting Information.2.3. Microarray Fabrication. Prior to printing, the polymer was

dissolved in deionized water at a concentration of 10 mg/mL. The printingsolution contained the copolymer mixed with the biological species to beimmobilized in a printing buffer. In the case of oligonucleotides, 100 mMsodiumphosphate buffer (Napi, pH7.4) was used for printing, and proteinswere diluted in 10 mM phosphate buffered saline (PBS (Sigma Aldrich, St.Louis, MO). The final polymer content in solution was 1 mg/mL. Themolar concentration of polymer and proteins in spotting solution was in thesame order of magnitude and slightly higher for oligonucleotide probes,respectively. This ratio of copolymer chains to biomolecules yielded bestresults in preliminary experiments.

For each individual material and biological species, separate micro-arrays were printed. Additionally, batches of 10 identical microarrayswhere printed on poly(methyl methacrylate) (PMMA),34,35 for eachbiological species. All chips had an on-chip redundancy of at least 4, andthey were fabricated in one single process run to ensure best homo-geneity and comparability between the individual materials to be tested.

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Solutions containing the probe molecules and copolymer chains werespotted together onto the substrates in a contactless printing process(SciFlexArrayer S3, Scienion AG, Germany). The volume per dispenseddrop was 400( 20 pL. The resulting spot diameter depended on the surfacechemistry of the usedmaterial andwas inmost cases around150μm.Printingof the arrays was performed under clean room conditions at a temperature of22 �C and a relative humidity of 40%. Surface immobilization is achievedthrough irradiationwithUV light (Stratalinker 2400, Stratagene, La Jolla,CA)at a wavelength of 254 nm (Figure 2). The energy used for the cross-linkingprocess was 0.5 J/cm2 for the attachment of proteins and 1.25 J/cm2 foroligonucleotides. Suitable energy levels were determined experimentally as acompromise between robustness of immobilization and nativity of the probemolecules.2.4. Assay Handling. Washing and Blocking. To reduce unspe-

cific binding of labeled analyte, protein microarrays were blocked with asolution of milk powder (5% (w/v)) and BSA (1%(w/v)) in 10 mMphosphate buffered saline (PBS) for 15 min in staining jars on a shaker(KS 260 basic, IKA GmbH, Germany). The speed was 50 rpm, whichprovided gentle agitation. Finally, the samples were dried with nitrogen.

DNA biochips were washed in 100 mM sodium phosphate buffer, pH7.0 (Napi-buffer) with 0.1% (v/v) Tween 20 for 5 min, speed 50 rpm instaining jars on a shaker. During this step, unbound probemolecules andpolymer chains are removed. Hybridization and staining steps were eachtime followed by a similar washing step in Napi-buffer. All chips weredried with nitrogen prior to readout.Incubation/Hybridization. Hybridization experiments were carried

out using biotin-dUTP labeled PCR product of HPV 16. PCR reactionswere carried out as described previously.34 Briefly, a plasmid templateconsisting of the vector pCR2.1 was generated and 10 ng was used in aPCR subsequently. The employed pair of primers results in a PCRproduct of nearly 405 bp (genotype-specific length variation). To obtainsingle stranded PCRproducts, the samples were denatured at 95 �C for 5min. Prior to hybridization, the sample was mixed in equal parts with200 mM Napi.

Incubation of fabricated microarrays was performed in a Slideboosterhybridization station (Advalytix, Germany). Lifter slips (22 � 25 mm2,Implen GmbH, Germany) were used as reaction chambers and filledwith an analyte containing solution of 25 μL. To minimize evaporationduring the incubation experiments, the buffer reservoirs within thehybridization station were filled with the same buffer used for incubationwith analyte.Mixing intensity was set to 15/27, pulse pause ratio was 5:5,and temperature was 30 �C. In the case of oligonucleotide microarrays,the duration of hybridization with PCR amplicons was 10 min followedby a labeling step of 5 min. One tenth of the PCR product diluted inNapi-buffer was used for hybridization In the case of the proteinmicroarrays, incubation with analyte was performed for 2 h followedby incubation with labeling antibody solution for 30 min. The proteinmicroarrays were incubated with a 1:100 dilution of human serumcontaining either of the two parameters TPO and Jo-1. The dilutingsolution consisted of PBS, 2.5% (w/v) milk powder, 0.5% (w/v) BSA,and 0.05% (v/v) Tween 20.Labeling/Staining. Labeling of bound analyte was performed with

Cy5-conjugated streptavidin (GE Healthcare, diluted 1:200 in Napi-buffer) binding to biotinylated probes in the case of oligonucleotidemicroarrays. For protein microarrays, a Cy5-conjugated secondary anti-human antibody (109-175-098, Jackson Immunoresearch) was used at aconcentration of 0.2 μg/mL binding to the autoimmune specific analytes.2.5. Fluorescence Measurements and Hybridization Anal-

ysis. Read-out of the microarrays was performed in a biochip readerbased on total internal reflection fluorescence (TIRF) technologysimilar to the system described by Lehr et al.41 Laser light (635 nm) iscoupled into the edge of the microarray substrate, which acts as awaveguide. Upon total internal reflection and adequate optical focusing,a homogeneous evanescent field is generated at the surface of the

microarray. Hence, only fluorophores located within the volume cov-ered by the evanescent field are excited and can emit a fluorescencesignal. Quantitative analysis of the fluorescence intensities was per-formed using the software Signalyse (Holger Klapproth Life Science,Germany). It provides automated background correction of spotintensities with regard to autofluorescence of substrate materials andfluorescence related to unspecific adsorption of fluorescent molecules.To assess unspecific signal contribution by the polymer network, spotscontaining polymer chains but no biological species were added to themicroarrays as negative controls.

3. RESULTS AND DISCUSSION

3.1. Fabrication of the Hydrogel Based Microarrays. Theimmobilization technique for biological macromolecules de-scribed here is based on the formation of small surface-attachedhydrogel networks, which are generated in specific locations of thesubstrate to covalently attach biomolecules. For the fabrication of themicroarrays, a solution consisting of a benzophenone group contain-ing photopolymer and the desired probemolecules in aqueous buffersolution is dispensed onto the substrate in a contactless printingprocess. An advantage of the used printingmethod is that the volumeof dispensed drops can be monitored and adjusted. It permitsdeposition of defined amounts of probe molecules per spot with aprecision of better than (10%, which is a crucial requirement forreliable quantitative analyses. Furthermore, contactless printingallows deposition of probe molecules on complex 3D structuredsurfaces typical for a broad area of bioanalytical applications such asmicrofluidic channels or wells in wellplates (Figure 7).The terpolymer employed for the generation of the gels

primarily consists of poly(dimethylacrylamide) (PDMAA) as ahydrophilic matrix. A second component contained in the polymeris the sodium salt of styrene sulfonate (SSNa) which is incorpo-rated in the polymer chains to introduce charges to the polymerand thus further enhance their solubility in water. Styrene sulfonateincorporation proved to be superior to the vinyl phosphoric acidused in previous work,8,34�36,42 as the corresponding polymer wassimpler to synthesize and better control over the exact molecularweight and composition of the copolymer could be achieved. Thethird component of the polymer is 4-methacryloxyl-oxy-benzo-phenone (MABP), which provides photo reactive groups to thepolymer (Figure 1).After printing of the microarray, the dispensed drops rapidly

dry and form dots consisting of a mixture of glassy polymer,crystallized buffer salt, and the biological compounds. However, itshould be denoted that the polymer network is not entirelydesiccated, since both the polymer network and the salt residuesare very hygroscopic. Upon irradiation (i.e., flood exposure) thephotopolymer contained in the dots is cross-linked. Simultaneouslyto the cross-linking of the polymer matrix, the forming network is

Figure 1. Chemical structure of the copolymer used for the fabricationof the microarrays.

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bound to the substrate and the probe molecules are covalentlylinked to the polymeric scaffold by the same photochemical process.The photochemical reaction is caused by the benzophenonemoieties in the copolymer chains as described by Toomey et al.:12

Upon irradiation with UV light, the photoreactive MABP segmentundergoes an n,π* transition into a biradicaloid triplet state. In thisstate, themolecule is capable to abstract a hydrogen atom fromalmostany neighboring aliphatic C�H group, which is in close vicinity. Thetwo resulting carbon-based radicals generated by the hydrogenabstraction process can recombine and form a covalent C�C bond.The neighboring aliphatic groups can be part of other polymer chains,can be part of the substrate, or canbe contained in the biomolecule. Inaddition, photoreactive groups of the biological species itself, such asthymine in the case of DNA strands43 or tryptophan or tyrosine forproteins,44,45 can participate in the immobilization process by under-going a similar photochemical reaction. Immobilization of biomole-cules to surfaces via photogenerated radical cross-linking reactionsis a well-described technique.8,46,47 The short time scale of the UV-exposure and the thus employed rather small light dose does notaffect the functionality of the biomolecules as could be shownexperimentally.8,34,42 Alternatively, light of lower energy/longer wa-velength, for example, 360 nm, could be used. However, this requiresconsiderably longer exposure time, which might reduce the perfor-mance in subsequent assays.Salt residues on the microarrays originating from the printing

buffer do not take part in the photo-cross-linking process. They arewashed out of the microarray spots in subsequent washing steps.

An interesting feature of the described process is that func-tionalization occurs only at locations where solution has beenprinted onto. This spatially confined functionalization of supportmaterials takes place by simultaneous deposition and immobili-zation of probe molecules in a single process step (Figure 2).3.2. Process Characterization. Efficiency of Immobilization.

As outlined above, robust attachment of biological probe mol-ecules is a crucial requirement for microarray based analytics. Todetermine the efficiency of attachment of probe molecules to thechip surface within the polymer network, coupling controls ofCy5-labeled IgG and Cy5-labeled oligonucleotide strands wereprinted on PMMA standard substrates in a series of spotscontaining dispensed volumes ranging from 400 pL up to 2.8nL per spot by deposition of multiple drops per spot. The totalamount of labeled species per spot ranged from 66 amol to 0.46fmol for proteins and 0.4 fmol to 2.8 fmol for oligonucleotides.The efficiency of the immobilization process is expressed as theratio of spot intensities before and after washing of the DNAmicroarrays (Figure 3) and a blocking procedure for proteins,respectively (Figure 4). For both oligonucleotides and proteinmicroarrays, about 60% of themolecules initially contained in theprinting solution were immobilized within the surface-attachedhydrogel spots. The loss during washing might be due to awashing out of biological species or polymer chains containingbiomolecules. During the cross-linking process, polymer chainsbecome part of the network only with a certain probability, whichis related to the number of cross-linking groups per chain, themolecular weight of the polymer chains, and the conversion of

Figure 2. Flowchart of the microarray fabrication process, including an artist’s view of the cross-linking reaction and a molecular scheme of thephotoreaction of the photoactive benzophenone moieties contained in the polymer.

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the photoreaction. A certain fraction of polymer chains couldsimply be too short and/or might not carry enough cross-linkingunits to be sufficiently connected to the network or to thesubstrate. Additionally, some loss of polymer chains might beassociated with ripping off of loosely attached material duringprocessing. The polymer solution contains a printing buffer,which upon drying leads to formation of salt crystals within thehydrogel network. These crystals dissolve during washing pro-cesses and might also mechanically remove neighboring parts ofthe polymer network together with attached biological species.The polymer distribution within such dots was analyzed in

preliminary experiments using microarrays which carried dotswith direct fluorescencetly labeled (Fluorescein) polymer. Fluor-escence images obtained for this microarray with epifluorescencemicroscopy showed a rather homogeneous intensity distributionacross the area of the microarray dots directly after printing andcross-linking and after washing. This indicates that the polymer issufficiently homogeneously distributed. However, this is noindication for the uniformity of the network’s cross-linkingpoints, which is not determined yet.

Fluorescence intensities measured directly after printing andcross-linking and after washing showed a linear dependence onthe dispensed volume of labeled species per spot for both labeledoligonucleotides as well as proteins. This linear dependencypermits a tuning of the signal intensity of a dot in the array byprinting of an appropriate volume of biological species. Theability to adjust the signal intensity significantly simplifies theread-out of different analytes in one biological sample. Typicallybiological analytes cover several orders of magnitude in concen-tration, while the dynamic range of read-out devices is usuallylimited. Using the technique described here, the volume of dotsgiving only low intensity signals can be increased to enhance thesignal intensity. In contrast to an increase of the probe concen-tration in the dot, an increase in volume will not affect the spatialorientation of probes and the intermolecular distances. This isimportant with regard to nativity and accessibility of biomole-cules especially for high concentrations.7

To assess possible quenching effects, microarrays were printedwith varying amounts of probe molecules per spot. Anti-humanIgG antibodies were immobilized on PMMA substrates inamounts ranging from 4.2 to 66.7 amol per spot. The measuredsignals (Figure 5) after incubation with Dy647 conjugatedhuman IgG showed a linear dependency on the amount of probemolecules, which indicates that no significant quenching isoccurring. This is in good agreement with similar experimentsby Zubtsov et al.7

Biofunctionality and Assay Performance. To elucidate theinfluence of hydrogel immobilization on the biofunctionality ofthe surface attached species, binding (hybridization) experi-ments with clinically relevant analytes were performed for botholigonucleotides and proteins. In the case of DNA based micro-arrays, chips were fabricated with spots containing probes for theidentification of HPV genotypes 16 and 6. These probes werehybridized with biotinylated PCR amplicons of HPV 16 specificDNA sequences. Fluorescence intensities of microarray spotsmeasured after staining with Cy5-labeled streptavidin yieldstrong, specific signals for spots containing HPV 16 specificprobes while signals of spots specific for HPV 6 showeddistinctively lower intensities, equal to those of the negativecontrols. This holds for all tested chip materials as shown inTable 1. Signals were calculated as the average intensity of threedifferent probes per parameter.

Figure 4. Signal intensities measured for Cy5-conjugated anti-mousesecondary IgG as a function of the number of drops deposited per spot.(9) Signals measured prior to blocking of the microarrays; (O) signalsafter blocking with milk powder (5% (w/v)) and BSA (1% (w/v)) inPBS (1�). Amount of labeled species per disposed drop was 66 amol.Volume per drop was 400( 20 pL. Error bars show standard deviationfor 10 microarrays with an on-chip redundancy of 8.

Figure 5. Fluorescence signal intensities as a function of the amount ofanti-human IgG capture molecules after incubation with solutioncontaining 15 μg/mL Cy5-conjugated human IgG. Displayed areaverage intensities measured for 4 microarrays with an on-chip redun-dancy of 8. Error bars show standard deviation of the individual sets ofchips. Details are described in the text.

Figure 3. Signal intensities of spots containing immobilized Cy5-conjugated oligonucleotides (coupling controls) as a function of thenumber of drops deposited per spot. (9) Signals measured prior towashing of themicroarrays; (O) signals after washing with 100mMNapi(0.1% (v/v) Tween). The amount of labeled species per deposited dropwas 0.4 fmol. Volume per drop was 400 ( 20 pL. Error bars showstandard deviation for 10 microarrays with an on-chip redundancy of 8.

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Similar experiments were carried out for protein microarrays,where two clinically relevant parameters TPO and Jo-1 were usedexemplarily. After incubation of the chips with serum obtainedfrom real patient samples, which contained specific antibodiesagainst TPO or Jo-1, the microarrays showed specific signals forthe particular parameter present as shown in Figure 6. Similarlyto the results for oligonucleotide microarrays, signals measured forthe nonexpressed, unspecific parameter as well as the negativecontrols reveal distinctively lower and rather equal intensities. Hence,cross reactions as well as unspecific binding can be excluded for thesetwo clinically relevant protein factors (Table 1).However, it should be noted that it is beyond the scope of this

contribution to draw conclusions about the suitability of a certainmaterial for a specific diagnostic application. This would require

considerable experimental effort, process optimization, anddedicated handling protocols for each material.Here, the aim was to show the principle applicability of

this immobilization approach for a representative selection ofsupport materials based on handling protocols and read-outtechnology which had been specifically adapted for PMMAsubstrates.Signal-to-Noise Ratio and Signal-to-Background Ratio. To

obtain a good signal-to-noise ratio in the chip analysis process,low nonspecific adsorption of analyte molecules and low back-ground fluorescence of both the substrate and the hydrogel dotsare required.In conventional microarray analysis, the signal-to-noise value

is frequently taken as the ratio of signal intensities of the areas ofinterest (i.e., the spots of the microarray) and their surroundings(i.e., the blank substrate).11 However, in the case of spatiallyconfined surface functionalization such as in this case of polymercontaining spots, it has to be taken into account that the surface areaaround the hydrogel spots does not reflect the background contribu-tion of the polymer itself. To assess the latter, we additionally printednegative controls, which are spots that contain the same polymer butno probe molecules. In all following experiments, we define thesignal-to-background ratio as the quotient of the signal intensities ofprobe containing hydrogel and “empty” control spots. A similarmethod was reported by Angenendt et al.48

Typically, the fluorescence intensity measured for such nega-tive controls was very small compared to intensities of spotscontaining specific probe molecules (Table 1). Since the func-tionalization reaction occurs only in areas of interest, namely, atthe spot locations, the overall background signal is stronglyreduced. In addition, the hydrogel is strongly protein repellentand no nonspecific adsorption occurs. It is a characteristic featureof PDMAA based polymer networks to prevent nonspecificadsorption of biological macromolecules to a large extent. Thisis related to the highly swollen state of such surface-attachednetworks in aqueous solutions, which show “entropic shielding”against protein adsorption.8,12,36,49

Immobilization on 3D Structured Supports. Besides immo-bilization on conventional planar substrates, the immobilizationtechnique described here was also employed successfully to

Table 1. Examples for Substrate Materials Used for Photo-polymer Based Microarray Productiona

oligonucleotidemicroarrays

proteinmicroarrays

material specificitybsignal/

backgroundc specificitybsignal/

backgroundc

PMMA þþþ þþþ þþþ þþþPC þ þ þþþ þþþPS 0 (*) 0 (*) 0 (*) 0 (*)Topas þþþ þþþ þþ þþPP þ (**) þ (**) 0 (**) 0 (**)COP þþ þþ þþþ þþþNexterion B þþ þþ þþþ þþþNexterion E þ þþ þþ þþþ

aClassification:þþþ, values > 20;þþ, 20g valuesg 10;þ, values >5; “0” values < 5; (*) PS showed comparatively high auto fluorescence;(**) PP is not compatible with the TIRF based read-out techniquebecause of its opacity. b Specificity is defined as the ratio of signalsmeasured for spots containing specific probe molecules and spotscontaining other probes than those specific to the analyte. c Signal/background is defined as the ratio of signal intensities of spots containingspecific probes and signal intensities of negative control spots.

Figure 6. (A) White light image showing a typical microarray onPMMA substrate. (B) False color image of a microarray after a direct assaywith Cy5-conjugated IgG; shown are in alternating order from top tobottom dilution series of coupling controls and spots with immobilized anti-IgG antibodies (from left to right, dilution factor of 2). (C) Fluorescenceimage of microarrays (subarrays) after assays with serum containing anti-TPO antibodies and serum containing JO-1 specific antibodies (D).

Figure 7. Polymer hydrogel dots on 3D structured surfaces. (A)Glucoseoxidase immobilized within microwell electrodes (diameter100 μm) using the polymer hydrogel technology. (B�D) Microarray dotsimmobilized within microfluidic channel structures. Inset in (D) shows afluorescence image of a microarray after a DNA hybridization assay.

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attach probe molecules to substrates with more complex topo-graphies such as microtiter plates, microwells, or microfluidicchannels (Figure 7). Especially the latter might be interesting, asit allows one to combine the format of classic microarrays withmore sophisticated microfluidic applications. Integration ofmicroarray based bioassays within fluidic designs such as lab-on-a-chip assemblies will permit automatization and reduction ofassay time by using enhanced mass transport of analyte mol-ecules to its corresponding binding partners, which at lowconcentrations frequently is a strongly limiting factor of micro-analytical processes.50�52

4. CONCLUSION

Printing of solutions of biomolecules together with a benzo-phenone group containing photopolymer followed by brief UV-irradiation allows for a simple and spatially confined functiona-lization of planar and structured substrates in a single processstep. During the irradiation process, photo-cross-linking of thedeposited polymer takes place and a highly water-swellablehydrogel is formed. Simultaneously to cross-linking of the matrix,the biomolecules become chemically attached to the hydrogelmatrix and the whole forming network becomes covalentlybound to the substrate. The described process can be used forthe immobilization of biomolecules such as DNA or proteinsonto a wide spectrum of different substrates, ranging frominorganic substrates modified with self-assembled monolayers(SAMs) to a large variety of different polymer materials. Itrequires no sophisticated pretreatment of the microarray sub-strates. This “all plastic” biochip technology can be combinedwith biological standard procedures such as PCR and immu-noassays and shows a comparable performance to commonlyused glass slides. This immobilization technique is ideally suitedfor plastic materials commonly used in microfluidic structuresand lab-on-a-chip applications, rendering it a promising platformfor high-throughput biomedical applications.

’ASSOCIATED CONTENT

bS Supporting Information. Synthesis and characterizationof the copolymer; H NMR, FTIR, and UV�vis spectra of thecopolymer and a table listing sequences of all used oligonucleo-tide probes specific for HPV subtypes 6 and 16. This material isavailable free of charge via the Internet at http://pubs.acs.org.

’ACKNOWLEDGMENT

We thank Alexander Dietz for valuable technical support. Wegratefully acknowledge the valuable contributions from Dr.Richard Kneusel and Dr. Armin Neininger from DIARECTAG, Freiburg, Germany, who also provided the autoimmuneproteins. Microfluidic ChipShop, Jena, Germany is thanked forproviding the various plastic microscope slides as well as themicrofluidic substrates. This work was supported by the BMWi-AIF (“Channel & Spots”; KF2162002UL9).

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