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Simple One-Step Process for Immobilization of Biomolecules on Polymer Substrates Based on Surface-Attached Polymer Networks Martin 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 ABSTRACT: For the miniaturization of biological assays, especially for the fabrication of microarrays, immobilization of biomolecules at the surfaces of the chips is the decisive factor. Accordingly, a variety of binding techniques have been developed over the years to immobilize DNA or proteins onto such substrates. Most of them require rather complex fabrication processes and sophisticated surface chemistry. Here, a comparatively simple immobilization technique is presented, which is based on the local generation of small spots of surface attached polymer networks. Immobilization is achieved in a one-step procedure: probe molecules are mixed with a photoactive copolymer in aqueous buffer, spotted onto a solid support, and cross-linked as well as bound to the substrate during brief flood exposure to UV light. The described procedure permits spatially confined surface functionalization and allows reliable binding of biological species to conventional 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 is simple and cheap and seems to be promising for a variety of microdiagnostic applications.
1. INTRODUCTION Microarrays are versatile tools in biomedical research, as they permit highly parallel analytics of various samples such as blood, urine, saliva, or tissues.1 However, despite considerable effort, this technology still suffers from rather complex and cost-intensive manufacturing processes, tedious handling, and poor reproducibility2,3 which still prevents a wider acceptance in today’s clinical routine diagnostics. The development of a suitable strategy, which allows the simple and efficient immobilization of probe molecules to the chip surfaces, plays a key role in the successful design and implementation of a microarray.2 High surface concentration of probe molecules and retention of their biological activity, high sensitivity, and signal to background ratio as well as low unspecific binding are crucial parameters of such a process. To permit quantitative or semiquantitative bioassays and to allow for low intraand interchip variance, control over the amount of probe molecules per area, their orientation, and reliable and robust immobilization are decisive requirements. To meet these requirements, a wide spectrum of different immobilization strategies has been developed and published over the years. Commonly, biomolecules such as DNA strands or proteins are immobilized on substrates through binding to a monolayer for the functionalization of surfaces. To achieve efficient coupling, r 2011 American Chemical Society
several different chemistries have been developed. Most technologies are based on surface modification of the substrate with active groups reacting with more or less specific parts of the biomolecule itself or conjugated linker groups such as biotinylated or amino-modified DNA or proteins.3,4 The strategies for the immobilization of biomolecules can be divided into three categories, in which the surface attachment of the biomolecules is based on either covalent, physical, or affinity based binding interactions.5 Strategies which use covalent binding are often based on amine chemistry, where, for example, aminosilanes or lysine moieties are used as anchoring groups for biomolecules. Probably the most commonly used binding mechanisms rely on succinimidyl esters (NHS), epoxy, aldehyde, or carbodiimide containing compounds, which covalently bind to amine groups of the biomolecule.2 Covalent binding strategies yielding oriented immobilization of proteins are based on maleimides and disulfide derivates binding to cysteines of disulfide bridges in the hinge region of antibodies. Amino groups or hydrazines binding to carbohydrate residues of the Fc portion of antibodies provide Received: December 23, 2010 Revised: March 16, 2011 Published: April 14, 2011 6116
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Langmuir an alternative covalent, oriented binding mechanism.6 Physical adsorption of biomolecules onto substrates via intermolecular forces, ionic bonds, and hydrophobic and polar forces mainly occurs with porous materials such as polyaniline modified polypropylene membranes or poly-L-lysine coated substrates. Bioaffine attachment of biomolecules often relies on biotinavidin interactions. This system is known to form one of the strongest noncovalent bonds.5,6 All of these techniques are limited by the surface area available for binding. Due to the inherent limitation to a monomolecular (and thus two-dimensional) layer, only a limited amount of biologically active species can be immobilized per spot of the microarray. The maximum surface density of the immobilized molecules is further limited, as the density of capture molecules cannot be increased too strongly without running into the danger of losing their specific binding capabilities.7 For higher concentrations, intermolecular distances are a crucial factor as they influence the nativity of the bound species and the accessibility for target molecules as too dense binding results in strong steric hindrance. Additionally, the efficiency of immobilization is drastically 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 on microarray substrates, novel immobilization strategies based on polymer hydrogels have been developed.7,1023 The established methods can be divided into laminar coatings of the entire substrate10,15,16,19,20,2224 and local modifications of the surface by means of dispensing,2528 contact printing,29 or microstructuring.17,3032 An increased binding capacity of polymer compared to planar spots was reported, coming along with improved activity of the immobilized biological species.17,23 This was related to an increased intermolecular distance providing reduced steric hindrance and hence better accessibility, as well as better retention of nativity.7,11,33 The latter is a crucial factor especially for immunoassay applications. However, most approaches rely on rather complex surface chemistries, highly sophisticated fabrication processes, and complex, time-consuming handling procedures. In the following, a simple one-step immobilization process for the fabrication of microarrays is presented. It is based on the photogeneration of surface attached polymer networks with simultaneously covalent anchoring of oligonucleotides and proteins. We demonstrate that the process generally does not require a complex surface pretreatment and can be used on standard plastic and glass surfaces as well as on more complex three-dimensional (3D) structured surfaces such as microfluidic channels or microwells. Furthermore, we study the compatibility of such functionalized surfaces with biological standard procedures such as polymerase chain reaction (PCR)34,35 and direct as well as sandwich immunoassays. The performance of this immobilization technique for typical microarray applications was determined for DNA assays, genotyping human papilloma virus (HPV) and protein assays detecting autoimmune factors TPO and Jo-1.
2. MATERIALS AND METHODS 2.1. Materials. Substrates. Injection molded microscopy slides (75 25 1 mm3) fabricated from polycarbonate (PC-08), polystyrene (PS-04), cyclic olefin copolymer (TOPAS-02), polypropylene (PP02), and cyclic olefin polymer (COP-01) were obtained from microfluidic ChipShop GmbH, Jena, Germany. Bulk commercially available transparent foils of acrylic glass with a thickness of 1 mm were laser-cut
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to microscope slide dimensions, and the protective adhesive foil was removed. All plastic substrates were ultrasonically cleaned as previously described by Neumann et al.36 Glass slides Nexterion Glass B (Schott AG, Germany) and epoxysilane modified glass substrates (Nexterion Slide E, Schott AG, Germany) were used as obtained. Oligonucleotides/DNA. Three specific oligonucleotide probes against HPV genotypes 6 and 16 as well as control oligonucleotides (coupling control and detection control) were chosen exemplarily. The same sequences have been used in previous work for microarray based genotyping of human papilloma virus.34,35 Oligonucleotides as well as HPV specific primers were synthesized by TIB MOLBIOL GmbH, Berlin, Germany. The sequences of all used oligonucleotides are listed in the Supporting Information. Proteins. For immunoassay tests, a sandwich immunoassay specific for thyroid peroxidase (TPO, 101 kDa) and Jo-1 (58 kDa) was performed. These factors were chosen as clinically relevant parameters for the indication of certain autoimmune diseases.3740 Human sera containing antibodies against these parameters were used as analytes. Cy5-conjugated secondary anti-human IgG antibody (109-175-098, Jackson Immunoresearch, West Grove, PA) was used for labeling of bound species. All parameters were obtained from Diarect AG (Freiburg, Germany). Cy5conjugated anti-mouse secondary antibodies (ab6563, abcam, U.K.) were used 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 (009000-003, Jackson Immunoresearch, West Grove, PA) was used as analyte. The latter was labeled with DY647 fluorophores using a Fluoro Spin 647 labeling kit (emp biotech GmbH, Berlin, Germany). The labeling procedure was carried out according to the recommendations of the manufacturer. Likewise, the degree of labeling and the resulting effective concentration of labeled protein were determined.
2.2. Synthesis and Characterization of the Photoactive Copolymer. The copolymer was synthesized using a standard free radical 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 under nitrogen 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 for 20 h. After completion of the polymerization reaction, the polymer was precipitated by dropwise addition of the solution to a large excess of diethylether, filtered off, and purified by reprecipitation from methanol. After freeze-drying, the copolymer was obtained as a white powder in a typical yield of 50% and Mw of around 300 000 g/mol.12 The synthesized and freeze-dried copolymer was stored at 4 °C protected from light. A detailed 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 printing solution contained the copolymer mixed with the biological species to be immobilized in a printing buffer. In the case of oligonucleotides, 100 mM sodium phosphate buffer (Napi, pH 7.4) was used for printing, and proteins were diluted in 10 mM phosphate buffered saline (PBS (Sigma Aldrich, St. Louis, MO). The final polymer content in solution was 1 mg/mL. The molar concentration of polymer and proteins in spotting solution was in the same order of magnitude and slightly higher for oligonucleotide probes, respectively. This ratio of copolymer chains to biomolecules yielded best results in preliminary experiments. For each individual material and biological species, separate microarrays were printed. Additionally, batches of 10 identical microarrays where printed on poly(methyl methacrylate) (PMMA),34,35 for each biological species. All chips had an on-chip redundancy of at least 4, and they were fabricated in one single process run to ensure best homogeneity and comparability between the individual materials to be tested. 6117
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Langmuir Solutions containing the probe molecules and copolymer chains were spotted together onto the substrates in a contactless printing process (SciFlexArrayer S3, Scienion AG, Germany). The volume per dispensed drop was 400 ( 20 pL. The resulting spot diameter depended on the surface chemistry of the used material and was in most cases around 150 μm. Printing of the arrays was performed under clean room conditions at a temperature of 22 °C and a relative humidity of 40%. Surface immobilization is achieved through irradiation with UV light (Stratalinker 2400, Stratagene, La Jolla, CA) at a wavelength of 254 nm (Figure 2). The energy used for the cross-linking process was 0.5 J/cm2 for the attachment of proteins and 1.25 J/cm2 for oligonucleotides. Suitable energy levels were determined experimentally as a compromise between robustness of immobilization and nativity of the probe molecules. 2.4. Assay Handling. Washing and Blocking. To reduce unspecific binding of labeled analyte, protein microarrays were blocked with a solution of milk powder (5% (w/v)) and BSA (1%(w/v)) in 10 mM phosphate buffered saline (PBS) for 15 min in staining jars on a shaker (KS 260 basic, IKA GmbH, Germany). The speed was 50 rpm, which provided gentle agitation. Finally, the samples were dried with nitrogen. DNA biochips were washed in 100 mM sodium phosphate buffer, pH 7.0 (Napi-buffer) with 0.1% (v/v) Tween 20 for 5 min, speed 50 rpm in staining jars on a shaker. During this step, unbound probe molecules and polymer chains are removed. Hybridization and staining steps were each time followed by a similar washing step in Napi-buffer. All chips were dried with nitrogen prior to readout. Incubation/Hybridization. Hybridization experiments were carried out using biotin-dUTP labeled PCR product of HPV 16. PCR reactions were carried out as described previously.34 Briefly, a plasmid template consisting of the vector pCR2.1 was generated and 10 ng was used in a PCR subsequently. The employed pair of primers results in a PCR product of nearly 405 bp (genotype-specific length variation). To obtain single stranded PCR products, the samples were denatured at 95 °C for 5 min. Prior to hybridization, the sample was mixed in equal parts with 200 mM Napi. Incubation of fabricated microarrays was performed in a Slidebooster hybridization station (Advalytix, Germany). Lifter slips (22 25 mm2, Implen GmbH, Germany) were used as reaction chambers and filled with an analyte containing solution of 25 μL. To minimize evaporation during the incubation experiments, the buffer reservoirs within the hybridization station were filled with the same buffer used for incubation with 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 followed by a labeling step of 5 min. One tenth of the PCR product diluted in Napi-buffer was used for hybridization In the case of the protein microarrays, incubation with analyte was performed for 2 h followed by incubation with labeling antibody solution for 30 min. The protein microarrays were incubated with a 1:100 dilution of human serum containing either of the two parameters TPO and Jo-1. The diluting solution 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 Napibuffer) binding to biotinylated probes in the case of oligonucleotide microarrays. For protein microarrays, a Cy5-conjugated secondary antihuman antibody (109-175-098, Jackson Immunoresearch) was used at a concentration of 0.2 μg/mL binding to the autoimmune specific analytes.
2.5. Fluorescence Measurements and Hybridization Analysis. Read-out of the microarrays was performed in a biochip reader based on total internal reflection fluorescence (TIRF) technology similar to the system described by Lehr et al.41 Laser light (635 nm) is coupled into the edge of the microarray substrate, which acts as a waveguide. Upon total internal reflection and adequate optical focusing, a homogeneous evanescent field is generated at the surface of the
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Figure 1. Chemical structure of the copolymer used for the fabrication of the microarrays. microarray. Hence, only fluorophores located within the volume covered by the evanescent field are excited and can emit a fluorescence signal. Quantitative analysis of the fluorescence intensities was performed using the software Signalyse (Holger Klapproth Life Science, Germany). It provides automated background correction of spot intensities with regard to autofluorescence of substrate materials and fluorescence related to unspecific adsorption of fluorescent molecules. To assess unspecific signal contribution by the polymer network, spots containing polymer chains but no biological species were added to the microarrays as negative controls.
3. RESULTS AND DISCUSSION 3.1. Fabrication of the Hydrogel Based Microarrays. The immobilization technique for biological macromolecules described here is based on the formation of small surface-attached hydrogel networks, which are generated in specific locations of the substrate to covalently attach biomolecules. For the fabrication of the microarrays, a solution consisting of a benzophenone group containing photopolymer and the desired probe molecules in aqueous buffer solution is dispensed onto the substrate in a contactless printing process. An advantage of the used printing method is that the volume of dispensed drops can be monitored and adjusted. It permits deposition of defined amounts of probe molecules per spot with a precision of better than (10%, which is a crucial requirement for reliable quantitative analyses. Furthermore, contactless printing allows deposition of probe molecules on complex 3D structured surfaces typical for a broad area of bioanalytical applications such as microfluidic channels or wells in wellplates (Figure 7). The terpolymer employed for the generation of the gels primarily consists of poly(dimethylacrylamide) (PDMAA) as a hydrophilic matrix. A second component contained in the polymer is the sodium salt of styrene sulfonate (SSNa) which is incorporated in the polymer chains to introduce charges to the polymer and thus further enhance their solubility in water. Styrene sulfonate incorporation proved to be superior to the vinyl phosphoric acid used in previous work,8,3436,42 as the corresponding polymer was simpler to synthesize and better control over the exact molecular weight and composition of the copolymer could be achieved. The third component of the polymer is 4-methacryloxyl-oxy-benzophenone (MABP), which provides photo reactive groups to the polymer (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, it should be denoted that the polymer network is not entirely desiccated, since both the polymer network and the salt residues are very hygroscopic. Upon irradiation (i.e., flood exposure) the photopolymer contained in the dots is cross-linked. Simultaneously to the cross-linking of the polymer matrix, the forming network is 6118
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Figure 2. Flowchart of the microarray fabrication process, including an artist’s view of the cross-linking reaction and a molecular scheme of the photoreaction of the photoactive benzophenone moieties contained in the polymer.
bound to the substrate and the probe molecules are covalently linked to the polymeric scaffold by the same photochemical process. The photochemical reaction is caused by the benzophenone moieties in the copolymer chains as described by Toomey et al.:12 Upon irradiation with UV light, the photoreactive MABP segment undergoes an n,π* transition into a biradicaloid triplet state. In this state, the molecule is capable to abstract a hydrogen atom from almost any neighboring aliphatic CH group, which is in close vicinity. The two resulting carbon-based radicals generated by the hydrogen abstraction process can recombine and form a covalent CC bond. The neighboring aliphatic groups can be part of other polymer chains, can be part of the substrate, or can be contained in the biomolecule. In addition, photoreactive groups of the biological species itself, such as thymine in the case of DNA strands43 or tryptophan or tyrosine for proteins,44,45 can participate in the immobilization process by undergoing a similar photochemical reaction. Immobilization of biomolecules to surfaces via photogenerated radical cross-linking reactions is a well-described technique.8,46,47 The short time scale of the UVexposure and the thus employed rather small light dose does not affect the functionality of the biomolecules as could be shown experimentally.8,34,42 Alternatively, light of lower energy/longer wavelength, for example, 360 nm, could be used. However, this requires considerably longer exposure time, which might reduce the performance in subsequent assays. Salt residues on the microarrays originating from the printing buffer do not take part in the photo-cross-linking process. They are washed out of the microarray spots in subsequent washing steps.
An interesting feature of the described process is that functionalization occurs only at locations where solution has been printed onto. This spatially confined functionalization of support materials takes place by simultaneous deposition and immobilization 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 molecules is a crucial requirement for microarray based analytics. To determine the efficiency of attachment of probe molecules to the chip surface within the polymer network, coupling controls of Cy5-labeled IgG and Cy5-labeled oligonucleotide strands were printed on PMMA standard substrates in a series of spots containing dispensed volumes ranging from 400 pL up to 2.8 nL per spot by deposition of multiple drops per spot. The total amount of labeled species per spot ranged from 66 amol to 0.46 fmol for proteins and 0.4 fmol to 2.8 fmol for oligonucleotides. The efficiency of the immobilization process is expressed as the ratio of spot intensities before and after washing of the DNA microarrays (Figure 3) and a blocking procedure for proteins, respectively (Figure 4). For both oligonucleotides and protein microarrays, about 60% of the molecules initially contained in the printing solution were immobilized within the surface-attached hydrogel spots. The loss during washing might be due to a washing out of biological species or polymer chains containing biomolecules. During the cross-linking process, polymer chains become part of the network only with a certain probability, which is related to the number of cross-linking groups per chain, the molecular weight of the polymer chains, and the conversion of 6119
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Figure 3. Signal intensities of spots containing immobilized Cy5conjugated oligonucleotides (coupling controls) as a function of the number of drops deposited per spot. (9) Signals measured prior to washing of the microarrays; (O) signals after washing with 100 mM Napi (0.1% (v/v) Tween). The amount of labeled species per deposited drop was 0.4 fmol. Volume per drop was 400 ( 20 pL. Error bars show standard deviation for 10 microarrays with an on-chip redundancy of 8.
Figure 4. Signal intensities measured for Cy5-conjugated anti-mouse secondary IgG as a function of the number of drops deposited per spot. (9) Signals measured prior to blocking of the microarrays; (O) signals after blocking with milk powder (5% (w/v)) and BSA (1% (w/v)) in PBS (1). Amount of labeled species per disposed drop was 66 amol. Volume per drop was 400 ( 20 pL. Error bars show standard deviation for 10 microarrays with an on-chip redundancy of 8.
the photoreaction. A certain fraction of polymer chains could simply be too short and/or might not carry enough cross-linking units to be sufficiently connected to the network or to the substrate. Additionally, some loss of polymer chains might be associated with ripping off of loosely attached material during processing. The polymer solution contains a printing buffer, which upon drying leads to formation of salt crystals within the hydrogel network. These crystals dissolve during washing processes and might also mechanically remove neighboring parts of the polymer network together with attached biological species. The polymer distribution within such dots was analyzed in preliminary experiments using microarrays which carried dots with direct fluorescencetly labeled (Fluorescein) polymer. Fluorescence images obtained for this microarray with epifluorescence microscopy showed a rather homogeneous intensity distribution across the area of the microarray dots directly after printing and cross-linking and after washing. This indicates that the polymer is sufficiently homogeneously distributed. However, this is no indication for the uniformity of the network’s cross-linking points, which is not determined yet.
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Figure 5. Fluorescence signal intensities as a function of the amount of anti-human IgG capture molecules after incubation with solution containing 15 μg/mL Cy5-conjugated human IgG. Displayed are average intensities measured for 4 microarrays with an on-chip redundancy of 8. Error bars show standard deviation of the individual sets of chips. Details are described in the text.
Fluorescence intensities measured directly after printing and cross-linking and after washing showed a linear dependence on the dispensed volume of labeled species per spot for both labeled oligonucleotides as well as proteins. This linear dependency permits a tuning of the signal intensity of a dot in the array by printing of an appropriate volume of biological species. The ability to adjust the signal intensity significantly simplifies the read-out of different analytes in one biological sample. Typically biological analytes cover several orders of magnitude in concentration, while the dynamic range of read-out devices is usually limited. Using the technique described here, the volume of dots giving only low intensity signals can be increased to enhance the signal intensity. In contrast to an increase of the probe concentration in the dot, an increase in volume will not affect the spatial orientation of probes and the intermolecular distances. This is important with regard to nativity and accessibility of biomolecules especially for high concentrations.7 To assess possible quenching effects, microarrays were printed with varying amounts of probe molecules per spot. Anti-human IgG antibodies were immobilized on PMMA substrates in amounts ranging from 4.2 to 66.7 amol per spot. The measured signals (Figure 5) after incubation with Dy647 conjugated human IgG showed a linear dependency on the amount of probe molecules, which indicates that no significant quenching is occurring. This is in good agreement with similar experiments by Zubtsov et al.7 Biofunctionality and Assay Performance. To elucidate the influence of hydrogel immobilization on the biofunctionality of the surface attached species, binding (hybridization) experiments with clinically relevant analytes were performed for both oligonucleotides and proteins. In the case of DNA based microarrays, chips were fabricated with spots containing probes for the identification of HPV genotypes 16 and 6. These probes were hybridized with biotinylated PCR amplicons of HPV 16 specific DNA sequences. Fluorescence intensities of microarray spots measured after staining with Cy5-labeled streptavidin yield strong, specific signals for spots containing HPV 16 specific probes while signals of spots specific for HPV 6 showed distinctively lower intensities, equal to those of the negative controls. This holds for all tested chip materials as shown in Table 1. Signals were calculated as the average intensity of three different probes per parameter. 6120
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Table 1. Examples for Substrate Materials Used for Photopolymer Based Microarray Productiona oligonucleotide microarrays
protein microarrays
material
specificityb
signal/ backgroundc
specificityb
signal/ backgroundc
PMMA PC PS Topas PP COP Nexterion B Nexterion E
þþþ þ 0 (*) þþþ þ (**) þþ þþ þ
þþþ þ 0 (*) þþþ þ (**) þþ þþ þþ
þþþ þþþ 0 (*) þþ 0 (**) þþþ þþþ þþ
þþþ þþþ 0 (*) þþ 0 (**) þþþ þþþ þþþ
Classification: þþþ, values > 20; þþ, 20 g values g 10; þ, values > 5; “0” values < 5; (*) PS showed comparatively high auto fluorescence; (**) PP is not compatible with the TIRF based read-out technique because of its opacity. b Specificity is defined as the ratio of signals measured for spots containing specific probe molecules and spots containing other probes than those specific to the analyte. c Signal/ background is defined as the ratio of signal intensities of spots containing specific probes and signal intensities of negative control spots. a
Figure 6. (A) White light image showing a typical microarray on PMMA substrate. (B) False color image of a microarray after a direct assay with Cy5-conjugated IgG; shown are in alternating order from top to bottom dilution series of coupling controls and spots with immobilized antiIgG antibodies (from left to right, dilution factor of 2). (C) Fluorescence image of microarrays (subarrays) after assays with serum containing antiTPO antibodies and serum containing JO-1 specific antibodies (D).
Similar experiments were carried out for protein microarrays, where two clinically relevant parameters TPO and Jo-1 were used exemplarily. After incubation of the chips with serum obtained from real patient samples, which contained specific antibodies against TPO or Jo-1, the microarrays showed specific signals for the particular parameter present as shown in Figure 6. Similarly to the results for oligonucleotide microarrays, signals measured for the nonexpressed, unspecific parameter as well as the negative controls reveal distinctively lower and rather equal intensities. Hence, cross reactions as well as unspecific binding can be excluded for these two 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 certain material for a specific diagnostic application. This would require
Figure 7. Polymer hydrogel dots on 3D structured surfaces. (A) Glucoseoxidase immobilized within microwell electrodes (diameter 100 μm) using the polymer hydrogel technology. (BD) Microarray dots immobilized within microfluidic channel structures. Inset in (D) shows a fluorescence image of a microarray after a DNA hybridization assay.
considerable experimental effort, process optimization, and dedicated handling protocols for each material. Here, the aim was to show the principle applicability of this immobilization approach for a representative selection of support materials based on handling protocols and read-out technology which had been specifically adapted for PMMA substrates. 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 background fluorescence of both the substrate and the hydrogel dots are required. In conventional microarray analysis, the signal-to-noise value is frequently taken as the ratio of signal intensities of the areas of interest (i.e., the spots of the microarray) and their surroundings (i.e., the blank substrate).11 However, in the case of spatially confined surface functionalization such as in this case of polymer containing spots, it has to be taken into account that the surface area around the hydrogel spots does not reflect the background contribution of the polymer itself. To assess the latter, we additionally printed negative controls, which are spots that contain the same polymer but no probe molecules. In all following experiments, we define the signal-to-background ratio as the quotient of the signal intensities of probe containing hydrogel and “empty” control spots. A similar method was reported by Angenendt et al.48 Typically, the fluorescence intensity measured for such negative controls was very small compared to intensities of spots containing specific probe molecules (Table 1). Since the functionalization reaction occurs only in areas of interest, namely, at the spot locations, the overall background signal is strongly reduced. In addition, the hydrogel is strongly protein repellent and no nonspecific adsorption occurs. It is a characteristic feature of PDMAA based polymer networks to prevent nonspecific adsorption of biological macromolecules to a large extent. This is related to the highly swollen state of such surface-attached networks in aqueous solutions, which show “entropic shielding” against protein adsorption.8,12,36,49 Immobilization on 3D Structured Supports. Besides immobilization on conventional planar substrates, the immobilization technique described here was also employed successfully to 6121
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Langmuir attach probe molecules to substrates with more complex topographies such as microtiter plates, microwells, or microfluidic channels (Figure 7). Especially the latter might be interesting, as it allows one to combine the format of classic microarrays with more sophisticated microfluidic applications. Integration of microarray based bioassays within fluidic designs such as labon-a-chip assemblies will permit automatization and reduction of assay time by using enhanced mass transport of analyte molecules to its corresponding binding partners, which at low concentrations frequently is a strongly limiting factor of microanalytical processes.5052
4. CONCLUSION Printing of solutions of biomolecules together with a benzophenone group containing photopolymer followed by brief UVirradiation allows for a simple and spatially confined functionalization of planar and structured substrates in a single process step. During the irradiation process, photo-cross-linking of the deposited polymer takes place and a highly water-swellable hydrogel is formed. Simultaneously to cross-linking of the matrix, the biomolecules become chemically attached to the hydrogel matrix and the whole forming network becomes covalently bound to the substrate. The described process can be used for the immobilization of biomolecules such as DNA or proteins onto a wide spectrum of different substrates, ranging from inorganic substrates modified with self-assembled monolayers (SAMs) to a large variety of different polymer materials. It requires no sophisticated pretreatment of the microarray substrates. This “all plastic” biochip technology can be combined with biological standard procedures such as PCR and immunoassays and shows a comparable performance to commonly used glass slides. This immobilization technique is ideally suited for plastic materials commonly used in microfluidic structures and lab-on-a-chip applications, rendering it a promising platform for high-throughput biomedical applications. ’ ASSOCIATED CONTENT
bS
Supporting Information. Synthesis and characterization of the copolymer; H NMR, FTIR, and UVvis spectra of the copolymer and a table listing sequences of all used oligonucleotide probes specific for HPV subtypes 6 and 16. This material is available free of charge via the Internet at http://pubs.acs.org.
’ ACKNOWLEDGMENT We thank Alexander Dietz for valuable technical support. We gratefully acknowledge the valuable contributions from Dr. Richard Kneusel and Dr. Armin Neininger from DIARECT AG, Freiburg, Germany, who also provided the autoimmune proteins. Microfluidic ChipShop, Jena, Germany is thanked for providing the various plastic microscope slides as well as the microfluidic substrates. This work was supported by the BMWiAIF (“Channel & Spots”; KF2162002UL9). ’ REFERENCES (1) Hoheisel, J. D. Microarray technology: beyond transcript profiling and genotype analysis. Nat. Rev. Genet. 2006, 7 (3), 200–210. (2) Kusnezow, W.; Hoheisel, J. D. Solid supports for microarray immunoassays. J. Mol. Recognit. 2003, 16 (4), 165–176.
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