Multiplexed Protein Patterns on a Photosensitive Hydrophilic Polymer Matrix

Multiplexed Protein Patterns on a Photosensitive Hydrophilic Polymer Matrix

(Parte 1 de 2)

Multiplexed Protein Patterns on a Photosensitive Hydrophilic Polymer Matrix

By Parijat Bhatnagar,* George G. Malliaras, Il Kim, and Carl A. Batt*

Multiplexed functional proteins immobilized on microfabricated sensors and surfaces[1–3] have found applications in highthroughput screening[4] of drug molecules,[5] early disease detection,[6] organ printing, and complex tissue engineering.[7–10] Complex biological integrated patterns emulating physiological microenvironments have been used to engineer tissue junctions from stem cells by selective differentiation[2,1] and study interaction with the extracellular matrix (ECM).[1–13] Parallel developments in lab-on-a-chip (LOC) platform technologies have been identified for label-free biosensing[14] with faster analysis using less reagent and analyte volumes.[15–17] If LOC technology is to take advantage of the developments in the semiconductor industry,[16] efforts are needed to create biologically friendly microfabrication processes to allow integration of microelectronic circuitry with protein patterns.[17–19] Currently used methods for multiplexed protein patterns include softlithography,[1,20] inkjet printing,[21] and dip-pen nanolithography.[2] However, none of these have been integrated with complementary metal–oxide–semiconductor (CMOS) processing for high-volume manufacturing.[23] Soft-lithography and inkjet printing have proven to be versatile for protein patterning, however, resolution and hence alignment of the protein patterns with pre-existing features remains a challenge.[1,19] Dip-pen nanolithography, an analogue of scanning probe microscopy, can achieve high resolution but is extremely slow and has not been adopted by industry. Here we demonstrate a photolithographic process[24,25] on hydrogel-based biomaterial[26] for patterning three different types of proteins. The technique is scalable and capable of patterning a multitude of proteins aligned with respect to each other and surface microstructures. UV light (365nm), benign to proteins and DNA,[27,28] was used. This strategy allowed us to integrate harsh upstream CMOS processing involving extreme pH, vacuum processes, and organic solvents, with downstream aqueous biomolecular processing at neutral pH.

We have earlier demonstrated methods to array single oligonucleotides or proteins.[29,30] Lift-off-based photolithography[2] and oxygen-plasma-etch-based patterning of two proteins[31] has also been demonstrated and is capable of scaling up to more proteins, but due to the subtractive nature of these processes none can be adopted with multiple layers in 3D.[8,9] Bochet et al. have described solution-based photochemistry of orthogonal photolysis of inter- and intramolecular acid groups using two different photolabile protecting groups (PLPGs) with differential sensitivity to 254-nm and 420-nm UV light.[25] This was further developed by Campo et al. who illustrated photopatterning to create chemically diverse areas for patterning colloidal particles and different biomolecules.[24] Photogenerated functional groups have also been used for solid-phase synthesis of multiplexed gene chips[32] and peptide chips,[3] which utilizes synthetic nucleotides or amino acid residues, respectively, protected by PLPGs.

Photochemical immobilization strategies can be categorized into two groups: photocatalyzed reactions and photodeprotection of reactive groups.[34] The former involves a single-step reaction by creating short-lived reactive groups on the surface by photoexposure. Although advantageous in facilitating a singlestep reaction, this technique cannot be integrated with semiconductor industry equipment because it requires the substrate to be present in a liquid environment inside the photolithographic equipment. Due to the aforementioned limitations we resorted to a photodeprotection strategy to generate either an amine- (photogenerated base, PGB) or a carboxylic-acid- (photogenerated acid, PGA) functionalized surface[24,25] followed by subsequent immobilization of proteins.[35]

Cr microstructures, which serve as alignment marks in downstream protein patterning, were first patterned on a wafer using electron-beam evaporation of Cr, standard projection photolithography, and subtractive wet-etching of Cr. A selfassembled monolayer (SAM) of [3-(methacryloyloxy)propyl]- trimethoxysilane with a polymerizable terminal group was formed on the wafer surface from solution-phase (MOPSAM).[30] A functional-group-containing monomer (FGM) (amine or protected carboxylic acid) was then polymerized with a thin film of acrylamide (AAm)–methylenebisacrylamide (Bis) copolymer [poly(AAm–Bis–FGM)][30] (Figure 1). 2-Nitrobenzyl succinimidyl carbonate (NBSC), a PLPG adduct prepared as described elsewhere,[36] was subsequently used to protect surface amine groups as 2-nitrobenzyl-derived carbamate (Scheme 1). In the case of 2-nitrobenzyl-derived ester groups (that yield surface

Dept. of Biomedical Engineering Cornell University, Ithaca, NY 14853 (USA) E-mail:

Prof. C. A. Batt Dept. of Food Science Cornell University, Ithaca, NY 14853 (USA) E-mail:

Prof. G. G. Malliaras Dept. of Materials Science & Engineering Cornell University, Ithaca, NY 14853 (USA)

Prof. I. Kim Dept. of Polymer Science & Engineering Pusan National University, Busan 609-735 (Korea)

[+] Present Address: Intel Corp., 5200 NE Elam Young Pkwy, RA3-355, Hillsboro, OR 97124, USA

DOI: 10.1002/adma.200903255

carboxylic acid groups upon photoexposure), the monomer itself contained acid groups protected as 2-nitrobenzyl-derived esters.

The photosensitive hydrogel surface was then exposed in selective regions to 365-nm UV light (730mWcm 2 intensity) to create spatial patterns of PGB (Scheme 1) or PGA (Scheme 2) as a result of photochemical cleavage of 2-nitrobenzyl-derived carbamate or 2-nitrobenzyl-derived ester, respectively. Proteins were covalently immobilized on the PGB or PGA surface through stable amide linkages with primary amines available on lysine residues of protein molecules using bis(sulfosuccinimidyl)suberate (BS3) (Scheme 1) or carbodiimide chemistry (Scheme 2), respectively.[35] After protecting the functional groups on the immobilized protein molecules (amine-group protection for PGB-based protocol and acid-group protection for PGA-based protocol), the process was sequentially repeated using different photomasks with a previously patterned Cr alignment mark to create a multiplexed protein surface. It was necessary to wash the surface with mildly acidic (pH 4) sodium citrate buffer for 3h after every protein immobilization step to promote positive charges on nonspecifically adhered protein molecules, resulting in their electrostatic repulsion from the aminated surface.

Our initial efforts used photogenerated surface amine groups for protein immobilization (Scheme 1). Figure 2 shows an early effort to immobilize IgG antibodies from rat, rabbit, and mouse separately on individual chips with PGB, however, nonspecific adhesion was a challenge. It has been reported that photocleavage of 2-nitrobenzyl protecting groups to generate amine groups is accompanied by generation of an aldehyde-based side product (2-nitrosobenzaldehyde) that can react with primary amines to form imines,[24,27,37,38] thus rendering photogenerated primary amine groups unavailable for further conjugation. Semicarbazide hydrochloride has been used as an aldehyde scavenger in solution,[24,27,38] but due to CMOS equipment compatibility issues we were unable to use any solution-phase protocol during photoexposure. This led us to explore photogenerated surface acid groups[24,25] (Scheme 2). We believed that this approach would be a better choice since the photochemistry of this system is similar to the widely used i-line photoresists in the semiconductor industry, where acid functional groups are photogenerated by exposure to 365-nm UV light.

Carbodiimide chemistry was then utilized for surface immobilization of each protein on PGAregions (Scheme 2).Carboxylic groupson attached proteins were transformed to primary hydroxyl groups using carbodiimide chemistry.[35] This step prevented a chemical reaction between primary amines of subsequent proteins with carboxylic acid groups of proteins already immobilized on surface. Rat, rabbit and mouse IgG were immobilized sequentially as per Scheme 2. Proteins were detected using fluorescently labeled probe antibodies on a single chip and are shown in Figure 3a–c. Figure 3d shows an overlay image of the three images. Considerable nonspecific adhesion was still observed, which did not reduce after treatment with acidic or alkaline buffers. However, nonspecific physical adhesion is also an inherent property of IgG antibodies[39] and in fact this property of IgG molecules is utilized to block surfaces in biological assays (e.g., western blots) and eliminate nonspecific adhesion of proteinspecific IgG molecules in subsequent steps. We argued that this property of IgG may have resulted in our observation of background fluorescence from nonspecific adhesion and may not interfere when we immobilize other proteins of interest. We might also be able to prevent nonspecific adhesion of labeled probe antibodies towards the protein of interest by blocking the

Figure 1. Fabrication of multiplexed protein patterns on a photosensitive surface. i) Electron-beam evaporation of Cr, projection photolithography (spin, 365-nm UV exposure, develop), subtractive wet etch of exposed Cr, MOP-SAM formation on SiO2 surface; i) formation of crosslinked polymer thin film containing FGM; i) 365-nm UV exposure of selective areas, aligned to Cr patterns,on polymer thinfilm to cleave PLPG resultinginto PGB or PGA; iv) useof Scheme1 (for PGB) or Scheme 2 (for PGA) for covalent immobilization of proteins P1 on the exposed surface; v) chemical protection of functional groups on proteins P1 ((v-1) amine group protection for PGB based protocol and (v-2) acid group protection for PGA based protocol); vi) 365-nm UV exposure of selective areas on polymer thin film resulting into PGB or PGA; vii) immobilization of proteins P2 on newly exposed areas. Steps (vi–vii) are repetitions of steps (i–v) for immobilization of second protein P2. vi) Steps (i–v) are again repeated for immobilization of third protein P3. All three proteins are aligned to Cr patterns and hence also to each other.

2 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Mater. 2009, 21, 1–5 Final page numbers not assigned

hydrogel surface using buffers containing unlabeled IgG molecules.

To validate if the photosensitive hydrogel upon selective exposure could be used as a generic platform to array non-IgG proteins without interference from nonspecific adhesion, we repeated the procedure using fibronectin protein (from bovine plasma) (Fig. 3e). Although we are unable to explain incomplete fluorescence in 100-m patterns, we could faithfully reproduce 5-m protein patterns over the entire 100-m wafer from 25-m projection mask patterns (5:1 pattern reduction) with minimal background noise due to nonspecific physical adhesion.

In summary, we have developed an additive method based on photolithography to pattern multiple proteins in alignment with each other and with surface microstructures at 5-m resolution. This method can seamlessly incorporate biomolecular patterning with new advancements in high-resolution photolithography and materials processing for next generation integrated circuits. Further applications include creation of microenvironments with multiple differentiation signals including growth factors and ECM proteins confined to certain parts of the cell population,[40] and engineering vascularized tissues from undifferentiated stem cells.[10,1,13] Due to its additive nature, this methodology can also be integrated with rapid-prototyping methods[8,41] for 3D tissue or organ scaffolds.


Surface Polymerizat ion of the Poly(AAm–Bis–FG M) Hydrogel

Film: MOP-SAM was formed on the silicon oxide surface followed by the poly(AAm–Bis–FGM) layer [30].AAm (80%),Bis (15%),and FGM(5%) (w/w) were dissolved in a suitable solvent for the total monomer content to be 20% (w/v). The solution was then diluted 1:1 with 80% glycerol (v/v). Free radicals were generated by addition of 25% (w/v) ammonium persulfate (APS) and N,N,N0,N0-tetramethylethylenediamine (TEMED) (Bio-Rad Laboratories, Hercules, CA). This solution (0.35mL) was dispensed on 100-m-diameter wafer, covered with a sacrificial 100-m wafer, and polymerized for 4h. It was then soaked in tris buffered saline (TBS) with 0.5% Triton X-100 (TBST) solution overnight. The sacrificial wafer was lifted-off mechanically by hand to obtain the thin hydrogel film attached to the MOP-SAM-functionalized wafer.

Surface Polymerization with Protected Amine Groups in the Hydrogel

Matrix: 2-Aminoethyl methacrylate hydrochloride (Polysciences, Inc., Warrington, PA) was used as the FGM in water. APS and TEMED at 0.004mL each per mL of gel precursor solution were used. The wafer containing surface amine groups was treated with NBSC [36] overnight in the dark.

Surface Polymerization with Protected Acid Groups in the Hydrogel

Matrix: 2-Nitrobenzyl methacrylate (Polysciences, Inc., Warrington, PA) was used as the FGM in dimethyl sulfoxide (DMSO). APS and TEMED at 0.04mL each per ml of gel precursor solution were employed. The higher initiator amount was required due to the free-radical scavenging activity of nitro-aromatic groups [42].

Protein Immobilization on Photogenerated Amines: Wafer with PGB was treated with BS3 (30mM) in phosphate buffered saline (PBS) for 30min. After rinsing the surface with PBS, wafer was then treated for 3h with protein solution (1mgmL 1 in PBST (PBSþ0.5% Triton X-100), pH 7.3).

Protein Immobilization on Photogenerated Acids: Wafer with PGA was treated with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) (0.15M) and N-hydroxysulfosuccinimide (sulfo-NHS) (0.03M) dissolved in 2-[morpholino]ethanesulfonic acid (MES) buffer (pH 4.7) (0.1M) for 30min. The wafer was then treated for 3h with protein solution (1mgmL 1 in PBST).

Chemical Protection of Carboxylic Acid Groups on Surface-Immobilized

Proteins: Wafer with surface- immobilized proteins was treated with EDC (0.3M) and ethanolamine hydrochloride (0.5M) in

MES buffer (pH 4.7) (0.1M) for 30min.

Detection of Immobilized Proteins: Immobilized

IgG antibodies were treated with ELISA grade bovine albumin serum (BSA) (Product #A7030, Sigma-Aldrich, St. Louis, MO) (40mgmL 1 in TBST) and then with a cocktail solution of anti-rat, anti-rabbit, and anti-mouse IgG probe antibodies labeled with Alexa 647, Alexa 546, and Alexa 488, respectively (Invitrogen, Carlsbad, CA) (0.004mgmL 1 each in TBST).

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