Parijat Bhatnagar

Multiplexed Protein Patterns on a Photosensitive Hydrophilic Polymer Matrix

2010 WILEY-VCH Verlag Gmb Multiplexed functional proteins immobilized on microfabricated sensors and surfaces have found applications in highthroughput screening of drug molecules, early disease detection, organ printing, and complex tissue engineering. Complex biological integrated patterns emulating physiological microenvironments have been used to engineer tissue junctions from stem cells by selective differentiation and study interaction with the extracellular matrix (ECM). Parallel developments in lab-on-a-chip (LOC) platform technologies have been identified for label-free biosensing with faster analysis using less reagent and analyte volumes. If LOC technology is to take advantage of the developments in the semiconductor industry, efforts are needed to create biologically friendly microfabrication processes to allow integration of microelectronic circuitry with protein patterns. Currently used methods for multiplexed protein patterns include softlithography, inkjet printing, and dip-pen nanolithography. However, none of these have been integrated with complementary metal–oxide–semiconductor (CMOS) processing for high-volume manufacturing. 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. 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 on hydrogel-based biomaterial 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 (365 nm), benign to proteins and DNA, 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. Lift-off-based photolithography and oxygen-plasma-etch-based patterning of two proteins 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. Bochet et al. have described solution-based photochemistry of orthogonal photolysis of interand intramolecular acid groups using two different photolabile protecting groups (PLPGs) with differential sensitivity to 254-nm and 420-nm UV light. This was further developed by Campo et al. who illustrated photopatterning to create chemically diverse areas for patterning colloidal particles and different biomolecules. Photogenerated functional groups have also been used for solid-phase synthesis of multiplexed gene chips and peptide chips, 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. 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 followed by subsequent immobilization of proteins. 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). 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)] (Figure 1). 2-Nitrobenzyl succinimidyl carbonate (NBSC), a PLPG adduct prepared as described elsewhere, 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

Multiplexed electrospray deposition for protein microarray with micromachined silicon device

Multiplexed electrospray deposition device capable of delivering picoliter volumes made by silicon micromachining technology has been developed as a deposition tool for making protein microarrays in a noncontact mode. Upon application of potential difference in the range of 7–9kV, biomolecules dissolved in suitable buffer with nonionic surfactant and loaded on the electrospray tips were dispensed on the substrate with microfabricated hydrogel features (1–10μm) in cone-jet mode. Schiff base chemistry followed by reductive amination was utilized for covalent immobilization.

Integrated non-SO2 underlayer and improved line-edge-roughness dielectric etch process using 193nm bilayer resist

We present an integrated reactive ion etch (RIE) process using bilayer (a top imaging layer and a bottom underlayer) thin film imaging system to push the limits of 193nm wavelength photolithography. Minimizing the line-edge roughness (LER) and maintaining the critical dimension (CD) of the transferred pattern are important in high-resolution RIE. Along with LER and CD issues and shrinking ground rules, deleterious effects of SO2 in the underlayer etch chemistry necessitated the development of non-SO2 chemistry. Thus a N2–H2–CO chemistry was developed and integrated with the etch process of underlying borophosphosilicate glass using Ar–O2–C4F8–CO–CH3F chemistry.

Controlling line-edge roughness and reactive ion etch lag in sub-150 nm features in borophosphosilicate glass

We have developed a reactive ion etch (RIE) process in borophosphosilicate glass (BPSG) for 150 nm line-and-space features, where line-edge roughness (LER) complemented with RIE lag becomes a major issue. Effect of flow rates and carbon-to-fluorine atomic ratio of fluorohydrocarbon gases was utilized to achieve acceptable process window allowing lower radio frequency powers therefore obtaining acceptable LER and RIE lag in the high-resolution features etched into BPSG.

Integrated reactive ion etching to pattern cross-linked hydrophilic polymer structures for protein immobilization

Patterning of cross-linked hydrophilic polymer features using reactive ion etching (RIE) capable of covalently immobilizing proteins has been achieved. Projection photolithography was used to pattern photoresist to create micromolds. Vapor phase molecular self-assembly of polymerizable monolayer in molds allowed covalent binding of hydrogel on surface during free-radical polymerization. Excess hydrogel blanket film was consumed with oxygen RIE resulting into hydrogel pattern of 1μm size aligned to prefabricated silicon oxide structures. Proteins were finally coupled through their primary amine groups selectively to acid functionalized hydrogel features through stable amide linkages using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysulfosuccinimide.