© Copyright 1998 American Chemical Society
JULY 21, 1998 VOLUME 14, NUMBER 15
Letters Direct Protein Microarray Fabrication Using a Hydrogel “Stamper” Brett D. Martin,* Bruce P. Gaber, Charles H. Patterson, and David C. Turner Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, D.C. 20375-5348 Received December 3, 1997. In Final Form: March 12, 1998 Micropatterned arrays of active proteins are vital to the next generation of high-throughput multiplexed biosensors and advanced medical diagnostics. We have developed a simple method for fabricating antibody arrays using a micromolded hydrogel “stamper” and an aminosilylated receiving surface. The stamping procedure permits direct protein deposition and micropatterning while avoiding cross-contamination of separate patterned regions. Three different antibodies were stamped in adjacent arrays of 50-80 µm circular areas with retention of activity. 125I labeling and atomic force microscopy studies showed that the stamper deposited protein as a submonolayer. The fluorescent signal-to-background ratio of labeled bound antigen was greater than 25:1.
Introduction We have created a new “microstamper” that delivers a molecular film of protein onto a surface in a single step. This enables production of protein microarrays suitable for use in miniature biosensors and medical diagnostics. The stamper is a micromolded, elastic hydrogel that is loaded with protein solution and briefly brought into contact with a receiving surface, leaving a protein film in a manner analogous to the action of an ink stamper on paper (Figure 1). Using the stamper, we have formed arrays of several active antibodies arranged in elements of 10-80 µm circular spots. As described in the literature, Kumar et al. showed that a poly(dimethylsiloxane) stamper could be used to form ultrafine patterns of alkanethiol monolayers on gold.1 This approach is well suited for deposition of small inorganic molecules. Our approach is dramatically different because it accomplishes direct delivery of active biomolecules such as proteins from an aqueous polymeric reservoir. For multiplex array fabrication this direct process can be superior to other * To whom correspondence should be addressed. (1) Kumar, A.; Whitesides, G. M. Appl. Phys. Lett. 1993, 63, 2002. Lopez, G. P.; Albers, M. W.; Schreiber, S. L.; Carroll, R.; Peralta, E.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 5877.
methods that may subject the entire sensor surface to multiple protein solutions during construction. The improvement of multiplex, high-throughput assays is of importance for the continued development of small, point-of-care medical devices enabling rapid detection and identification of pathogens, cytokines, and drugs.2 In most common designs for array fabrication, capturing biomolecules are placed on the sensing surface in dedicated microregions and ligand is bound from sample solution and identified according to its position on the x-y plane. With this approach many investigators have built miniature high-throughput arrays that can function as multiplex sensors when optically interrogated. Those containing sequences of DNA homologues have been the most successful.3 There is an additional need for miniature sensors that are able to rapidly detect and identify explosives and chemical/biological warfare agents. Detection systems based upon immobilized antibodies (IgGs) are leading candidates.4 Key advantages of this approach are the exquisite specificity of antibody-antigen interactions, resulting in few false alarms, as well as unusually (2) Baines, W.; Smith, G. C. J. Theor. Biol. 1988, 135, 303. Byfield, M. P.; Abuknesha, R. A. Biosens. Bioelectron. 1994, 9, 373.
S0743-7463(97)01331-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/03/1998
3972 Langmuir, Vol. 14, No. 15, 1998
Letters
Figure 2. The hydrogel polymer poly(6-acryloyl-β-O-methylgalactopyranoside-co-methylene-bis-acrylamide). The average molar ratio of m to n is 100.
Figure 1. General scheme for antibody “stamping” followed by exposure to labeled antigen. IgG orientation is idealized.
strong association constants (as high as 1010 M-1), that allow detection of antigenic molecules in dilute samples, i.e., at concentrations down to 10-9 to 10-14 M.5 Construction of antibody-based devices often involves formation of surface protein bilayers and/or use of photolithographic or ink-jet printing techniques. For example, streptavidin has been micropatterned and allowed to associate photobiotin,6 which subsequently binds solute proteins in patterns via activation by directed UV or laser light. This procedure and other photolithographic methods can be successful.7 Unfortunately, when applied to multiplex microarray fabrication the entire surface must be sequentially exposed to a series of solutions, each containing a different protein to be patterned. The result is often an undesirably high level of nonspecific protein adsorption8 or cross reaction9 into a previously patterned region. Alternatively, jet-printing methods have been applied but have met with limited success because they cannot produce small (
