Multicolor microcontact printing of proteins on nanoporous surface for patterned immunoassay
Appl Nanosci (2011) 1:79–85
DOI 10.1007/s13204-011-0009-0
ORIGINAL ARTICLE
Multicolor microcontact printing of proteins on nanoporous
surface for patterned immunoassay
Elaine Ng • Ashwini Gopal • Kazunori Hoshino •
Xiaojing Zhang
Received: 10 April 2011 / Accepted: 11 April 2011 / Published online: 27 April 2011
Ó The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract The large scale patterning of therapeutic proteins is a key to the efficient design, characterization, and
production of biologics for cost effective, high throughput,
and point-of-care detection and analysis system. We
demonstrate an efficient method for protein deposition and
adsorption on nanoporous silica substrates in specific patterns using a method called ‘‘micro-contact printing’’.
Multiple color-tagged proteins can be printed through
sequential application of such micro-patterning technique.
Two groups of experiments were performed. In the first
group, the protein stamp was aligned precisely with the
printing sites, where the stamp was applied multiple times.
Optimal conditions were identified for protein transfer and
adsorption using the pore size of 4 nm and thickness of
30 nm porous silica thin film. In the second group, we
demonstrate the patterning of two-color rabbit immunoglobin labeled with fluorescein isothiocyanate and tetramethyl rhodamine iso-thiocyanate on porous silica
substrates that have a pore size 4 nm, porosity 57% and
thickness of the porous layer 30 nm. A pair of protein
stamps, with corresponding alignment markings and
coupled patterns, were aligned and used to produce a twocolored stamp pattern of proteins on porous silica. Different colored proteins can be applied to exemplify the diverse
protein composition within a sample. This method of
multicolor microcontact printing can be used to perform a
fluorescence-based patterned enzyme-linked immunosorbent assay to detect the presence of various proteins within
a sample.
E. Ng (&) A. Gopal K. Hoshino X. Zhang
Department of Biomedical Engineering, The University
of Texas, Austin, TX 78758, USA
e-mail:
Keywords Multicolor Microcontact printing
Patterned immunoassay Nanoporous
Introduction
Microcontact printing (lCP) is a method in which a stamp
is ‘‘inked’’ with specific molecules with subsequent deposition of those molecules onto a substrate with wellcontrolled pattern. Such a technique was first demonstrated
in the patterning of alkanetholate self-assembling monolayers (SAMs) on gold substrates (Kumar et al. 1994).
Since then, lCP has been extended to incorporate many
applications, including the patterning of functional proteins
for immunoassays and biosensors (Bernard et al. 2000;
Pattani et al. 2008). Furthermore, to enhance the diagnostic
power of immunoassays, multiple proteins can be patterned
on the same substrate and has been demonstrated in a
variety of ways (Inerowicz et al. 2002; Crozatier et al.
2006; Ghosh et al. 2008). The ability to print multiple
proteins on a single substrate and on such a small scale is
important in creating an efficient point of care detection
and analysis systems.
Enzyme-linked immunoabsorbent assay (ELISA) is a
technique used to detect the presence of specific antibodies
or antigens in a sample (Li et al. 2008). There are many
different types of ELISA, however, the sandwich ELISA
technique described here will be most applicable to the
investigation. It involves affixing an amount of antigen to a
surface specifically via a capture antibody and then washing the surface with a specific primary antibody. The
antigen binds to the primary antibody. The surface is
washed with a second solution containing an enzymelinked secondary antibody. The primary antibody is linked
to the enzyme-linked secondary antibody. Upon the
123
80
Appl Nanosci (2011) 1:79–85
addition of a particular substrate, the enzyme is converted
to a detectable signal. For example, in fluorescence ELISA,
when light of a specific wavelength is shone upon the
sample, the antigen–antibody complexes will fluoresce and
the amount of antigen in the sample can be inferred by the
magnitude of the fluorescence. Typical ELISA testing
requires long procedures; involving the use of microliter
well plates as well as generous amounts of reagents for
each immunoassay performed, and quantified using spectrophotometry comparisons with standards (Li et al. 2008;
Rolland et al. 2008; Stephan and Vieths 2004; Sun et al.
2010). Applications of ELISA include use in the food
industry to detect food allergens such as milk, nuts, and
eggs (Rolland et al. 2008; Stephan and Vieths 2004).
In this paper, we demonstrate an efficient method for the
deposition and adsorption of two different color-tagged
proteins on a single nanoporous silica substrate through
sequential application of the lCP technique. Furthermore,
we demonstrate the application of multicolor lCP for
multiple antigen detection in immunoassays by performing
a sandwich ELISA for the simultaneous detection of two
common allergens, ovomucoid found in egg white, and
casein found in milk. The ability to microcontact print and
detect multiple antigens in a single sitting, coupled with
fluorescence optical detection and quantification, has the
potential to lay grounds for more efficient immunoassays
and biosensors. The ability to also miniaturize and microscale the immunoassay itself enables wider, more cost
efficient, varieties of biomedical point of care applications.
Experimental procedures
Fabrication of porous silica substrates
It has been shown that substrates functionalized with a
nanoporous silica thin film can be used to enhance protein
adhesion and adsorption onto the substrate surface through
physical means (Hu et al. 2009), more so than surfaces
functionalized by chemicals such as 3-aminopropyltriethoxysilane (APTES) or glutaraldehyde (GA) (Blinka
et al. 2010). All experimentations described in this paper
used nanoporous silica thin film functionalized substrates
characterized by 4 nm pores, 56% porosity, and 30-100 nm
thickness for optimal protein adsorption and adhesion.
These characterizations along with the nanoporous silica
substrate fabrication process are detailed in a paper by
Blinka et al. (2010). Figure 1 shows the schematic for
nanoporous silica substrate fabrication. The nanoporous
and rough surface of the substrate enhances absorption of
proteins by increasing the surface area of contact between
protein and substrate. In general, a polymer-silicate coating
solution was prepared and spin coated onto glass or silicon
123
Fig. 1 Schematic of the fabrication of nanoporous silica substrates
(Hu et al. 2009)
substrate. The evaporation of solvent during spin coating
drives the formation of a silica-copolymer SAM of thin
film nanoscale dimensions. Pore sizes, thickness of the
film, and porosity of the film could be tuned through the
molar ratio of silicate to polymer and deposition rates.
Multicolor microcontact printi (...truncated)
