Multicolor microcontact printing of proteins on nanoporous surface for patterned immunoassay

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.

Easy Fabrication of Thin Membranes with Through Holes. Application to Protein Patterning

“…As an example, we created two spreading assays on a glass surface patterned by 10×10 arrays of 30 µm discs. The first assay is intended to probe the immune response of macrophages to patterns of opsonin [29] . Following the protocol described in Materials and Methods , we locally patterned mouse anti-BSA antibodies onto a homogenously BSA coated surface ( Figures 3A ).…”

Multi-Dimensional Nanostructures for Microfluidic Screening of Biomarkers: From Molecular Separation to Cancer Cell Detection

“…Microfluidics contributes several advantages in as biological analytical systems, as previously mentioned. Microfluidic devices are already capable of providing a platform for continuous hydrodynamic-driven size-based filtration of cells 14,25,86,87 , and molecular separation including, but not limited to, protein 38,47,57 and DNA 41,42,78 separations and detection. So what more would the integration of nanostructures into microfluidic channels provide?…”

“…Physical properties of mesoporous films provide a variety of advantages which have been utilized in biological molecular separations 20,28,33,35,40,57 and size-based filtration of molecular species 21,70 . Current size-based molecular separation involves gel-based techniques such as gel electrophoresis or gel-filtration (size-exclusion) chromatography for DNA, RNA, or protein separation.…”

“…Mesoporous films provide physical structures that increase roughness and surface area of substrate surfaces, allowing size-selective trapping of proteins and enhance protein adsorption 4,57 . Blinka et al .…”

“…Ng et al . previously used the microcontact printing technique, a protein transfer technique for producing self-assembled monolayers (SAMs) 81 , to physically transfer a uniform monolayer of capture antibodies onto nanoporous silica substrates and demonstrated a system for performing multiplexed ELISAs for protein separation and detection 57 . Nanoporous silica film substrates were fabricated through self-assembly of surfactant micelles by polymer units mixed with soluble silicates, tetraethyl orthosilicate (TEOS) and tetraethoxysilane, in homogenous, hydro-alcoholic solutions.…”