Protein A‐based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme‐linked immunosorbent assay

Abstract

Highly efficient antibody immobilization is extremely crucial for the development of high-performance polymeric microdevices for enzyme-linked immunosorbent assay (ELISA). In this article, a site-selective tyrosinase (TR)-catalyzed protein A strategy for antibody immobilization was developed to enhance the sensitivity of ELISA in poly-(methyl methacrylate) (PMMA) microchannels for interferon-gamma (IFN-gamma) assay. To effectively immobilize the target antibodies, oxygen plasma was first used to activate the inert PMMA. This is followed by poly(ethyleneimine) (PEI) coating, an amine-containing functional polymer. For comparison, protein A was also immobilized through the commonly used amine-glutaraldehyde (GA) chemistry. Oxygen plasma treatment effectively increased the amount of PEI attachment and subsequent binding efficiency of the primary antibody. The antibody immobilized via TR-catalyzed protein A was able to provide much better specific antigen capture efficiency than GA chemistry due to the optimal spacing and orientation. Consequently, by using this new method, the detection signal and the signal-to-noise ratio of the ELISA immunoassay in microdevices were all significantly improved. In comparison to the standard assay carried out in the 96-well microtiter plate, the treated microchannels exhibited a broader detection range and a shorter detection time. And the detection limit was also decreased to 20 pg/mL, much lower than that obtained in other microdevices.

Protein immobilization techniques for microfluidic assays

“…Thus, involved chemical surface preparation is generally required to induce surface functional groups for protein immobilization. 9,35,[77][78][79][80] As immobilization on planar surfaces yields limited protein density, three dimensional (3-D) structures have been employed inside microfluidic channels for higher protein capture capacity, 21,22,81 resulting in improved immunoassay sensitivity or enzyme conversion rates (Figure 1(b)). 3-D structures have been created by patterning microstructures (e.g., microposts 29,82,83 and micropits 60 ) or through insertion of porous membranes 67,84,85 before assembly of microfluidic chips.…”

“…Inert plastic surfaces lack functional groups, so chemistry is employed to prepare the surface to immobilize proteins. 29,80,124 Oxygen plasma 77,79 or strong bases/oxidizers 35,77,78 are often used to introduce functional groups.…”

“…However, electrostatic interactions can be studied by measuring the binding isotherm while changing the buffer ionic strength. 141,145 35,[77][78][79]124 or silicon surfaces. 35,40,59,68,77,79,124 Proteins are immobilized on the composite layer directly by electrostatic interaction, 146 covalent chemistry, 40,68 or bioaffinity interaction.…”

“…141,145 35,[77][78][79]124 or silicon surfaces. 35,40,59,68,77,79,124 Proteins are immobilized on the composite layer directly by electrostatic interaction, 146 covalent chemistry, 40,68 or bioaffinity interaction. 35,40,68,77,79,124 Electrostatic interaction has also been used to pack microbeads (with bead-surface immobilized proteins) into beds in microfluidic channels.…”

“…Lee's group reported antibody immobilization on a PMMA surface using TR enzyme (Figure 14(c)). The PMMA surface of a simple straight channel 79 or a CD (compact disc) microfluidic device 124 was first treated with oxygen plasma to introduce negative charge, then positive PEI was adsorbed by electrostatic interaction. The PEI provided a plethora of amine functional groups, which were later covalently linked to tyrosine residues of protein A by a TR-catalyzed conversion into reactive O-quinones.…”