On-Chip Immunoassay Using Electrostatic Assembly of Streptavidin-Coated Bead Micropatterns

Abstract

We propose an original concept for a sandwich immunoassay that is completely performed on-chip using streptavidin-coated beads as substrate. The latter are electrostatically self-assembled on aminosilane micropatterns at the bottom of a microfluidic channel. We use mouse IgG diluted in phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA) solution as target antigen. The fluorescent sandwich immunocomplex is formed on the beads during the operation of the chip both in stop-flow and continuous-flow modes. Target mouse IgG antigen is detected down to a concentration of 15 ng/mL in stop-flow mode and 250 pg/mL in continuous-flow mode, using only 1300 nL of sample volume. We also demonstrate the possibility of simultaneous detection of two different antigens in a PBS-BSA solution using a dual microfluidic channel structure.

Protein immobilization techniques for microfluidic assays

“…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. In post-assembly approaches, microbeads 54,62,[86][87][88][89][90][91][92][93] can be packed into enclosed channels or various polymers such as hydrogels, [18][19][20][21][22][23][24]57,[94][95][96][97] sol-gels, 64,98,99 polymer monoliths, 61 or membranes 100 can be polymerized in situ. For silica-based 3-D structures such as silica beads 91 and alkoxysilane-based sol-gels, 63 a similar glass/silicon surface immobilization strategy can be used.…”

“…Surfaces are modified to have stronger charge or hydrophobicity to adsorb protein better than the native surface, with reports of such physisorption as practically irreversible. 92 In some cases, physisorption happens instantaneously (i.e., high k on ) compared to covalent or bioaffinity bonds that usually requires a substantial incubation time, and thus physisorption can be used as an intermediate immobilization step of a multistep assay sequence (e.g., Western blot). 20,24 Physisorption has a wider choice of buffer system, compared to widely used covalent bonding through primary amines where popular amine-based buffers (e.g., Tris, glycine) cannot be used.…”

“…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. 92 Protein has also been directly immobilized inside hydrogels via electrostatic interaction. Kim et al created negatively charge polyacrylamide (PA) gels to immobilize proteins after separation by electrophoresis to yield microfluidic Western blotting (Figure 3).…”

“…The authors reported a 1-pM detection limit. Sivagnanam et al patterned antibody functionalized microbeads on a glass surface 92 using dot and line patterns of APTES. The APTES pattern was created using a lift-off process on the glass substrate of a hybrid glass-PDMS microfluidic device (Figure 14(b)).…”

Study of Spatio-Temporal Immunofluorescence on Bead Patterns in a Microfluidic Channel

“…We recently reported a method, based on electrostatic self-assembly, for realizing an onchip immunoassay using streptavidin-coated beads as assay substrate for the detection and quantification of target Ag [13]. Bead immobilization occurred through electrostatic force between the negatively-charged bead surface and a positively-charged aminosilane micro-pattern applied to a glass substrate [13][14]. The receptor Ab was coupled to the bead surface for specific binding with the target Ag.…”