Size-dependent self-assembly of submicron/nano beads-protein conjugates for construction of a protein nanoarray

Abstract

A protein nanoarray is created when submicro and nano beads, varying in their size and each conjugated with different proteins, self-assemble to specific locations depending on the diameter matching the surface electron beam patterns created. Protein binding is confirmed from the fluorescence attenuation of the beads upon antigen-antibody binding on the bead surface. This method, called size-dependent self-assembly, allows control of the location of each type of bead, and thus, control of the location of multiple proteins. It provides fast multi-component patterning with a high binding resolution, which can be detected using a fluorescent light microscope. This method is developed to be a simple stand-alone tool for analysis of protein interactions. In addition, it has the potential to be used in conjunction with other methods protein analysis methods, such as enzyme-linked immunosorbent assay (ELISA) and atomic force microscopy (AFM).

Figure 1

Concept illustration showing the size-dependent self-assembly of the beads on the e-beam patterned surface.

Figure 2

Fluorescent image showing size-dependent self-assembly with 70% saturation rate of the patterns with beads. Green = 300 nm beads. Blue = 140 nm beads. The blue beads in the second and third column are indicated with arrows.

Figure 3

SEM image at 30000× magnification showing size-dependent self-assembly of 140 nm beads and 30 nm AuNPs in 180 nm and 130 nm well patterns created by electron beam lithography.

Figure 4

SEM image of 140 nm and 80 nm beads and AuNP bound within the patterns. The upper left pattern contains both an AuNP and an 80 nm bead.

Figure 5

Figure showing edge of left droplet containing unconjugated 140 nm fluorescent beads and the right droplet containing the same type of beads conjugated with mIgG and bound to anti-mIgG. The unbound beads fluoresced brightly, while the conjugated beads showed signal attenuation when bound to anti-mIgG.

Figure 6

Images showing 140 nm fluorescent beads bound to a patterned array and tables showing pixel intensity. Beads are first conjugated with mIgG, and subsequently with anti-mIgG antibodies tagged with FITC. The presence of the FITC (column A) confirms the binding of proteins, which attenuates the fluorescent signal from the beads (column B). Based on pixel intensity measurement, mIgG conjugated beads have approximately 50% the fluorescent intensity of the unconjugated fluorescent beads (Column C).