Imaging of Chemical Micropatterns on Polycarbonate for Peptide Immobilization

Topics Covered

Abstract
Introduction
Preparation of Slides
Results
Fluorescence Results
SARFUS Results
Discussion of Results
Conclusion
Advantages of SARFUS

Abstract

In this study, a new method is described for micropatterning polycarbonate surfaces for biomolecular interaction studies. The Sarfus technique allows rapid characterisation of the micropatterns and reveals a doughnut effect which hopefully has a minor effect on the fluorescence image.

Introduction

Polycarbonate (PC) is widely used as a substrate in the preparation of microfluidic devices. Due to the potential use of compact discs as platforms for high-throughput analysis of biomolecular interactions, the utility of PC for bioanalysis has recently attracted much attention.

In this article, a new method for the chemical micropatterning of a polycarbonate substrate is reported (figure 1). Silica nanoparticles (1) functionalized with semicarbazide groups were printed on PC by using a noncontact piezoelectric microarrayer to give micropatterns (2). The semicarbazide groups present on the micropatterns were site-specifically ligated with unprotected peptides (3) derivatized by an -oxo aldehyde group, to give substrate (4): peptides linked to the micropatterns through semicarbazone bonds. The surface between the spots was left unchanged.

The use of nanoparticles of different diameters (27 to 304 nm) permitted the influence of surface curvature on signal strength and capture specificity to be studied. The nanoparticle layer on PC substrate was characterized by using Sarfus.

Figure 1. Chemical micropatterning of polycarbonate surface for the site specific immobilization of peptides.

Preparation of Slides

Semicarbazide silica nanoparticles (1) were prepared from silica nanoparticles of different diameters (27, 82, 151, 192, 256 and 304nm) and were printed on PC slides (75 x 25 x 1 mm) by using a noncontact piezoelectric arrayer (three drops, ~1 nL total).

The printed PC slides were incubated under cover-glass with peptides COCHO-HA or COCHO-FLAG, and then anti-HA or anti-FLAG antibodies followed by tetramethylrhodamine-labelled secondary antibodies.

Results

Fluorescence Results

Fluorescence analysis (data not shown) shows the high specificity of the capture of anti-HA or -FLAG antibodies by immobilized peptides and that highest signals were obtained for 82- and 27-nm nanoparticles, probably due to a higher specific surface area.

SARFUS Results

The micropatterns formed by printing semicarbazide nanoparticles on a PC substrate were also characterized by Sarfus.

For this analysis, a Surf with a polycarbonate toplayer (termed ‘Surf PC’) was used. Preliminary experiments demonstrated the inertia of solvent on the toplayer.

Sarfus analysis showed no significant changes in the size or height of the micropattern before and after the different incubations, meaning that no nanoparticle desorbtion occurred during the different washing and incubation steps, and that the thickness of the micropattern is mainly dictated by the thickness of the nanoparticle layer.

For the Sarfus measurement, a calibration is performed from 4’-noctyl- 4-cyanobiphenyl (8CB) liquid crystal that forms spontaneously well-defined multi-layer structures with step height of 32Å (Figures 2A & 2B). Figure 2C shows the Sarfus image of a micropattern incubated with COCHO-FLAG peptide, anti-FLAG antibody and finally tetramethylrhodaminelabelled goat antimurine antibodies.

Discussion of Results

Figure 2. A) Sarfus image of 8CB drop on PC surf. B) Extracted profile along the dotted line in (A). C) Sarfus image of 27nm semicarbazides nanoparticles micropattern. D) Extracted profile along the dotted line in (C). E) left gives correspondence between fluorescence intensity and colors. Fluorescence image of the micropattern shown in C). Scale on left gives correspondence between fluorescence intensity and colors.

In Figure 2C, the spot displays a ring-like deposit along its perimeter probably due the migration of dispersed solids to the periphery of the drop during liquid evaporation. The 27 nm-nanoparticle layer inside the micropattern has a mean height of 5.3 nm.

Like other optical techniques, the Sarfus technique is sensitive to the quantity of matter per unit surface area. Thus, Sarfus measurement of a compact particle (radius R) monolayer (compacity ratio of 0.74) would give a layer thickness of 0.74R. The result obtained in this study suggested a compacity ratio of about 0.4 (5,3/13.5) leading to a mean distance between nanoparticles close to their diameter (27nm).

By comparing both images in Sarfus (figure 2C) and fluorescence (figure 2E) mode, one can see that the doughnut distribution of the nanoparticles visualized using Sarfus has a minor effect on the homogeneity of the fluorescence image. This observation suggests that only the external layer of the micropattern is accessible to the peptide or to the antibody.

Conclusion

A new method for the chemical micropatterning of polycarbonate based on the printing of functionalized silica nanoparticles is reported. Specific captures of antibody is proved.

The Sarfus technique allowed the easy characterization of an entire micropattern and the determination of the nanoparticle layer thickness.