Cytophobic surface modification of microfluidic arrays for in situ parallel peptide synthesis and cell adhesion assays

Abstract

A combination of PEG-based surface passivation techniques and spatially addressable SPPS (solid-phase peptide synthesis) was used to demonstrate a highly specific cell-peptide adhesion assay on a microfluidic platform. The surface of a silicon-glass microchip was modified to form a mixed self-assembled monolayer that presented PEG moieties interspersed with reactive amino terminals. The PEG provided biomolecular inertness and the reactive amino groups were used for consequent peptide synthesis. The cytophobicity of the surface was characterized by on-chip fluorescent binding assays and was found to be resistant to nonspecific attachment of cells and proteins. An integrated system for parallel peptide synthesis on this reactive amino surface was developed using photogenerated acid chemistry and digital microlithography. A constant synthesis efficiency of >98% was observed for up to 7mer peptides. To demonstrate specific cell adhesion on these synthetic peptide arrays, variations of a 7mer cell binding peptide that binds to murine B lymphoma cells were synthesized. Sequence-specific binding was observed on incubation with fluorescently labeled, intact murine B lymphoma cells, and key residues for binding were identified by deletional analysis.

Figure 1

Schematic of surface modification of microchip by mixed SAM of PEG-silane and aminosilane

Figure 2

Schematic of experiments to characterize surface passivity and chemical reactivity of microchip surface

Figure 3

Stepwise synthesis of peptides using photogenerated acid chemistry. The O group indicates acid labile BOC protecting group on amino acids. (a) BOC-protected Leucine (L) is attached to the whole chip surface (b) Selected areas are irradiated with digital light patterns. (c) BOC group is removed in these select areas and terminal amino groups (N) are available for reaction (4) The next protected amino acid Valine (V) is coupled to these amino terminals to build the desired peptide. This cycle is repeated to generate site specific peptide sequences.

Figure 4

Scanned image of microchip at 635 nm where chambers 1 and 3 have acetyl capped Glycine, chambers 2 and 4 have deprotected Gly-Gly-Gly-NH₂, labeled with amine reactive Alexa 647.

Figure 5

Scanned image of microchip at 532 nm, derivatized with mixed SAM of PEG and amino silane. Trimer peptide synthesized in chambers 2 and 4. (a) incubated with fluorescently labeled cells. (b) After extensive washing with 1% Tween solution in PBS.

Figure 6

Scanned image of chip with 1mer to 7mer synthesized in sets of 4 spots. The top right set has a 1mer and a growing chain is synthesized step wise with a 7mer in the bottom right.

Figure 7

Plot of relative fluorescence intensities, where Ac = Acetyl capped and FR = fluorescein labeled. (1) G-FR (2) G-G-Ac (3) G-G-FR (4) G-G-G-Ac (5) G-G-G-FR (6) G-G-G-G-Ac (7) G-G-G-G-FR (8) G-G-G-G-G-Ac (9) G-G-G-G-G-FR (10) G-G-G-G-G-G-Ac (11) G-G-G-G-G-G-FR (12) G-G-G-G-G-G-G-Ac (13) G-G-G-G-G-G-G-FR

Figure 8

Scanned image of Chip A at 532 nm: Positive control DLWYDAV in chambers 1 and 3, deletion DLWYV in chamber 2, negative control GGG in chamber 4. Incubated with 1 × 10⁵ cells/ml and washed extensively.

Figure 9

Scanned image of Chip B at 532 nm: Positive control DLWYDAV in chambers 1 and 3, deletion DLDAV in chamber 2, negative control GGG in chamber 4. Incubated with 2 × 10⁶ cells/ml and washed extensively.

Figure 10

Plot indicating sequence specific binding of murine B lymphoma cells to various deletional sequences of DLWYDAV. All data is from the same selected area in the chips and normalized to initial concentration of cells.