Independently controlling protein dot size and spacing in particle lithography.

Particle lithography is a relatively simple, inexpensive technique used to pattern inorganics, metals, polymers, and biological molecules on the micro- and nanometer scales. Previously, we used particle lithography to create hexagonal patterns of protein dots in a protein resistant background of methoxy-poly(ethylene glycol)-silane (mPEG-sil). In this work, we describe a simple heating procedure to overcome a potential limitation of particle lithography: the simultaneous change in feature size and center-to-center spacing as the diameter of the spheres used in the lithographic mask is changed. Uniform heating was used to make single-diameter protein patterns with dot sizes of approximately 2-4 or 2-8 µm, depending on the diameter of the spheres used in the lithographic mask, while differential heating was used to make a continuous gradient of dot sizes of approximately 1-9 µm on a single surface. We demonstrate the applicability of these substrates by observing the differences in neutrophil spreading on patterned and unpatterned protein coated surfaces.

Patterning of quantum dot bioconjugates via particle lithography.

We present a simple technique to fabricate hexagonally ordered quantum dot bioconjugate (QDBC) dot arrays on glass coverslips. We used particle lithography to create periodic holes in a layer of methoxy-poly(ethylene glycol)-silane and then adsorbed QDBCs into the holes. To demonstrate the versatility of this technique, we made separate periodic arrays of quantum dots (QDs) conjugated to three different biologically important molecules: biotin, streptavidin, and anti-mouse IgG. The diameters of the regions where the QDBCs adsorbed were 500-600 nm and independent of the QDBC patterned. The site density of the QDBCs in the patterned holes could be varied by simply adjusting the coating concentration of the QDBC solution. We demonstrate the applicability of these substrates by designing a QDBC-based binding assay with a working concentration range of several orders of magnitude and a sub-picomolar detection limit.

The role of fibrinogen spacing and patch size on platelet adhesion under flow.

Platelet adhesion to the vessel wall during vascular injury is mediated by platelet glycoproteins binding to their respective ligands on the vascular wall. In this study we investigated the roles that ligand patch spacing and size play in regulating platelet interactions with fibrinogen under hemodynamic flow conditions. To regulate the size and distance between patches of fibrinogen we developed a photolithography-based technique to fabricate patterns of proteins surrounded by a protein-repellant layer of poly(ethylene glycol). We demonstrate that when mepacrine labeled whole blood is perfused at a shear rate of 100 s ⁻¹ over substrates patterned with micron-sized wide lines of fibrinogen, platelets selectively adhere to the areas of patterned fibrinogen. Using fluorescent and scanning electron microscopy we demonstrate that the degree of platelet coverage (3-35%) and the ability of platelet aggregates to grow laterally are dependent upon the distance (6-30 µm) between parallel lines of fibrinogen. We also report on the effects of fibrinogen patch size on platelet adhesion by varying the size of the protein patch (2-20 µm) available for adhesion, demonstrating that the downstream length of the ligand patch is a critical parameter in platelet adhesion under flow. We expect that these results and protein patterning surfaces will be useful in understanding the spatial and temporal dynamics of platelet adhesion under physiologic flow, and in the development of novel platelet adhesion assays.

Fabrication of protein dot arrays via particle lithography.

The ability to pattern a surface with proteins on both the nanometer and the micrometer scale has attracted considerable interest due to its applications in the fields of biomaterials, biosensors, and cell adhesion. Here, we describe a simple particle lithography technique to fabricate substrates with hexagonally patterned dots of protein surrounded by a protein-repellent layer of poly(ethylene glycol). Using this bottom-up approach, dot arrays of three different proteins (fibrinogen, P-selectin, and human serum albumin) were fabricated. The size of the protein dots (450 nm to 1.1 microm) was independent of the protein immobilized but could be varied by changing the size of the latex spheres (diameter=2-10 microm) utilized in assembling the lithographic bead monolayer. These results suggest that this technique can be extended to other biomolecules and will be useful in applications where arrays of protein dots are desired.