Overview of microdispensing

Non-contact microdispensing systems offer accurate and high-throughput deposition of bioactive fluids for many biological and life science applications. The high uniformity and small size of piezoelectric droplet generation makes the technology desirable for creating micro features for high throughput analysis, fabricating precision microstructures and depositing materials in MEMS and BioMEMS devices that cannot be achieved by other means. Ink-jet based deposition is flexible, requires no tooling, is non-contact, and is data-driven, as the printing pattern is created directly from CAD information and stored digitally. As an additive process with minimal waste, it is economical and environmentally friendly. Ink-jet printing technology can dispense spheres of fluid with diameters of 15-200µm (2pl to 5nl) at rates of 0-25,000 per second for individual on-demand droplets. Piezoelectric dispensing technology is adaptable to a wide range of material dispensing applications, such as biological reagents, biomaterials, and biocompatible polymers.

Drop formation sequence in MicroFab's glass dispenser when dispensing Isopropyl Alcohol at 2000 drops per second. A movie illustrating the drop formation is shown here.

Fluid properties considerations

The general fluid property requirements for a fluid to be used in a piezoelectric demand-mode ink-jet device are as follows: Viscosity = 0.5-40cp (Newtonian); Surface Tension = 20-70 dynes/cm. Fluids with properties outside these ranges may be dispensed using ink-jet devices, but with increased difficulty / decreased performance (see figure to the right). The left image of the figure shows an unstable inkjet microdispensing associated with high frequency deposition of a low surface tension, low viscosity solvent. The right image presents a stable inkjet microdispensing obtained using a higher surface tension and higher viscosity fluid at the same frequency. Combinations of the extreme values may also have poorer performance. If the fluid is heated or cooled, the above properties are required at the orifice. The viscosity and surface tension values described are appropriate for fluids with a specific gravity near 1. For high-density fluids, such as molten metals, the values above should be converted to kinematic values using the density of water. Newtonian behavior is not strictly required, but the fluid properties at the orifice flow conditions must be in the above range. Thus, as shear thinning fluid could have a low shear rate viscosity much higher than the 40cp. Viscoelastic behavior causes significant performance problems. Fluids properties can be modified to better match the demand-mode ink-jet requirements by changing the concentration of solute and/or solvent or by adding viscosity and surface tension modifying agents, such as glycols, glycerin, alcohols and surfactants.

Particle suspensions are acceptable as long as the particle / agglomerate size and density do not cause the suspension to depart from the fluid properties range given above. Particles that are >5% of the orifice diameter will cause at least some instability in drop generation behavior, but still may be acceptable in low concentrations. However, problems associated with the particles settling out during microdispensing idle time may occur.

Fluids containing polymeric and proteinaceous components may exhibit problems with the material drying and filming over on the orifice during idle time between dispenses. Such filming over may result in dispensing failure or drop instability. Corrective methods include increasing the relative humidity in area adjacent to the orifice, decreasing the concentration of the polymer or proteinaceous material, adding humectants to the fluid (glycols, glycerin) or using higher boiling point solvents.

MicroFab has dispensed a wide variety of biologically relevant fluids and reagents as presented below.

Biocompatible polymers

Biopolymer microdispensing has applications in tissue engineering and drug delivery, such as creation of cell attachment sites, scaffolds for tissue engineering, and coatings and drug delivery systems for controlled drug release. Polymeric materials can also be dispensed to create and/or modify solid support structures, such improving adhesion / wetting or repulsion / non-wetting at very high resolution.

Example of 450um diameter acrylamide gel pads containing different concentrations of catalyst printed onto a microscope glass slide; polymerization was UV initiated.

Examples of 50um wide lines of Poly(lactic acid) (PLLA) microdeposited by ink-jet onto a polystyrene substrate. Note in the right image that overprinting can be used to build up layered structures.

In the images above, on demand fibers have been created at MicroFab Technologies using bioabsorbable polymers and single channel microdispensing device. (US Patent Pending). Traditionally, polymer fibers are created through wet or molten extrusion process. Ink-jet dispensing allows digitally controlled, on-the-fly fiber dispensing.

Shown in the above image is a two part printed bioabsorbable polymer conduit Poly(lactic-co-glycolic acid) created as a nerve regeneration implant. This conduit contains ribs of a more rigid polymer formulation that have been ink-jet printed over the underlying softer inner tube. These ribs are intended to improve the compressibility resistance of the conduit when implanted. This conduit can be incorporated with growth regulators (NGF) to elicit neurite extension of seeded cells.

Left image - a solution of 3:1(w:w) PLGA (85:15 PLA to PGA) and paclitaxel in 1,2 dichlorethane being microdispensed in atmosphere; middle image: the PLGA / paclitaxel solution being dispensed into a solution of 0.1% (w:v) poly(vinyl alcohol) (PVA) resulting in microsphere formation (dispensing device tip is under the PVA solution). Right image - results of microsphere formation from the microdispensing shown in the middle image (mean diameter is 100um +/- 1.0um).

Biofluids / Bio-inks

Ink-jet microdispensing can be used to accurately deposit biological fluids, ranging from single molecules (proteins, DNA, lipids) to tissue extracts and cells. Because these biological relevant fluids are dispensed by ink-jet they are often referred to as bio-inks. For example, human serum has been microdeposited for the purposes of Western Blot analysis. Cells have been microdispensed into wells for drug screening assays and for cell seeding and patterning in tissue engineering applications. DNA and proteins have been dispensed to create gene and protein expression, immunoassay and diagnostic microarrays. BSA and other blocking agents (casein, non-fat dried milk, other animal serums, commercial formulations) can be printed to selectively block areas on a membrane or other substrate. Lipid microspheres for drug delivery have also been created using inkjet microdispensing. In addition, lipids have been microdeposited onto solid substrates to selectively control location of cell attachment to the substrate.

Human embryonic kidney cells (HEK293e) being dispensed at 240Hz using an ink-jet device with a 55mm orifice. The immediate cell viability was unchanged after being microdispensed. A movie illustrating the drop formation is shown here.

Above is an example of Boehringer Mannheim (now Boehringer Roche) Diagnostic’s MicroSpot™ Dispo. Up to 196 distinct reactions sites (i.e., antibody or oligonucleotide spots) would fit into their disposable reaction well and could be imaged using a fluorescence confocal scanning microscope. The initial pilot line used ten separate MicroFab Technologies ink-jet microdispensers to deposit a total of ten fluids. Each fluid was printed into multiple spots to provide redundancy, and a real-time inspections system imaged the printed dots using a secondary fluorophore.

Displayed is a sandwich immunoassay ink-jetted microarray for detection of different concentrations of Cytochrome C using rabbit anti-cytochrome C; spots are 40um in diameter.

Cholesterol microspheres (50um) created by inkjet microdispensing for the controlled release of hormones.

Example of the inkjet microdeposition of phosphatidylcholine over a frequency sweep of 480-4800Hz. Phosphatidylcholine is a major constituent of cell membranes.

Active molecules

Active components can be incorporated into a 3D matrix for tissue engineering applications. Materials, such as collagen, laminin and extracellular matrix can be microdeposited to create bioresorbable scaffold structures for implantation. Growth factors (nerve growth factor, connective tissue growth factor, vascular endothelial growth factor, platelet-derived growth factor, epidermal growth factor, basic fibroblast growth factor, etc.) can be incorporated into these microdeposited matrices to stimulate cell attachment, proliferation, differentiation, orientation and migration patterns. For example, piezoelectric microdispensing offers a method to create concentration gradients of growth factors for controlling neurite growing patterns in nerve regeneration scaffolds. Further examples of active molecules dispensed by ink-jet technologies are given in the reagents section.

Example of a step-wise gradient of NGF microdeposited onto a flat substrate. This example demonstrates the possibility of obtaining a gradient by simply changing the pitch of the drop placement during printing.

Orthogonal gradients obtained by changing the amount of fluid deposited at each location. The volume of fluid per spot increases in the direction of the arrows.

Miscellaneous microdispensing

Left - portion of a 20x20 array of the spots printed on a 250mm pitch having red and blue Inspeck 2.5um diameter microspheres and the 0.02um diameter green Flurospheres (60X). Right - 200X magnification of one of the spots containing the fluorescent microspheres

Commassie blue stained proteins on a PVDF membrane transferred from a 2D PAGE gel. The inset on the left shows spots (200-300um in diameter) that have been treated with the inkjet microdeposition of trypsin to obtain a micro-scale digestion of the protein spot of interest. A matrix material is then inkjet microdeposited onto the spots after the micro-scale digestion is complete. The protein blot is then transferred to a MALDI-TOF MS instrument for analysis in the process of peptide mass fingerprinting for protein identification. Multiple endoproteinase digestions can also be performed on the protein spots of interest in a similar manner. The use of a second protease not only independently confirms the proteins identity, but the combined peptide mass fingerprints increase sequence coverage. More information can be found in the Chemical Ink-Jet Printer (ChIP) brochure.

Reagents

Piezoelectric inkjet dispensing devices can be utilized to microdispense a wide variety of reagents and materials for numerous applications including combinatorial synthesis, surface modification for hydrophilic or hydrophobic patterning, creating reactive sites, performing chemical reactions on membranes, microwells or other substrates. The table below lists some reagents and materials that have been microdispensed using inkjet technology at MicroFab Technologies. This list is not all-inclusive.

alpha-cyano-4-hydroxycinnamic acid Acetone Acetonitrile Acrylamide Adiponitrile Anti-cytochorme C IgG Anti-goat FITC IgG Anti-rabbit Biotin IgG Avidin BSA 0.1% - 10%, 10 mM TRIS Butanol Cells HEK293e at 106 in media Collagen (calf skin) 1mg/ml Cyanoacrylate

DMF, Dimethylformamide DMSO Ethanol Ethyl Acetate Ethylene Glycol 5-30% Glycerol to 50% Heptane Human Serum Isopropanol Lithium Chloride to Saturated Methanol NMP, N-Methylpyrrolidone Octylglucoside 1% PEG 8000 0.1-30%

PLGA Polystyrene 8um in Buffer Polyvinylpyrrolidone 0.25% Potassium Carbonate 5M Propylene Carbonate Propylene Glycol 0.1-30% Sodium Bicarbonate 100mM Sodium Hydroxide, 80mM Trifluroacetic acid 0.1% Triton X 0.05-1.0% Tween 20 0.05-1.0% Trypsin Xylenes

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