PROCESSING TECHNOLOGIES

The benchtop low-pressure plasma system developed by PVA TePla America Inc. (Corona, CA) (left) is designed for cellular manufacturing and laboratory use. The company’s atmospheric Plasma Pen (right), engineered to keep voltages and current safely inside its body, is used for in-line applications and for spot treatment.

The technology of energizing a gas to produce a glow discharge, or plasma, has become a powerful tool in solving surface preparation problems in the medical device industry. Plasma is used to ultraclean and sterilize surfaces and also to promote the adhesion of biocompatible coatings to in vivo devices and biological materials to in vitro diagnostic platforms. Indeed, plasma can either activate surfaces for purposes of cell or biomolecule immobilization or, conversely, produce nonstick surfaces for antibiofouling or metered drug-dispensing applications. The functioning of microfluidic devices can be greatly enhanced by plasma, too.

Plasma treatment makes microchannels on clinical diagnostic devices wettable to biofluids without having an effect on the properties of the analyte itself. Plasma is also used for low-technology applications such as promoting marking with ink on catheters and bonding syringe needles to hubs with adhesive. And, since plasma is a dry surface treatment technique, there are no waste chemicals to dispose; this makes it an environmentally friendly process involving very few consumables.

This article discusses how gas plasma can be an enabling technology in the manufacture of IVD platforms. It focuses on the way gas plasma controls surface energies and tailors surface chemistry to promote the attachment of biological materials.

The Nature of Plasma

Understanding the science behind gas-plasma surface modification requires first knowing exactly what plasma is. Plasma is a state of matter, equivalent to a solid, liquid, or gas (see Figure 1). When enough energy is added to a gas, the gas becomes ionized and enters a plasma state. Electrons break free from the pull of their atoms or molecules and are then available to transfer energy to other moieties present through electronic collisions. The active components of plasma include ions, electrons, radicals, excited species (also called metastables), and photons, among others. The collective properties of these active species can be controlled and harnessed so as to perform a variety of surface treatments, including nanoscale cleaning, activation for surface wettability, chemical grafting, and coating deposition.

(click to enlarge) Figure 1. Basic states of matter. The principal difference between the plasma and gaseous states is that the plasma state is electrically conductive.

Chemically, plasma is a highly reactive environment that can be employed to change the properties of surfaces without affecting the bulk material. The energy carried by this partially ionized gas in fact can be controlled so that it contains low heat energy. This is achieved by coupling the energy into the free electrons rather than the heavier ions, and it allows heat-sensitive polymers such as polyethylene and polypropylene to be treated.

The energy is coupled into the gas most commonly by the creation of an electrical field between two electrodes at low pressure in a vacuum chamber. This is the operating principle behind fluorescent lighting.

Plasma can also be generated at atmospheric pressure. At one time, atmospheric plasmas were too hot to be used as a surface treatment tool, but the technology has advanced recently. Low-temperature plasmas produced at atmosphere now can be suitable for treating even the most heat-sensitive polymers. More and more applications using atmospheric plasma are appearing as this technology gains ground on low-pressure plasma generation.

Effects on Surface Properties

A solid contaminated by hydrocarbon molecules adsorbed on its surface can be cleaned by plasma-excited oxygen species that readily attack the organic contaminants. The oxygen removes the adsorbed hydrocarbon material by converting it to carbon dioxide (CO₂) and water through a basically simple mechanism (see Figure 2). Many initialization mechanisms involving different excitation states of oxygen as both a free radical and diatomic molecule are possible. The surface-adsorbed hydrocarbon can itself be excited by electronic collision with the plasma, thus providing an additional set of possible reaction pathways.

(click to enlarge) Figure 2. Plasma-generated oxygen radicals attack surface-adsorbed hydrocarbons.

For surfaces sensitive to oxidation, plasma-activated hydrogen is an alternative for surface cleaning. Not only can hydrogen reduce organic surface moieties to volatile hydrocarbons, it can reduce oxides of copper, nickel, silver, and other metals, as well.

The chemical characteristics of a plasma are mostly determined by the feed gas. Oxidizing atmospheres are created by molecular oxygen and nitrogen, nitrous oxide (N2O), and CO₂, for example. These gases are used to render surfaces wettable, or hydrophilic, with respect to polar solutions. This is achieved through plasma-induced covalent bonding of oxygen, which produces functional groups such as carbonyls, carboxylics, and hydroxyls on the surface. These polar groups increase the surface energy so that, for example, tissue cells adhere better, or analytes dispensed onto diagnostic platforms will flow more easily through microfluidic vias.

Reductive plasma atmospheres are created by molecular hydrogen, a mixture of hydrogen, argon, ammonia (NH₃), etc. Reducing plasmas have proved useful in the activation of fluorocarbon substrates such as polytetrafluoroethylene (PTFE). PTFE is a material well suited for implantable medical devices because of its inertness and biocompatibility.

Its properties become disadvantageous, however, when PTFE needs to be processed—for example, for the attachment of synthetic scaffolds to encourage tissue growth on in vivo devices. But reductive plasmas resolve issues such as these by reducing the overall fluorine concentration at the device surface through the replacement of fluorine atoms with functional groups such as hydroxyls. In the example cited, surface hydroxyl groups provide anchor points for support of the synthetic scaffolds.¹

Some applications require the subject material to be etched. Fluorine-containing gases such as nitrogen trifluoride (NF₃), sulfur hexafluoride (SF₆), and carbon tetrafluoride (CF₄) are very good for etching hydrocarbon polymers, silicon and its oxides, and nitrides, among other materials.

In addition to the strong chemical effects of a plasma, the technology produces directional effects that also play an important role. Particles carrying momentum to the surface can physically remove more-inert surface contaminants such as metal oxides and other inorganics and can cross-link polymers to lock the results of surface treatment in place.

Figure 3. A scanning electron microscope image of a cross section of polycarbonate PECVD-coated with a 1-µm layer of silicon oxide (SiOx), showing how well the PECVD process contours the varied topography of the polymer surface. The pit toward the right side of the image is approximately 4 µm deep and 4 µm wide.

Polymer coatings can be grown on surfaces through a process called plasma-enhanced chemical vapor deposition (PECVD). PECVD works by activating species, such as monomers, in the plasma and inducing their polymerization on the workpiece substrate (see Figure 3). Barrier, nonstick, and scratch-resistance properties are among those that some PECVD coatings can introduce. Other coatings may be designed to contain chemical functional groups, such as NH₂ (amine), OH (hydroxyl), or COOH (carboxyl), that provide specific binding sites for further grafting (for example, to immobilize proteins or sensor agents for biological materials) or increasing the affinity of antithrombogenic, lubricious, type IV collagen, or other functional coatings. Surface chemical properties of the deposited coating are determined within the first few tens of nanometers of the surface.

Processing IVD Platforms

The full scope of gas plasma applications in the medical device industry is very large indeed. This article focuses primarily on applications that relate to the manufacture of diagnostic device platforms. In that arena, plasma technology is employed to clean surfaces in preparation for downstream processing and to activate surfaces to promote biomolecule and cell adhesion. The latter end is achieved by changing surface polarity, by grafting specific functional groups to the substrate, or by polymerizing a coating onto the surface. To better understand how plasma tunes surfaces to make them suitable for the application requirement, some important cases might be considered.

Microfluidic Devices and Hydrophilicity. Surface energy is a property of materials that determines such factors as their wettability and their susceptibility to biofouling. In general, materials with high surface energies are hydrophilic and wettable to such fluids as blood, bacterial-cell suspensions, buffers, inks, adhesives, and various other adsorbates and coatings. Low-energy surfaces, on the other hand, are termed hydrophobic and characteristically exhibit nonstick behavior.

Typically, microfluidic devices have to have hydrophilic surfaces so that the analyte will flow smoothly and consistently through the microchannels to the detection and processing elements. Such flow is accomplished by means of a variety of pumping methods, including electroosmotic, thermal, and mechanical techniques. However, microfluidic platforms usually are made from polymeric materials—for example, acrylic, polystyrene, or polydimethylsiloxane (PDMS)—that are inherently hydrophobic.

One of the major problems caused by the hydrophobic nature of these materials is bubble trapping in the microchannels, which inhibits fluid flow. Even when the channels are primed with an alcohol-and-buffer solution, air bubbles can pose a problem. Gas plasma treatment, by oxidizing the surfaces of the microchannels to make them hydrophilic, can prevent the formation of air bubbles.

Flow rates are also affected during electrokinetic pumping by surface-charge densities. Electrokinetic pumping, used to drive fluids through microchannels, wors on the principle of electrokinesis, the conversion of electrical energy into kinetic energy. A charged surface will attract particles of opposite charge in the electrolyte. This allows the particles remaining in the fluid to be pumped electrophoretically through the vias with greater ease. Charging surfaces with plasma has been shown to be effective in supporting electrophoretic or electroosmotic flow.²

Immunoassays, Microarrays, and Tissue Culture Media. Platform substrates for such clinical diagnostics as immunoassays, microarrays, and cell culture media are predominantly fabricated from synthetic polymers. While such materials are ideal for this industry by virtue of being inert, mechanically stable, and inexpensive, their surface properties introduce inherent limitations. Specifically, binding sites for bioactive molecules or cells are inadequate. Bioactive entities cannot anchor themselves effectively to these surfaces. Strong and uniformly dispersed binding sites are important prerequisites for the immobilization of biological analytes and for in vitro cell cultivation. Synthetic-polymer platforms must be surface-modified so as to improve their properties to support cell proliferation and biomolecular adsorption. Application of gas plasma to the surface of these analytical devices effects the necessary changes.

Increased Cell Growth Yields

Animal- or plant-derived tissue cultures grown in vitro have to be supplied with nutrients, hormones, and other growth factors that are provided naturally when the cells grow in vivo. Tissue cells attach to solid surfaces, where they then proliferate in a liquid nutrient medium. In the case of animal cells, this would be serum. The surface properties of the growth medium must be conducive to uniform cell attachment and growth. Before these properties are tuned, however, the surface must be free of contamination. Mold release agents, volatile hydrocarbons, and other contaminating species can be removed from cell culture platforms by exposure to the energetic, yet cool, environment of plasma to ensure the proper pattern of cell attachment and proliferation (see Figure 4).

(click to enlarge) Figure 4. Not treating polystyrene wells may lead to nonuniform cell attachment and cell clumping (a) or areas with no cell attachment (b). A plasma-treated substrate promotes uniform cell attachment and proliferation (c).

The inherently hydrophobic nature of the polymeric materials from which culture media are manufactured does not favor tissue-cell attachment. Hydrophilic surfaces are needed instead. Oxidizing plasmas can be used to add oxygen groups to surfaces, thus increasing their polarity and rendering them hydrophilic.

Hydrophilic surfaces are attractive to tissue cells, inducing them to ad-sorb. Where a significant concentration of specific chemical functionality is required on a surface (as discussed in the next section), the application of gas plasma to the substrate can achieve the chemical graft or polymerization of monomer necessary to produce the desired functionality.

Roughened surfaces present a higher overall surface area than a smooth surface of equivalent extent. In theory, this equates to a higher number of potential cell-binding sites. Since cells typically are on the order of 10 µm in size, surface microroughening provides the greatest enhancement of cell adhesion.³ Surface roughening on a nano scale could not be expected to increase cell adhesion though, because the comparatively larger cells cannot take advantage of increased surface area on that scale.

However, nanoscale roughening can increase drug-induced differentiation and apoptosis. The reasons for this are unclear. An increased number of cell receptors might be the explanation. Or, perhaps enhanced signaling pathways to the nucleus account for this performance benefit. Whatever the mechanism behind it, this effect has important implications for improving tissue scaffold development on implantable devices.

Surface topography can be selectively altered in a plasma environment by either accelerating ions toward the surface or chemically etching it.

Capacitively coupled radio-frequency plasmas generally exhibit a net directional flow of ions toward the substrate. This is an effect of the relative response times of ions and electrons to the polarity change in the electric field producing the plasma. Since electrons are much lighter than the ions, they respond more quickly. Therefore, substrates lying in the path of the electrons will be charged negatively. The positive ions subsequently are accelerated toward the negatively charged surface through the agency of electrostatic attraction. The impact of these ions on the surface to which they are drawn removes surface material.

Argon gas is well suited for microroughening surfaces in this manner. The energy of the accelerated ions can be controlled by adjusting the power and pressure settings of the plasma-generating equipment. For example, increasing the pressure parameter above 1 mTorr can greatly reduce the ion impact energy, if not eliminate it completely. This enables the surface-roughening effect of plasma to be switched off.

(click to enlarge) Figure 5. Atomic-force-microscopy images showing the nanoroughening effect of an oxygen plasma on polyethylene terephthalate, or PET. The untreated surface is on the left.

The application of oxygen plasma is a much milder process than the one just described. It can thus be used to nanoroughen polymeric materials by the action of mild chemical etching (see Figure 5).

The cumulative effect of the combination of surface cleaning, surface activation, and nanoroughening by means of plasma treatment is an increase in cell attachment of as much as 30% in comparison with untreated substrates, and more-uniform cell coverage.

Improved Biomolecule Adhesion

Gas plasma technology is able to solve problems involving the adhesion of biomolecules to such diagnostic substrates as immunoassay and microarray platforms. It does this by providing particular chemical functionality at the surface that allows covalent coupling of biochemical species to occur.

Carboxylic, hydroxylic, and amino functionalities are important and common types of chemical functionality that are readily obtainable through gas plasma processing. For example, in the manufacture of microarrays, amino functionality provides binding sites for the direct attachment of nucleotides and oligonucleotides to the working surface.

If steric hindrance interferes with direct binding of these large biomolecules, then primer molecules, sometimes called linkers, are used. Linkers provide space for the biomolecule to adsorb to the surface in the proper configuration. As it happens, linker molecules themselves require that surfaces be activated to help them anchor to the substrate.

Most often, straightforward treatment with oxygen plasma is enough to promote the binding of these molecules. However, in some cases, specific functionality is required. Some capture agents work more efficiently in either an acidic or a basic environment, for example. If the capture agent is linked via a carboxylic group, an acid environment is provided. Amino functionality will produce a more basic environment.

(click to enlarge) Figure 6. Gas plasma surface treatment adds chemical functionality either by exposing the surface to plasma containing the specific functionality (a) or by growing a coating on the surface via PECVD (b) using a monomer that already has the desired functionality.

There are two basic ways to functionalize a surface with specific chemical groups. One is to deposit a PECVD coating that introduces the desired functionality, and the other is to generate plasma in the presence of the functional group and allow the group to bind to the surface (see Figure 6). While the latter is the simpler method, the former results in a higher surface concentration of the functionality (10–20%). Using ammonia in the feed gas will result in NH₂ groups binding to the surface. Methanol is used to functionalize with hydroxyl groups, and a combination of methanol and carbon dioxide provides carboxylic functionality.

Unfortunately, during the deposition of these groups, some fragmentation reactions occur that can transform the primary functionality. For example, ammonia plasma will deposit primary amino groups along with secondary and tertiary amines, nitriles, imines, and other derivative compounds. The relative amounts of these groups that are deposited vary depending on the type of plasma system used and the parameter settings. However, this method can be expected to produce 2–8% of the desired functionality.

(click to enlarge) Figure 7. The morphology of a gel droplet deposited onto a polymer slide via ink-jet is the desirable one when the surface energy is controlled through use of the right plasma feed gases (a) and exhibits deformation when placed on a too-hydrophilic surface such as a PECVD coating containing amino groups (b).

Sometimes, providing the right chemical functionality alone is not enough. Amine groups, for example, increase surface energies, rendering surfaces hydrophilic. Overly hydrophilic surfaces may not be desirable when, say, depositing gel drop arrays onto a microarray platform, because the microdroplets may wet the surface inappropriately (see Figure 7). This type of wetting results in malformed droplets.

Again, gas plasma can solve this problem and preserve proper droplet morphology by controlling the surface energy, even in the presence of the amino groups. During plasma amination of the microarray platforms, fluorine chemistry can be added to the process in a controlled manner. The fluorine binds to the floor of the platform and increases its hydrophobicity such that the droplets retain their spherical shape. Fortunately, this process does not affect the surface concentration of deposited primary amines, nor does it interfere with the covalent binding of the gel to the platform.

Immunoassay platforms are evolving constantly, undergoing transformation into a variety of shapes, sizes, and configurations. The most common forms of these substrates are 96- and 384-well microplates. Gas plasma treatment is an established method of hydrophilizing such plates in order to promote the immobilization of antigens, antibodies, and other biologically active small molecules.

(click to enlarge) Figure 8. Bubbles of trapped air are common when fluid is dispensed into untreated wells whose surfaces are hydrophobic (a), but essentially nonexistent in plasma-treated wells whose walls can be completely wetted by the dispensed fluid (b).

The formation of bubbles in wells during fluid dispensing is a potential problem that plasma is used to control (see Figure 8). A bubble trapped in a well with a hydrophobic surface will produce erroneous spectrophotometer readings and may even cause spillage into neighboring wells owing to the well volume it occupies. Plasma treatment ensures complete wetting of the analyte in the wells and can virtually eliminate the possibility of bubble formation.

While the hydrophobicity of microplate reservoirs can result in trapped bubbles in the analyte, there are circumstances where reservoirs that are too hydrophilic can create problems as well. Excessive hydrophilicity results sometimes in the analyte wicking up the sides of the well onto the platform floor and potentially contaminating neighboring reservoirs.

One such case known to the author involved an immunoassay platform fabricated from a proprietary hydrophobic polymer. This polymeric platform accommodated a number of reservoir wells that had gold detector plates at their base. The gold had to be cleaned prior to deposition of the biosensor material, so the platforms were exposed to oxygen plasma. However, while this cleaned the gold nicely, it affected the reservoir sides such that the adverse result just mentioned occurred: the dispensed biosensor solution wicked up. The challenge for the plasma process engineer was to clean the gold plates while preserving the optimal level of hydrophilicity on the reservoir walls. This was achieved by supplying a combination of feed gases to the plasma generator, one to remove the hydrocarbon contaminants from the gold and a second to render the reservoir walls hydrophobic through the addition of fluorine groups. This success demonstrated the versatility and adaptability of the plasma surface modification process.

Resistance to Adsorption

Applications for nonstick fluoropolymer technology extend well beyond cookware. In vivo and in vitro medical devices may need to have surfaces that resist the adhesion of proteins or cells in order to maximize their hemocompatibility. Antithrombic activity can be controlled, for example, by coating surfaces with materials similar to PTFE.

A surface’s suitability for adsorbtion is reduced by lowering its surface free energy—that is, the energy the surface has available to it for the formation of chemical bonds. One way of doing this for a medical device is to apply to it a thin coating that has an inherently low surface energy. Polymeric fluorocarbon coatings with nonstick properties readily adhere to a wide range of materials when deposited on their surface by PECVD. Gas plasma processing provides a reliable, biocompatible, and environmentally friendly method of reducing the surface energy of materials by polymerizing fluorocarbons onto a surface in a highly controlled environment. Any fluorocarbons present in the exhaust are captured by a scrubber at the pump outlet.

It has been reported that the interaction of DNA with polypropylene polymerase chain reaction plates can result in denaturation over time.⁴ This has implications for the storage of DNA in polypropylene vessels, possibly resulting in a reduction in both the quality and the quantity of DNA as it is stored over time. Studies have shown that oxygen plasma–treated polypropylene plates have a lowered adsorption affinity for DNA.⁵ Oxygen plasma generates a negatively charged surface. It is believed that this negative charge repels the phosphate backbone of the synthetic DNA and thus prevents the DNA from adsorbing to the surface.

Process Validation

Contact-angle measurements are used extensively as a measure of surface bondability. Untreated polymeric surfaces are low in energy, so water droplets applied to these surfaces bead up; they have high contact angles. This is because the cohesive forces of the water are stronger than the adhesive forces of the surface. On plasma-treated surfaces, water contact angles are very low because of the energy that has been added to the surface in the form of polar chemical groups (see Figure 9). This energy is used to bind to the water molecules, which causes the droplets to spread out across the surface. These are hydrophilic surfaces. Therefore, low surface contact angles can be taken as good indicators that a surface is wettable.

(click to enlarge) Figure 9. A bead of water on an untreated hydrophobic surface has a high contact angle (a), while the same surface following plasma treatment becomes hydrophilic, resulting in a low water contact angle (b).

X-ray photoelectron spectroscopy (XPS) and surface derivatization techniques are used to quantify the percentage of the surface that has been functionalized with the desired chemical groups. For example, the surface polymerization of allylamine will result in amino functionalization. To quantify the primary amine present, reagents selectively tag the primary amine groups with fluorine. Fluorine is used because it is easily detected by XPS and its chemical environment does not have to be distinguished. This is in contrast with the case of nitrogen, where many nitrogen-containing functionalities may coexist. Once the surface fluorine concentration has been determined by means of XPS, the original primary amine concentration can be derived.

Conclusion

The semiconductor industry has been using gas plasma technology in the manufacture of microchips for years. Its plasma processes were known to require a high level of sophistication, and plasma systems were tailored to that industry. More recently, however, plasma technology has expanded into the arena of surface-treating polymeric materials. But despite the merits and enabling capabilities of plasma technology in this new industrial application, the expansion of its use has been slow. One reason for this is that plasma solutions often have been associated with high cost and limited flexibility in terms of integration into the manufacturing process.

Demetrius Chrysostomou, PhD, is director of technology at PVA TePla America Inc. (Corona, CA). He can be reached at demetri@ pvateplaamerica.com.

Today, though, plasma technology companies are value-engineering their equipment in an effort to keep costs down. Not only that, they are also designing products to be highly flexible and versatile. Systems these days are available in batch and in-line configurations, and may operate at low pressures or at atmospheric pressure. They can easily be integrated into existing manufacturing lines, are quite easy to use, and can be operated by relatively low-skilled, and thus less costly, personnel.

Plasma technology is gaining greater recognition within the medical device industry because of its success as a surface cleaning and modification tool and its attractiveness as a dry, environmentally friendly process. It is no longer considered an excessively costly technology for filling surface preparation needs. Rather, it is seen as an effective process that facilitates manufacturing and provides a stepping stone to future technologies.