Application of plasma surface treatment technology in medical devices

The technique of applying voltage to a gas to produce a glow discharge, or “plasma,” has become a powerful tool for solving surface pretreatment problems in the medical device industry. Not only can plasma be used for extreme cleaning and disinfection of surfaces, it can also improve the adhesion of biomaterials to in vitro diagnostic platforms and biocompatible coatings to in vivo devices. Indeed, plasma not only activates surfaces to facilitate the fixation of cells or biomolecules, but can also conversely produce smooth surfaces that resist biofouling or are used for metered drug formulation. Plasma can also greatly improve the efficacy of microfluidic devices. Microchannels on clinical diagnostic devices can be made more “wet” to biofluids without affecting their own analytical performance. Plasma is also used in some low-end technical areas, such as improving ink marking on catheters and improving the adhesion of syringe needles to syringe barrels. In addition, because plasma is a dry surface treatment technology, there is no need to deal with waste chemicals, making it a green process with minimal consumables. In this article, we will discuss the capabilities of plasma technology in the in vitro diagnostic platform industry. We will focus on how plasma can control surface energy and modify surface chemistry to improve adhesion to biomaterials. What is the science behind the magical effects of plasma on surfaces?

What is plasma?

Figure 1: Schematic diagram of the four states of matter. The fundamental difference between the plasma state and the gas state is that the plasma state can be electrically conductive. The electrons are freed from the gravitational pull of atoms or molecules and energy can be transferred through collisions between electrons.

Plasma is a state of matter, just like solid, liquid or gas. Sufficient energy is applied to the gas to ionize it into a plasma state. The "active" components of plasma include: ions, electrons, active groups, excited nuclides (metastable state), photons, etc. Controlling and harnessing the performance of these active components after aggregation can be used for various surface treatments, such as nano-level cleaning, activation of surface wettability, chemical grafting, coating deposition, etc.

The high chemical activity of plasmas is used to modify the properties of surfaces without affecting the substrate. The energy carried by these partially ionized gases can actually be controlled to contain very low "heat" energy. This is achieved by coupling the energy to free electrons rather than to heavier ions, which allows the treatment of heat-sensitive polymers such as polyethylene and polypropylene. How is the energy coupled to the gas? In most cases, it is by applying an electric field between two electrodes at low pressure. This is similar to how a fluorescent lamp works, only the light is not emitted. We direct its chemical properties to treat the surface of the material. Plasmas can also be generated at atmospheric pressure. In the past, atmospheric pressure plasmas were too hot to be used as a tool for surface treatment. Recently, improved techniques have made it possible to generate low temperature plasmas at atmospheric pressure, which can be used to treat the most temperature-sensitive polymers.

How do plasmas change the properties of surfaces?

Figure 2: Plasma as a surface treatment tool is mostly generated in a low-pressure vacuum chamber. With the advancement of technology, plasma generation at atmospheric pressure has become popular and is being used more and more. Figure 2a is a desktop low-pressure plasma system from PVA Tepla. This type of system has advanced performance and is very suitable for unit industry and laboratories. Figure 2b is a close-up of PVA Tepla's atmospheric pressure plasma pen. This design safely controls the voltage and current inside the plasma pen body, which can be used for online applications or selective local treatment.

Suppose a solid surface is adsorbed with hydrocarbon contaminants. These contaminants react easily with plasma-generated oxygen. Oxygen attacks the adsorbed hydrocarbons, converting them into CO2 and H2O. Figure 3 is a simple reaction mechanism. For surfaces that are easily oxidized, plasma-generated hydrogen can be used for surface cleaning. Hydrogen can not only convert some of the organic matter on the surface into volatile hydrocarbons, but also reduce the oxidation of metals such as copper, nickel, and silver.

The chemical properties of the plasma are almost entirely dependent on the feed gas. For example, O2, N2, N2O, CO2, etc. can generate oxidative plasmas. These gases are used to make the surface more wettable, or hydrophilic, to polar solutions. This is achieved by plasma-induced covalent oxygen bonds to carbonyl, carboxyl, hydroxyl, etc. functional groups. These polar functional groups can increase the energy of the surface, thereby allowing tissue cells to adhere better or allowing analytes dispensed onto a diagnostic platform to flow more easily through microfluidic channels.

Ar/H2, NH3, etc. can generate reducing plasmas. These gases have been shown to effectively activate fluorocarbons, such as PTFE. Because of its inertness and biocompatibility, PTFE is an ideal material for manufacturing in vivo medical devices. However, these characteristics are disadvantageous factors in processing PTFE, such as the need to adhere to synthetic scaffolds to promote tissue growth on in vivo devices. Reducing plasma can solve these problems by reducing the fluorine concentration across the surface and replacing fluorine atoms with functional groups such as hydroxyl groups. The surface hydroxyl groups can provide anchor points for supporting these synthetic scaffolds.

Some applications require the host material to be etched. Fluorine-containing gases such as NF3, SF6, CF4 are very suitable for etching hydrocarbon polymers, silicon, silicon oxide, silicon nitride and other materials. In addition to the strong chemical effect of plasma, the direct effect also plays an important role. The kinetic particles hitting the surface can remove more surface inert pollutants (such as metal oxides and other inorganic pollutants), and cross-link the polymer at the appropriate location to maintain the effect of plasma treatment.

Polymer coatings can be grown by plasma enhanced chemical vapor deposition (PECVD) process. PECVD works by activating nuclides such as monomers in plasma and inducing them to polymerize on the base surface of the workpiece. PECVD coatings have some properties such as protective layer, anti-sticking, and anti-scratch. In addition, some coatings contain some special functional groups, such as -NH3, -OH, -COOH. These functional groups provide suitable bonding sites for subsequent grafting (such as fixing proteins or sensors for biomaterials), or can improve the binding force of functional group coatings (such as antiprothrombin, lubrication, type IV collagen, etc.). The surface chemistry of the deposited coating is determined by the outer surface at a depth of tens of nanometers.

What can plasma do for IVD platforms?

The applications of plasma in the medical device industry are indeed very extensive. Therefore, this article will focus mainly on the application areas that have been confirmed by our R&D department and are relevant to the medical diagnostic platform industry. In this field, plasma is used to prepare surfaces for downstream processes and to activate surfaces to facilitate the adhesion of biomaterials. The latter is achieved by changing the surface polarity, grafting specific functional groups or polymerizing coatings on the surface. In order to better understand how plasma can adjust the surface to meet the needs of the application, let's look at some important examples.

Microfluidic devices and hydrophilicity

Surface energy is a material property that determines factors such as wettability and susceptibility to biofouling. Generally, materials with high surface energy are hydrophilic and wettable to fluids such as plasma, bacterial cell suspensions, buffers, inks, glues, and various adsorbates and coatings. On the other hand, surfaces with low energy are called hydrophobic and have "non-stick" properties. These "non-stick" surfaces are discussed below.

Typically, microfluidic devices require hydrophilic surfaces to allow for a steady and gentle flow of analytes through microchannels to the detection and processing components. This flow can be achieved by various methods, such as pumping, electroosmosis, heat, or mechanical methods. As with media (see below), microfluidic devices are made of hydrophobic polymers (acrylic, polystyrene, polydimethylsiloxane (PDMS)). A major problem caused by the hydrophobicity of these materials is that bubbles are trapped in the microchannels, inhibiting the flow of the fluid. Even when the channels are treated with alcohol and buffer, bubbles are still a problem. Plasma treatment can oxidize the surfaces of the microchannels, making them hydrophilic and thus preventing the formation of bubbles. Surface charge density during electrodynamic pumping also affects flow rate. Electrodynamic pumping drives fluid through microchannels by the principle of an electrical reaction that converts electrical energy into kinetic energy. Charged surfaces attract oppositely charged particles in the electrolyte. This allows these particles to remain in the fluid and be more easily moved through the channel by electrodynamic pumping. Plasma can effectively promote electrophoretic or electroosmotic flow over charged surfaces.

Figure 3: The reaction mechanism above is a simplified schematic of plasma generated oxygen radicals attacking hydrocarbons adsorbed on the surface. There are many other mechanisms involving different excited states of oxygen, such as free radical states and divalent molecules. Hydrocarbons adsorbed on the surface can be excited by electron collisions in the plasma, providing additional possible reaction pathways.

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Immunoassays, Microarrays, and Tissue Culture Media

Platforms used in clinical diagnostic substrates such as immunoassays, microarrays and cell culture media are mainly made of synthetic polymers. While these materials are industrially attractive for their inertness, mechanical stability and low cost, their surface properties have inherent limitations. In particular, they lack suitable binding sites for the effective attachment of bioactive molecules or cells to their surfaces. Strong and evenly distributed binding sites are an important prerequisite for the fixation of biomaterials and in vitro cell culture. In order to improve the performance of synthetic polymer platforms for cell proliferation and bimolecular adsorption, their surfaces must be modified. Here we discuss the role of plasmas in the surface modification of these analytical devices.

Plasma improves cell growth rate

Tissue culture (cells from animals or plants) requires nutrients, hormones, and other growth factors to grow in vitro, which are naturally provided in the body. Tissue cells adhered to a solid surface and then propagated into a nutrient-rich liquid medium, such as serum (in the case of animal cells). The surface properties of the medium must allow cells to adhere and grow evenly. However, before the surface properties can be adjusted, their contaminants must be removed. Cooling the cell culture platform to remove release agents, volatile hydrocarbons, and other contaminants is also a suitable environment for the use of plasma.

The inherent hydrophobicity of the polymeric materials used to make the culture medium is not conducive to the adhesion of tissue cells. Therefore, a hydrophilic surface is required. Oxidative plasma is used to increase the oxygen functional groups on the surface, thereby increasing their polarity and making them hydrophilic. Hydrophilic surfaces can induce the adsorption of tissue cells. Hydrophilic surfaces adsorb tissue cells and induce them to be adsorbed. When special chemical properties are required, chemical grafting or polymerization of some monomers containing the desired functional groups can be performed. We will discuss this in more detail in the following sections.

Figure 4: The left and center images show untreated polystyrene wells. The left image shows uneven cell adhesion and cell clumping. The center image shows a region with no cell adhesion. The right image shows uniform cell adhesion and proliferation in the plasma treated medium.

Rough surfaces have more surface area, which in theory means more sites for cell binding. Since cells are typically on the order of 10 μm in size, micro-roughening of the surface can significantly improve cell adhesion. Nanoscale surface roughening is not as effective in improving cell adhesion because larger cells cannot utilize the increased nanoscale surface area. However, a real example is that nanoscale roughening can induce drug differentiation and apoptosis. Although the specific reasons are not clear (possible reasons include increasing the number of cell receptors and improving the signaling pathway to the nucleus), this has important implications for the development of improved tissue scaffolds for implantation devices.

The morphology of a surface can be selectively altered in a plasma environment, either by increasing the acceleration of the ions that impact the surface or by chemical etching processes. Ions in a capacitively coupled RF plasma are usually directed toward the substrate in a network of directions. This is determined by the reaction time of the ions and electrons to the change in polarity of the electric field that generates the plasma. Electrons react more quickly, as they are much lighter than ions. Therefore, a substrate placed in the path of the electrons will have a negative charge while waiting for the positive ions to arrive. The positive ions will be accelerated toward the surface due to the electrostatic attraction of the negatively charged surface. By collision, these ions will be able to remove material from the surface. Argon is well suited for this method of micro-roughening surfaces. The energy of the accelerated ions can be controlled by setting the plasma energy and pressure. For example, increasing the pressure by one millitorr significantly reduces the collision energy of the ions (if the collision energy is not completely eliminated), thus removing the roughening effect of the plasma on the surface. Compared to the argon process just mentioned, the oxygen plasma process is much milder, and its mild chemical etching effect can be used to roughen polymer materials at the nanometer level.

In summary, the combined effects of surface cleaning, activation, and micro-roughening with plasma can increase cell adhesion (up to 30% compared to untreated substrates) and lead to more uniform cell distribution.

Improving the adhesion of biomolecules in immunoassay and microarray platforms using plasma

Plasma technology can solve the problem of adhesion of biomaterials to diagnostic substrates. It does this by providing the surface with specific chemical functional groups that allow the biochemical elements to couple into covalent bonds. Carboxyl, hydroxyl and amino groups are important examples of common chemical functional groups that can be easily obtained using plasma processing. For example, in the microarray industry, amino groups can provide bonding sites for direct attachment of nucleosides (DNA or RNA) and oligonucleotides to the working surface. If the spacing between the atoms prevents the binding of these large biomolecules, then primary molecules, sometimes called "bonds", can be used. Bonds provide space for the biomolecules to adsorb on the surface in the appropriate structure. Indeed, the bonded molecules themselves also need to be activated on the surface to help them attach to the substrate. Often, the direct action of oxygen plasma is sufficient to improve the binding of these molecules. However, sometimes specific functional groups are required. For example, some capture agents work well in acidic or basic environments. If the capture agent is bonded through the hydroxyl group, it can provide an acidic environment. In contrast, amino groups can provide an alkaline environment.

There are two basic methods for attaching specific chemical groups to a surface. One method is to deposit a coating containing the desired functional groups by PEC VD, and the other method is to generate a plasma with existing functional groups and allow them to bind to the surface. Although the latter method is simpler, the former has a higher concentration of surface functional groups (10%-20%). Using ammonia as a feed gas can bind -NH3 to the surface. Methanol is used to bind hydroxyl groups, and using methanol and CO2 together can provide carboxyl groups. Unfortunately, the deposition of these functional groups also causes some side reactions that change the primary functional group. For example, ammonia plasma deposits primary amino groups while also depositing quaternary amines, tertiary amines, nitriles, imines, etc. The ratio of these groups varies depending on the plasma system and the parameters used. Nevertheless, this method can provide 2-8% of the desired functional groups.

Sometimes simply providing the right chemical functional groups is not enough. Amino groups can increase the surface energy making it more hydrophilic. Sometimes an overly hydrophilic surface is not desirable, such as in the case of arrays of gel droplets on a microarray platform, because the droplets can wet the surface. This type of wetting results in unsightly droplets. Again, plasma can solve this problem by controlling the surface energy to maintain the droplet morphology, even in the presence of amino groups. When plasma ammoniation of microarray platforms, adding fluorine to the process is a controlled method. Fluorine constrains the platform substrate and increases its hydrophobicity, thus allowing the droplet to maintain its spherical shape. Fortunately, the process does not affect either the primary amine concentration deposited on the surface or the covalent bonding of the gel to the platform.

Figure 6: Plasma surface treatment to increase chemical functional groups: by exposing the surface to a plasma containing specific functional groups (can increase 2-8% of the desired functional groups (e.g., using ammonia plasma to increase amino groups)), or by PECVD using monomers containing the desired functional groups to grow a coating on the surface (can increase 10-29% of the functional groups).

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The shape, size, and structure of immunoassay platforms are subject to frequent changes and modifications. 96- and 384-well plates are the most common substrate types. Plasma treatment renders the wells hydrophilic, thereby facilitating the immobilization of antigens, antibodies, and other bioactive small molecules. One potential problem is the formation of bubbles during fluid dispensing, which can be controlled using plasma. In Figure 7 we compare two wells, (7a) without plasma treatment and (7b) with plasma treatment. Well (7a) contains a trapped bubble. This bubble can cause erroneous readings in the spectrophotometer and may even spill into adjacent wells depending on the space it occupies. Plasma ensures that the analyte in the well is completely wetted, virtually eliminating the possibility of bubble formation.

The above paragraphs explain how hydrophobic cells can trap bubbles in the analyte solution. However, cells that are too hydrophilic can cause the analyte solution to capillary climb onto the platform layer and potentially contaminate adjacent cells. In one such example, we have an immunoassay platform made of a hydrophobic polymer. This polymer provides a number of wells with gold detectors on the bottom. The gold surface needs to be cleaned before the biosensor is deposited, so the platform is exposed to an oxygen plasma. While the plasma does a good job of cleaning the gold surface, it has a negative effect on the sides of the wells, causing the dispensed biosensor solution to capillary climb onto the sides. The challenge for the plasma process engineer is to clean the gold plate while maintaining the hydrophilicity of the well walls. This can be achieved by using a plasma with a mixture of feed gases: on the one hand to remove hydrocarbon contaminants on the gold surface, and on the other hand to make the well walls hydrophobic by adding fluorine groups. Regardless of how difficult the task may be, the process has proven the versatility of plasma surface modification.

Medical devices require "non-stick" performance

Figure 7: Figure 7a shows trapped air bubbles after fluid was dispensed into an untreated well. Hydrophobic well surfaces often trap air. Figure 7b shows a fully wetted well surface during fluid dispensing after plasma treatment.

The concept of a "non-stick" surface is well known in the durable cookware industry. Coating a cooking pot with Teflon® prevents food from sticking to the cooking surface. The application range of "non-stick" has well extended to frying pans. In vivo and in vitro medical devices sometimes require surfaces that prevent protein or cell adhesion in order to improve blood compatibility. For example, the activity of antithrombin can be controlled by coating the surface with a PTFE-like material.

Reducing the surface free energy, which is the energy available on the surface to form chemical bonds, can reduce the adsorption force on the surface. One possible way to do this is to apply a low surface energy coating. Fluorocarbon polymer coatings have Teflon®-like properties and, like Teflon®, are composed of (CFx)n chemical units. Such coatings can be easily attached to various materials by PEC VD. Plasma treatment provides a reliable, biocompatible and green method to reduce the surface energy of materials by polymerizing fluorocarbons on the surface with high controllability. A purifier at the pump outlet can absorb all fluorocarbons at the outlet.

It has been reported that prolonged interaction of DNA with polypropylene PCR plates can cause DNA denaturation. This means that while polypropylene is used to easily store DNA, over time it can reduce the quality and quantity of the stored DNA. Studies have shown that polypropylene plates treated with oxygen plasma can reduce the adsorption of DNA. Oxygen plasma can impart a negative charge to the surface. It is believed that these negative charges repel the silicate backbone of the artificial DNA, thus preventing the DNA from adhering to the surface.

How to verify the effect of plasma?

Figure 8: The left photo shows a drop of water on an untreated hydrophobic surface. The right photo shows the same surface without plasma treatment. After plasma treatment, the surface becomes hydrophilic.

Contact angle measurement is a widely used method to measure the adhesion of a surface. Untreated polymer surfaces have low surface energy and water droplets on such surfaces show high contact angles. This is because the cohesive forces of the water droplet are stronger than the adhesive forces to the surface. The contact angle of a water droplet on a plasma treated surface is very low because the energy of the surface is increased in the form of polar chemical functional groups. This energy is used to bind water molecules, causing the water droplet to spread along the surface. This is a hydrophilic or wettable surface. Therefore a low surface contact angle indicates that the surface is wettable.

X-ray photoelectron spectroscopy (XPS) and surface derivatization techniques are used to determine the percentage of the surface modified with the desired chemical groups. For example, surface polymerization of allylamine can form amino groups. To determine the number of primary amines, the primary amines can be selectively fluorinated with reagents. Fluorine is used because it is easily detected by XPS and its chemical properties are not changed (for example, nitrogen can coexist with nitrogen-containing functional groups). The concentration of surface fluorine detected by XPS can be used to determine the concentration of original primary amines on the surface.

Conclusion

For many years, plasma technology has been used in the semiconductor industry for microchip manufacturing. These processes are known to be very complex, but plasma systems are well suited to this industry. More recently, plasma technology has been extended to polymer materials. Despite the advantages and operability of plasma technology in this area, this application area has been slow to expand. One reason is that the cost of conventional plasma methods is high and the flexibility of the production process is limited. Today's plasma companies require engineers to not only minimize the cost of their products, but also increase the flexibility and versatility of their products. Today's systems are available in batch and in-line configurations, as well as low-pressure or atmospheric pressure systems. They are easily integrated into existing production lines, are very easy to use, and require very low labor costs to operate.

Plasma technology has gained high acclaim in the medical device field because it can clean and modify surfaces very well, and it is actually a dry, green process. It is no longer considered a "witchcraft" or an expensive option that requires surface pretreatment. This efficient process makes manufacturing easier and lays the foundation for future technologies.