Gas Plasma Surface Chemistry for Biological Assays Protocol

Introduction of Gas Plasma Surface Chemistry

Gas plasma processes are utilized on a variety of materials for the purpose of engineering a working surface. A well-structured surface chemistry enables attachment of many biologically relevant molecules such as: antibodies, antigens, proteins, gels, and other macromolecules. The plasma based surface modification may be performed on a wide range of materials from metals to glass to polymers.

Plasma surface modification of polymers is a growing field. It is important to note that many of the first plasma systems entered into service for the purpose of surface etching. Surface etching generally refers to a method of removing or cutting into a surface. Such plasma technologies found wide adoption within the semiconductor industry for their nanoscale precision and uniformity. However these plasma etching tools were not especially suited for addition of surface chemistry on three-dimensional (3D) and thermally sensitive materials such as polymers. Thus the development of equipment configurations to allow for treatment of materials in a variety of forms from powders to extruded tubing to large rolls.

Figure 1. Plasma system chamber configurations.

These equipment and process advances have contributed to commercially scalable and reproducible stable surface modifications. Gas plasma technologies are increasingly innovating surface engineering through automation and process efficiencies.

Charged particles and disassociated chemical bonds make up gas plasma. These particles may become ionized with heat or a strong electromagnetic field. Sufficient energy must be applied to a system in order to produce plasma. The plasma may be generated under atmospheric pressure or under partial vacuum pressure. A few distinct advantages of an atmospheric plasma system are in-line processing, rapid treatment speeds, and compact line-of-sight automation. A system for producing plasma under partial vacuum pressure typically involves batch treatment within a vessel; however continuous treatments of transported profiles do exist. The distinct advantages of partial vacuum plasma are 3D surface treatments, environmental control, chemical versatility, and the modification of porous structure. Notably, a partial vacuum process also enables lower temperature plasma. Such plasma systems are able to produce tailored surface chemistry onto thermally sensitive materials. Additionally these methods provide atomic-level cleaning, stable surface wetting and deposition of thin organic films.

Plasma treatment technologies modify surfaces without affecting the bulk material properties ( see Note 1). These methods provide inexpensive and controlled alternatives to custom material formulation and to traditional wet chemistry processing. In many cases low cost or commodity lab ware surfaces may be reengineered into high-performance substrates for cell culture, selective filtration, and discovery.

Partial pressure plasma reactors offer the user a versatile tool for creating tailored working surfaces. Tools for design include the equipment, chemistry and control of the variables described herein. Since practiced in a controlled reduced pressure environment, once a process has been validated, and materials specified, the operator can expect reproducibility. While plasma processing has grown into a distinct field of practice, the user can evaluate and prepare research and production tools simply by evaluation of and consideration of the variables discussed in this chapter.

Methods of Gas Plasma Surface Chemistry

Plasma is referred to as the fourth state of matter. It is comprised of electrons, ions, and other excited meta-stables ( see Fig. 3). The color of the plasma is distinct for every gas ( see Note 6). The signature wavelengths of light are produced when excited electrons return back into a lower energy state. The spectra of light emissions will extend past the visible region into the UV and far UV. UV emissions can contribute to the surface modifications. This requires that chamber view ports are equipped with a UV filter. Substrates placed within the glow region are referred to as being within the “Primary” plasma. “Secondary” plasma refers to dark space regions outside the glow. The terms “downstream” or “remote” also refer to secondary plasma. The secondary plasma contains lower concentrations of charged species. The authors primarily practice surface modification within the primary plasma where there is a greater reactivity.

During the plasma process, energetic particles commence to cleave or form complexes with chemical bonds near the substrate surface. Unstable bonds and free radicals are produced on the substrate. They eventually recombine with molecular fragments or other charged moieties moving through the plasma. The dominant reaction on most polymer substrates occurs through hydrogen substitution along a polymer’s aliphatic linkages. In the presence of oxygen, many hydrogen bonds are abstracted from the carbon backbone and then substituted by oxygen to form polar compounds. Different chemical functionalities are produced by regulating the concentration of elements present within the plasma. Under these conditions the substrate material will form new covalent bonds and thereby the surface chemistry becomes reengineered. The chemical reactions generated by the plasma are generally confined to the substrate surface. The bulk properties are rarely impacted.

RF and microwaves sources efficiently generate plasma under partial vacuum pressure. Low gas pressures increase the mean free path allowing particles to accelerate with greater ease within the electromagnetic field. Collisions become less frequent translating to less heat generation from friction. Eventually particles do collide. There are both elastic and inelastic collisions. In an elastic collision the particle rebounds without losing or giving up its energy. It then continues to travel until its next collision. In an inelastic collision the particle gives up its energy and returns to its ground state. Within the primary plasma there is a continuous field for particles to accelerate within. This enables treatment of porous and microporous media such as filters, membranes, sintered parts, non-wovens, and foams. Reactive plasma species sustain enough energy to modify interstitial surfaces.

Plasma Process Design

There exists a misconception that plasma processing provides a standard or universal result. The outcome of the plasma process will actually vary as a function of substrate material, chemistry selection, and many other factors related to the plasma process and equipment design. The following section identifies a methodology for designing, testing, and scaling a plasma process which produces a set of objectives.

1. Problem definition: Identify the project goals with a detailed account of the desired surface properties. Surface chemistry alone may not address all the material challenges. Consider other factors which may influence your result: surface topography, environment, processing aids, packaging, and sterilization. It may be possible to simultaneously address multiple surface property needs using a plasma process ( see Notes 1 and 2)
2. Equipment selection: There are many options and system con- figurations which should be reviewed early in a project development. Note that a plasma process does not inherently scale or transfer from one equipment format to another. As an example, the research done in a small benchtop tool may not necessarily achieve an identical response in a reel-to-reel or larger batch system. The electrode size, spacing, orientation, and power source selection, influence the density and composition produced by the plasma. When possible utilize an equipment configuration that most closely matches the final size or desired equipment format. Realistically many opt for development or proof of concept in a small scale plasma system. In this scenario keep a detailed record of the equipment used and coordinate project milestones with equipment transfer.
3. Surface preparation: Plasma interactions described herein predominantly occur on the sub-micron or atomic scales. Physical debris and contaminants that migrate to the surface may compromise the outcome of a surface modification. Remove undesirable species from the surface using appropriate cleaning methods. Only use solvents or detergents that are compatible with the material system. Additionally the surface should be properly rinsed of residual cleaning agents (see Note 1.)
4. Plasma cleaning: A short plasma cleaning step will help to ensure that a substrate surface becomes atomically clean. Organic oligomers and low molecular weight molecules are quickly deconstructed and removed by oxygen plasma. The recommended times for a plasma cleaning step vary, from a few seconds to a few minutes. Different gases, or combination of gases, may be selected to create desired plasma energies for ablating species. For example, a mixture of oxygen and tetrafluoromethane produces more aggressive plasma. With judicious selection of conditions, expect no observable change to the substrate resulting from the plasma cleaning. Over treatment of polymeric materials could result in damage to the part. Surface ashing or hazing may be mitigated by reducing the power and/or treatment time. Furthermore a poor adhesion or a poor treatment stability could also be an indication of over treatment.
5. Surface chemistry: Plasma surface activation provides permanent and covalent attachment of surface chemistry. Chemical species are selectable by the gas and the vapor chemistry introduced into the plasma, resulting in unique functional species and further described below.
   1. Hydroxyl chemistry: Gas plasma containing oxygen gas or alcohol vapor such methanol produce a sufficient density of surface hydroxyl groups. Hydroxyl groups link biological molecules through simple dehydration reactions. These also impart surface polarity which enhances substrate wettability. The simplicity of hydroxylation makes it one of the most common plasma modifications.
   2. Carboxyl chemistry: Gas plasma containing, carbon dioxide gas, acetic acid vapor, or acrylic acid vapor is used to produce carboxyl groups. The carboxylic acid functionality is an integral part of amino acids and it is especially useful in biomolecule capture. Under high pH the carboxylic acid will disassociate to form a carboxylate ion. Carboxyl surfaces are hydrophilic, are negatively charged, and are capable of nonspecific binding.
   3. Amine chemistry: Gas plasma containing ammonia gas, diamine vapor, or allylamine vapor produce primary, secondary, and tertiary amine. Primary amines donate hydrogen atoms to form hydrogen bonds. Amines are commonly employed in the capture, immobilization, and retention of biological molecules such as proteins, lipids, and antibodies.
   4. Custom chemistry: Plasma processing enables many other possibilities in surface chemistry. Judicious selection of the gas and vapor species enables an operator to design and customize their surface chemistry to include deposition of thin film coatings, by the process known as Plasma Enhanced Chemical Vapor Deposition (PECVD). Example PECVD surfaces include hydrophobic and low retention coatings that may be used for dispensing components. The plasma contains many reactive and charged moieties that can change and alter the chemistries being input as gas or vapor. Ultimately the surface will form a distribution of chemical groups.

Process Development

The primary variables to study in a plasma process development program are: chemistry, gas and/or liquid vapor flow (resulting in specific pressures), plasma power, and process time. Keep in mind that polymers are complex and may express different affinities or susceptibilities to various plasma gases or plasma compositions (see Note 1). Therefore an implementation that worked with one material system will not inherently work with all material systems. A basic methodology for determining a successful plasma condition would be to screen plasma chemistry and then to optimize around a desirable surface response. A review of considerations in designing a process follows.

1. Chemistry screening: Process development begins with a screening of different plasma gas and vapor chemistries with the objective of determining which plasma chemistries provide the greatest improvement in surface properties. Process chemistries which exhibit the most notable improvements become process candidates for further development and optimization. For example, a development in designing surface amine might compare the effectiveness of the following different plasma compositions: ammonia, argon with allylamine, and argon with aminopropyltrimethoxysilane. All of these processes are expected to deliver amine to the surface however the chemistry screening will determine which plasma chemistries best modify the substrate for the particular application. The ammonia process might yield a sufficient density of amine but that amine might be difficult to access because it resides as a pendant side group close to the polymer back bone. The allylamine on the other hand may form short chains or even a branched structure influencing steric hindrance. The plasma chemistry screening practically tests the substrates susceptibility to plasma and provides insights into unique surface properties traits that might otherwise remain undefined.
2. Process optimization: The density of surface chemistry, the coating thickness, network structure and hydrophobic effect are some of the properties that may be fine-tuned during process optimization. Process improvements are made by investigating variations in the plasma parameters. For example, once the chemistry has been identified in the screening phase, a Box- Behnken design of experiment provides an effective architecture for experimental design. The primary variables affecting the process design at this stage are power, time and gas ratio. The plasma power and process time will usually impact the density of surface chemistry and stability of the treatment. If the plasma power becomes too intense then the gas species fragment and lead to performance loss. At low power the density or concentration of surface functionality might be too low and this would also manifest in decreased performance. In addition to the primary variables noted, pulsed power plasma and/or modified duty cycles may be employed for control of branching, fragmentation, surface roughness and temperature management. In one example of process development, an argon and acrylic acid plasma produced a functional density of carboxyl groups on a porous membrane. Although suitable surface conjugation was demonstrated during the screening study the treatment increased hydrophobicity of the membrane resulting in poor membrane flux. To optimize the process oxygen was added into the plasma. This improved the wettability of the surface treatment. An oxygen flow was determined that improved membrane flux. This did not negatively impact the effectiveness of the carboxyl species.
3. Gas or vapor flow: The concentration and composition of the plasma chemistry affects the generation of surface chemistry. Flow management systems will regulate and ideally monitor flow into the plasma chamber. Vapor chemistries are typically accompanied by a co-process gas. They will assist in sustaining a uniform glow. Inert gases such as argon and helium make good candidates for co-process gas because they do not form chemical bonds with the primary gas chemistry. Oxygen containing compounds will have a tendency to oxidize other species within the chamber. This may be undesirable in an example where the projected amine would be converted to an undesired nitrogen oxide. The gas flow into chamber volumes less than 5 cubic feet generally does not exceed one liter per minute. Liquid flows in vapor delivery systems do not generally exceed 20 ml/h.
4. Chamber pressure: The process pressure affects the process temperature and the plasma density. The process pressure usually results from the flow of species into the reactor chamber. Regulating pressure independent of flow is not necessary, however some process chemists favor controlling the chamber pressure by adjusting pump impedance at the vacuum port using a throttle valve ( see Note 5.) Higher pressure plasma processes generally contain more thermal energy whereas lower pressure plasma contains more energetic species.
5. Continuous vs. pulsed power: Plasma power is one of the most notable influences on the surface modification. Some plasma generators come with the ability to pulse the power and to employ a duty cycle. Pulse and duty cycle enable an operator to reduce plasma temperature. This is especially critical in the modification of thermally sensitive materials. Plasma temperatures may also introduce surface stress which could negatively impacts treatment stability in some material systems. Pulse and duty cycle are valuable tools in plasma film formation such as the deposition of ultra-thin coatings. This works by regulating kinetic events within the polymerization reaction. The power cycles have the ability to influence coating thickness, coating chemistry, and coating density.
6. Interstitial modification: Partial vacuum plasma processing is advantageous for modifying the interstices of porous media, in addition to the visible outer surfaces. This technique is ideal for changing the wicking and binding properties of sintered, woven, non-woven, and membrane materials that are commonly used for filtration. As described herein, the equipment used creates primary plasma. In primary plasma, there is a greater mean free path of the particles before a collision. This sustained energy is ideal for modifying the interstices of porous media, or for use inside complex nano-scale vias or channels. With atmospheric processes, on the other hand, the mean free path is short when the species are out of the direct energy field, thus the treatment area is limited. The first step of the process, where modification in either the inner dimensions or interstices is required, requires conditions to ensure that the polymer is out gassed. In all plasma processes, the reaction occurs between the plasma and surface. Removing barriers from the surface is critical for a successful modification. During an out gassing step, extended time for the pump down as well as a lower based pressure is important ( see Note 4). Following the pump down, the plasma may be introduced continuously or via multiple step process. An example of the wicking and wetting effect of a high density polyethylene, following an oxygen plasma process. With judicious selection of plasma processing parameters, surface stability may result in years of storage prior to use.

Plasma Treatment in Partial Vacuum System

Once a study has been outlined, the steps to partial vacuum processing are simply listed as follows:

1. The components, to be treated, are placed in the Reactor Chamber, which is vacated, via a pumping system, to a base pressure that may range from 25 to 100 mtorr ( see Note 4). During this initial processing step, the isolation valve is in the open position.
2. Process gas(es) and/or liquid vapors are introduced into the chamber at a desired flow rate to attain a specific pressure ( see Note 3).
3. RF energy (as used in the Plasma Science, PTS and Plasmatreat partial vacuum systems) is supplied to electrodes within the chamber and excites the vapors/gas (es) into a plasma ( see Note 3).
4. During processing, pressure is continuously monitored as an indicator of chemistry uniformity ( see Notes 6–8).
5. While under vacuum a sequence of multiple steps may be conducted as part of a single recipe. Between each step and after the last step the chamber is pumped down to the base pressure to evacuate gases from the previous step ( see Notes 4 and 8).
6. Following completion of the last step, there is a venting cycle in either room air or a neutral gas such as nitrogen, to bring the chamber to atmosphere. The venting speed is controlled to minimize disruption to fragile components, if required.
7. The parts are removed and packaged or transferred to a subsequent process ( see Note 2).

Evaluation of Plasma Treated Surface

Designing a validation method is as important as designing the plasma process. It is critical to validate the surface with a test that is representative and correlates to your application. A few popular techniques for quantifying and qualifying the effects of a plasma surface modification are described herein.

1. Surface energy: The alteration to the surface bonds will usually change the surface tension. Contact angle and dyne inks provide a means for rapid examination for surface tensions. Water will generally form lower contact angles on polar surfaces. The contact angles are measured between the substrate and the tangent where the droplet meets the surface. Only use distilled water when making a water contact angle measurement. Low surface wetting angles correlate well with oxygen functionalities. Surface dyne inks come in kits containing numerous fluids with differing surface tension. A dyne fluid must have a lower surface energy in order to wet onto a surface. Otherwise the fluid will bead up or retract. The surface dyne method and the water contact angle provide a facile methodology for probing the plasma treated surface. Note that these methods do not necessarily discriminate chemical functionality. Surface energy probes such as dyne level and water contact angle do not make successful engineering specification. The authors recommend using such surface analysis methods as process quality control tools if results may be validated by functional testing.
2. Chemical analysis: There are numerous analytical tools which are able to determine a surface’s chemical composition. Some examples include X-ray Photoelectron Spectroscopy (XPS), Nuclear Magnetic Resonance (NMR), Time-of-Flight Secondary Ion Mass Spectrometry (TOF SIMS) and Energy Dispersive Spectroscopy (EDS). These and similar methods are useful in attributing surface affects to the fundamental surface chemistry. Unfortunately many of these methods are not readily available due to cost and requirement of trained specialists, and their results do not necessarily predict success. Analytical methods make useful research tools in plasma surface engineering.
3. Markers/Stains: Staining and fluorescent probe methods are especially useful in evaluating the surface. Testing is highly functional and typically relevant to the application. A successful conjugation will show good retention after washing. Evaluate the surface for good selectivity and a low coefficient of variation. Low retention could be an indication of a poor surface chemistry or a problem with surface contamination. A high coefficient of variation could indicate poor chamber uniformity or again a surface contaminant.
4. Stability and Accelerated Aging: Treatment stability should be assessed for every application. Many of the reactive surface chemistries are sensitive to environmental effects. These effects could include moisture, temperature, light, exposure to solvents and oxidation. If the parts need to be stored use a clean packaging material. Most plastic bags contain plasticizer or anti- blocking agents that will confound a plasma surface treatment ( see Notes 1 and 2). The authors use heat sealable forensics bags for short term storage. Polymer substrates are especially prone to surface changes over time. In general the surface treatment will diminish quickly on low durometer elastomers and on materials with low glass transition temperatures. Mobile surfaces lose surface treatment to reorientation more quickly than a glassy surface. Accelerated aging may be used to simulate long term stability. Most polymer systems adhere to the rule of time- temperature superposition allowing thermal analysts to extrapolate time dependent behavior using heat. Large deviations occur from real world behavior when extrapolating too far out or when performing accelerated aging with improper conditions.

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