Each year, billions of multi-well plates, pipettes, bottles, flasks, vials, Eppendorf tubes, culture plates, and other polymer items are manufactured for use in research, drug discovery, and diagnostic testing.
Although many are simple and inexpensive consumables, an increasing percentage are now being surface treated using gas plasma or have functional coatings specifically designed to improve the quality of research and increase the sophistication of diagnostics.
Among the goals of surface modification is improved adhesion and proliferation of antibodies, proteins, cells, and tissue. Surface modification also improves signal-to-noise ratio, which enables more precise testing while requiring less target material or markers.
For some manufacturers, altering the properties of these devices can also make sense from a business perspective. More specialized offerings can create a competitive edge and increase the value of each consumable. For those creating next-generation medical diagnostic devices, coated or plasma treated devices optimized for the testing can improve the quality, specificity, and efficacy of the results as well.
Plasma is a state of matter, like a solid, liquid, or gas. When enough energy is added to a gas, it becomes ionized into a plasma state. The collective properties of these active ingredients can be controlled to clean, activate, chemically graft, and deposit a wide range of chemistries.
However, most of the applications of plasma for plastic components used in diagnostics can be categorized as simple treatments, such as O2 or argon for cleaning the substrate at the molecular level. The use of plasma is also well established for surface conditioning to make polymers more hydrophobic (water repellent) or hydrophilic (affinity to water).
However, in vitro diagnostic substrates may require more selective chemistries for the selective adhesion promotion and conjugation of bio active molecules. This can be achieved by providing particular chemical functionality at the surface, allowing covalent coupling of biochemical species to occur. Amino, carboxylic, hydroxyl, and epoxy functionalities are important examples of the chemistries that are readily obtainable using a gas plasma surface treatment.
Multi-well, or microtiter, plates are a standard tool in analytical research and clinical diagnostic testing laboratories. Most plates come with 96, 384, or 1536 sample wells that function as small test tubes.
The most common material used to manufacture microtiter plates is polystyrene because it is biologically inert, has excellent optical clarity, and is tough enough to withstand daily use. Most disposable cell culture dishes and plates are also made of polystyrene. Other polymers such as polypropylene and polycarbonate are also used for applications that must withstand a broad range of temperatures such as for polymerase chain reaction (PCR) for DNA amplification.
Untreated synthetic polymers, however, are extremely hydrophobic and so provide inadequate binding sites for cells to anchor effectively to their surfaces. To improve biomolecule attachment, survivability and proliferation, they must be surface modified using plasma to become more hydrophilic. Microtiter plates, for example, can be modified with hydroxyl, carboxyl, or amine groups to render them hydrophilic (or wettable) and to introduce a negative or positive charge.
Treating the surface in this manner has many benefits, including improved analyte wetting of wells; greater proliferation of cells without clumping; reduced amount of serum, urine, or reagents required for testing; and lower risk of overflow and cross-well contamination.
Improved Antibody Adhesion for Bioassays
Microtiter plates are commonly used for bioassays such as the enzyme-linked immunosorbent assay (ELISA) used broadly for diagnostic testing. ELISA is used to detect the presences of a substance, usually an antigen, in a liquid sample.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a microtiter plate via adsorption to the surface or via capture by another antibody specific to the same antigen. After the antigen is immobilized, the detection antibody is covalently linked to an enzyme or can be detected by a secondary antibody that is linked to an enzyme through bioconjugation. In a final step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.
To improve the bond and function of the antibody, plasma coatings can be applied to orient the Y-shaped IgG proteins that are used in the majority of these types of tests. Failure to do so can mean that some antibodies face down or deform and become essentially unavailable for bonding with antigens.
For most uncoated polymer surfaces, the orientation of the Y-shaped captured antibodies can't be controlled. However, a functional coating can be used to favor the proper upward orientation so that the entire surface is available for the assay. This can improve the signal-to-noise ratio and dynamic range of an assay.
For this application, amine coatings are commonly used because they have a middle surface energy, with water contact angles of approximately 60 degrees. So, the coating is hydrophilic enough that the liquid disperses well and hydrophobic enough to facilitate bonding of the material. Other alternatives including putting down a linker molecule such as an epoxide or carboxylic acid, or applying a quartz-like surface using plasma enhanced chemical vapor deposition. All of these approaches provide a similar surface energy, but have functional differences that may be important, depending on the application.
Surface Modification for Cell and Tissue Cultures
The enormous growth in studies of whole live cells has led to an entirely new range of microplate products. These microplates are cell- and tissue-culture treated for this work.
It is important to note that the issues of adhesion that apply to proteins used for ELISA can also apply to cells and tissue cultures.
Often constructed of high-density polyethylene or polypropylene, which tends to be hydrophobic, pipettes can still have difficulties with liquids sticking to the surface — particularly on, or around, the tip. To address this issue, some pipette manufacturers add fluorinated polymers within the polypropylene during the injection molding process. However, there can still be issues, such as phase separation or leaching.
To ensure that pipette tips sheet off any aqueous solution more effectively, a superhydrophobic surface can be created using nanotechnology. One such technique involves etching the surface to roughen it such that air, nitrogen, oxygen, and other gases are trapped in the recesses, allowing the liquid to float on the top in a lotus effect.
Another method involves applying a more hydrophobic coating on the inside and outside of the pipette tip. PVA TePla, for example, has designed special trays and fixtures capable of treating entire racks for 96 and 384 well microtiter plates. The process uses pulses of plasma that activate a specific monomer, causing it to diffuse and polymerize within the pipette tip.
Since plastic components are susceptible to leaching from plasticizers, stabilizers, and polymerization residues, plasma is sometimes also used to coat the inside of the containers with a quartz-like barrier material. These flexible quartz-like coatings are polymerized onto the plastic by plasma-enhanced chemical vapor deposition. The resulting coating can be very thin (100-500 cnm), highly conformal, noncrystalline, and highly flexible (180° ASTM D522) coating.
Markets for this barrier coating include drug discovery, drug delivery, biological storage, stem cell, and IVF culture wear. In addition to the barrier properties of this coating, SiO2 is also chemically resistant to solvents, making it ideal for use in the analytical wear.
Plasma treatment is now commonly used enough that leading equipment providers are able to modify existing, mature tools and technology, complete with fixturing, to deliver what are essentially drop-in solutions.
Some providers even provide access to on-site research and development equipment as well as engineering expertise. PVA TePla, for example, often invites manufacturers to visit its lab to run parts and conduct experiments on in-house equipment, with full customer involvement.
It is during these technical customer/supplier meetings that many of the best experimental matrices and ideas are produced. The elegance of these plasma treatment solutions is that they leverage existing technology and know-how, as opposed to creating something that is completely new. Access to this knowledge base facilitates new product entrants into the market.