Each year, billions of multi-well plates, pipettes, bottles, flasks, vials, Eppendorf tubes, culture plates, and other polymer labware items are manufactured for use in research, drug discovery, and diagnostic testing.
Although many are simple, 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/or proliferation of antibodies, proteins, cells, and tissue; as well as improved signal-to-noise ratio so testing is more precise with less target material or markers required.
For some labware manufacturers, altering the properties of these devices can also make sense from a business perspective. In a market dominated by several large labware manufacturers, more specialized offerings can create a competitive edge and drive up the value of each consumable. For those creating next generation medical diagnostic devices, coated or plasma treated labware optimized for the testing can improve the quality, specificity, and efficacy of the results as well.
“With polystyrene or polypropylene labware, if you can add a functional coating or use plasma to alter the surface properties you can turn a $2 item into a $50 item,” says Mic Barden of PVA TePla America, a leading system engineering firm that designs plasma systems for surface activation, functionalization, coating, ultra-fine cleaning, and etching.
To be sure, some surface modification of plastic labware using plasma is already pursued by the largest labware manufacturers.
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 labware 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 1,536 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 bio assays
A common usage for microtiter plates is for bio assays 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.
To improve the bond and function of the antibody, plasma coatings can be applied to orient the Y-shaped IgG proteins utilized in the majority of these types of tests. Failure to do so can mean some antibodies face down or deform and become essentially unavailable for bonding with antigens.
“With most uncoated polymer surfaces you can’t control how the Y-shaped ‘capture’ antibodies are oriented,” says Barden. “However, a functional coating can be used to favor the proper upward orientation so the entire surface is available for the assay. In this way, we can improve the signal-to-noise and dynamic range of an assay.”
For this, applying an amine coating is a popular solution.
“Amine coatings are commonly used because they have a middle surface energy, with water contact angles of approximately 60 degrees,” says Barden. “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.
According to Barden, all these approaches provide a similar surface energy, but have functional differences that may be important, depending on the application.
Surface modification of labware for cell, tissue cultures
The enormous growth in studies of whole live cells has led to an entirely new range of microplate products which are cell and tissue culture treated for this work.
According to Barden, the issues of adhesion that apply to proteins used for ELISA can also apply to cells and tissue cultures.
Pipettes are another common laboratory tool. Often constructed of high-density polyethylene or polypropylene that 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 pipette tips “sheet” off any aqueous solution more effectively, companies like PVA TePla can utilize nanotechnology to create a superhydrophobic surface.
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 affect.”
Another method involves applying a more hydrophobic coating on the inside and out of the pipette tip.
PVA has already designed special trays and fixtures capable of treating entire racks for 96 and 384 well microtiter plates. The process utilizes “pulses” of plasma that activate a specific monomer, causing it to diffuse and polymerize within the pipette tip.
Since plastic labware is 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 (100-500nm), highly conformal, non-crystalline, and highly flexible (180o 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.
According to Barden, plasma treatment is common 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 labware manufacturers to visit its lab in Corona, CA, 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,” says Barden. “Access to this knowledge base facilitates new entrants into the market.”