Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-02-18
2001-11-20
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S002000, C435S007100, C435S041000, C435S183000, C435S325000, C435S243000, C435S283100, C435S289100, C435S307100, C436S547000, C530S300000, C530S350000, C536S022100
Reexamination Certificate
active
06319664
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for reducing the binding of organic materials (e.g., peptides, proteins, nucleic acids, and cells) to hydrophobic surfaces (e.g., polymeric surfaces). The invention also relates to articles of manufacture (e.g., labware) having such low binding surfaces.
BACKGROUND OF THE INVENTION
Biological materials such as peptides, proteins, nucleic acids, and cells are often stored or transferred in containers such as centrifuge tubes and pipettes made of plastic or other hydrophobic materials. It is a common observation that biological compounds adsorb/bind to the surfaces of such containers. This is also true for organic materials which exhibit some hydrophobicity in an aqueous solution, e.g., acridinium compounds, PCBs, etc.
For many applications, such binding is undesirable. For example, the binding results in the loss of valuable materials, such as, enzymes and antibodies, and can result in variations in the dispensing of organic materials, especially when small volumes are involved. The binding of proteins, cells, and platelets to hydrophobic surfaces is also of concern in a variety of blood handling procedures.
As a result of these considerations, extensive efforts have been made to provide methods for reducing the binding of proteins and other organic compounds to hydrophobic surfaces. Examples of the approaches which have been considered can be found in Caldwell et al., U.S. Pat. No. 5,516,703; Ding et al., International Application Publication WO94/03544; Amiji et al.,
Biomaterials,
13:682-692, 1992; J. Andrade, “Principles of Protein Adsorption” in
Surface and Interfacial Aspects of Biomedical Polymers,
J. Andrade, editor, Volume 2, Plenum Press, New York, 1-80, 1985; Lee et al.,
Polymeric Mater. Sci Eng.,
57:613-617, 1987; Lee et al.,
Journal of Biomedical Materials Research,
23:351-368, 1989; Lee et al.,
Biomaterials,
11:455-464, 1990; Lee et al.,
Prog. Polym. Sci.,
20:1043-1079, 1995; Merrill et al.,
ASAIO Journal,
6:60-64, 1983; Okano et al.,
Journal of Biomedical Materials Research,
20:1035-1047, 1986; Okkema et al.,
J. Biomater. Sci. Polymer Edn.,
1:43-62, 1989; Owens et al.,
Journal of Cell Science,
87:667-675, 1987; Rabinow et al.,
J. Biomater. Sci. Polymer Edn.,
6:91-109, 1994; Schroën et al.,
Journal of Membrane Science,
80:265-274, 1993; Sheu et al.,
J. Adhesion Sci. Technol.,
6:995-1009, 1992; Shimada et al.,
Polymer Journal,
15:649-656, 1983; and Thurow et al.,
Diabetologia,
27:212-218, 1984.
The criteria which a successful technique for producing a low binding surface should satisfy include: 1) a sufficiently low level of binding; 2) substantial permanence; 3) ease of use; and 4) low cost. It is the goal of the present invention to provide methods for producing low binding surfaces which satisfy all of these criteria.
SUMMARY OF THE INVENTION
The present invention achieves the above criteria through the combination of specific coating materials and specific process steps, both of which are critical to the success of the technique.
The specific materials employed in the invention are non-ionic surfactants which have a hydrophilic element which can extend into an aqueous solution, e.g., a hydrophilic end group, and have a hydrophilic-lipophilic balance number (HLB number) which is less than or equal to 5. The term “non-ionic surfactant” is used herein in accordance with its classical definition as a molecule containing two structurally dissimilar groups having different solubilities in an aqueous solution. See
Kirk-Othmer Encyclopedia of Chemical Technology,
Third Edition, Volume 22, page 332, John Wiley & Sons, New York, N.Y., 1983.
As demonstrated in the examples presented below, a HLB number less than or equal to 5 has been found critical to achieve a durable low binding surface. Although non-ionic surfactants have been previously considered for use in producing low-binding surfaces (see the references cited above), the criticality of a HLB number less than or equal to 5 has not previously been recognized. As the present invention demonstrates, above this number, protein binding is either not substantially inhibited or is only temporally inhibited, while at or below the number, long term inhibition of protein binding is achieved.
One specific process employed in the invention comprises the steps of applying the non-ionic surfactant to the surface (substrate) in a solvent and then drying the surface (substrate) to remove the solvent and thereby bring the surfactant into direct contact with the surface so as to bind thereto. Preferably, the surface is fully dried. The applying and drying steps must be performed without an intermediate washing step with an organic solvent.
As demonstrated in Examples 7 and 8, the drying step is critical to obtaining a durable low-binding surface. Without this step, the non-ionic surfactant can be removed from the hydrophobic surface by aqueous solutions, thus causing the surface to lose its low-binding properties. Such removal occurs even if a non-ionic surfactant having a HLB number less than or equal to 5 is used. However, once the coating has been dried onto the surface, it becomes effectively permanent and is not substantially removed by contact with an aqueous solution. This is an important advantage of the invention since the low-binding surfaces which the art desires are for use with aqueous solutions.
The avoidance of any washing with an organic solvent prior to the drying step is important in view of the low HLB numbers of the non-ionic surfactants used in the practice of the invention. Those low HLB numbers make the non-ionic surfactant substantially soluble in organic solvents, so that washing with such a solvent will remove essentially all of the surfactant from the surface, thus preventing the surfactant from performing its low-binding function.
Those references which have employed non-ionic surfactants having HLB numbers less than or equal to 5 have not disclosed, suggested, or in any way recognized the criticality of the above process steps. Specifically, the Thurow et al. and Schroën et al. references cited above each use at least one non-ionic surfactant having a HLB number less than 5. In particular, Thurow et al.'s preferred Genapol PF-10 material has a HLB number of less than 5, as does Schroën et al.'s L-92 material. While Thurow et al report that Genapol PF-10 prevents adsorption of insulin to latex particles, Schoën et al. report that L-92 does not prevent adsorption of lipase to a polypropylene membrane. Significantly, neither reference describes drying the non-ionic surfactant onto a hydrophobic surface, and thus neither can produce a low-binding surface which is substantially permanent, as is required for a practical product.
Sheu et al. also use non-ionic surfactants having low HLB numbers (i.e., PLURONIC 121, 122, and 127), but employ a complicated argon glow discharge process to bind these surfactants to a hydrophobic surface, namely, low density polyethylene (LDPE). In certain experiments, they omitted the glow discharge treatment and instead merely applied the surfactants to LDPE and washed with chloroform (see their FIG.
2
). Under these conditions, they reported no reduction in protein binding compared to untreated LDPE (see their page 1006). Given this conclusion, Sheu et al. clearly did not recognize that low HLB surfactants could be successfully used to produce low-binding surfaces without the need for glow discharge treatment, as demonstrated by the present invention.
The process steps of the invention, i.e., applying the surfactant in a solvent and then drying to remove the solvent, are plainly easy to perform. The process is also inexpensive since only very low concentrations of surfactant are needed to achieve a low-binding surface. For example, one pound of surfactant which costs about a dollar (U.S.), can provide a micron thick coating on about 5,000 square feet (465 square meters) of hydrophobic surface. The invention thus satisfies each of the above four criteria for a practical process fo
Bookbinder Dana Craig
Fewkes, Jr. Edward John
Griffin James Arthur
Smith Frances M.
Tennent David L.
Beall Thomas R.
Corning Incorporated
Naff David M.
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