Coating processes – Medical or dental purpose product; parts; subcombinations;...
Reexamination Certificate
2001-10-10
2004-03-23
Beck, Shrive P. (Department: 1762)
Coating processes
Medical or dental purpose product; parts; subcombinations;...
C427S002110, C427S002130, C427S002300, C427S155000, C427S384000
Reexamination Certificate
active
06709692
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for reducing the adsorption of organic materials (e.g., peptides, proteins, nucleic acids, and cells) onto hydrophobic or hydrophilic surfaces (e.g., polymeric surfaces). The invention also relates to devices, vessels and apparatus (e.g., microtiter plates, microfluidic channels and kits) having been treated by such methods and methods of performing fluid operations therein.
BACKGROUND
Biological materials such as peptides, proteins, nucleic acids, and cells are often stored, transferred or reacted in devices and apparatus such as multiwell plates, microcentrifuge tubes and pipettes made of plastic or other non-polar materials. It is a common observation that biological compounds adsorb/bind to the surfaces of such devices. 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 various 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 WO 94/03544; 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; Schroen et al., Journal of Membrane Science, 80:265-274, 1993; Sheu et al., J. Adhesion Sci. Technol., 6:995-1009, 1992; Shinada et al., Polymer Journal, 15:649-656, 1983; and Thurow et al., Diabetologia, 27:212-218, 1984.
Of particular interest is the treatment of small volume reaction devices that allow for multiple reactions under a variety of conditions. Such advances have been made in microfluidics and microtiter plate technology, therefore, there is a need for methods of treating these devices to decrease contamination, increase reaction yields and save valuable reagents.
Microfluidics involves using microchannels instead of test tubes or microplates to carry out analyses and reactions. These microchannels or microcircuits are etched into silicon, quartz, glass, ceramics or plastic. The size of these channels is of micrometer order, while the reaction volumes are of nanolitre or of microlitre order. The principle is to guide the reaction media, which contain reagents and samples, over zones which correspond to the different steps of the protocol. The integration of reactors, chromatographic columns, capillary electrophoresis systems and miniature detection systems into these microfluidic systems allows the automation of complex protocols by integrating them into one single platform. These “laboratories on chips” have made it possible to obtain results which are efficient in terms of reaction speed, in terms of product economy and in terms of miniaturization which allows the development of portable devices. Remarkable results have also been obtained for the integration and automation of complex protocols, such as biochemical or molecular biology protocols which often require numerous manipulations. These manipulations comprise in particular mixing reagents and samples, controlling the reaction temperature, carrying out thermal cycling and detection. Wolley et al. (Anal. Chem., 68, 4081-4086, 1996), for example, described the integration of a PCR microreactor, a capillary electrophoresis system and a detector in a single device. A device on a chip which allows the integration of a step for mixing the reagents and an enzymatic reaction has been described by Hadd et al. (Anal. Chem., 69, 3407-3412, 1997). This device provides a microcircuit of channels and reservoirs etched into a glass substrate, and moving and mixing of the fluids takes place by electrokinetics. Numerous microfluidic systems for the integration of protocols and of analyses have thus been described in particular in international patent application WO 98/45481, the disclosure of which is incorporated herein by reference.
One of the great difficulties in implementing these devices resides in the high adsorption of samples and reagents to the channel surface during the movement of the fluids through the channel. In general, microfluidic devices contain channels in a micrometer size-range that have large surface-to-volume ratios (~10-100 times greater than a surface-to-volume ratio in conventional microtiter plates). This leads to an increased significance of the surface properties/quality/chemistry in microfluidic devices. At the same time, biological samples (e.g., protein samples, or reaction mixtures such as PCR mix, LCR mix, microsequencing (MIS) mix, etc.) are complex mixtures of large and small molecules of different polarities (e.g., DNA and protein molecules, dNTPs, ddNTPs, fluorescent labels, etc.) that may have a strong affinity to solid substrates, as well as for liquid/liquid and liquid/air interfaces. Proteins in particular are known to adsorb strongly to silica materials (Righetti, P. G., ed., 1996,
Capillary Electrophoresis in Analytical Biotechnology
, CRC series in Analytical Biotechnology, CRC Press, Boca Raton). For these reasons, surfaces in microfluidic devices are deactivated prior performing biological reactions in such microstructures. Deactivation of a surface reduces adsorption of organic materials onto the surface. Without deactivation of surfaces, biological reactions generally cannot be performed in silicon (silica) microchannels [Shoffner, M. A. et al., Nucleic Acids Res. 24: 375-379 (1996) and Cheng, J. et al., Nucleic Acids Res. 24: 380-385 (1996)].
Several possibilities for surface deactivation have been shown. Generally, the deactivation strategy depends on the material from which a microfluidic device is made. For example, silica surfaces (silicon chips) can be chemically functionalized, e.g., through silanisation reactions (Snyder, L. R., Kirkland, J. J., Introduction to modern chromatography, Wiley-Interscience, 1979, New York; Shoffner, M. A. et al., Nucleic Acids Res 24: 375-379 (1996); and Kopp, M. et al., Science, 280: 1046-1048 (1998)). The silanized chips can directly be used for biological reactions or may further be modified by preparation of a polymer coating layer on the surface.
Surface deactivation using coatings involves two approaches: covalent and non-covalent coatings. The stability of covalent coatings, some of which are referred to as polymer brushes, and adsorbed polymer layers depends on three factors: (a) the chemical stability of the surface, (b) the stability of the polymer-surface interaction, and (c) the chemical stability of the polymer that is used for a surface modification. Generally, with respect to the stability of a single polymer/surface interaction (a), a covalent bond (covalent coatings) is more stable than a non-covalent polymer/surface interaction (a fraction of a k
B
T unit, where k
B
is the Boltzman constant and T is temperature) in adsorbed polymer layers. However, due to a large number of segments (sometimes more than 10% of the total number of
Beck Shrive P.
Genset S.A.
Kolb Michener Jennifer
Saliwanchik Lloyd & Saliwanchik
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