Langmuir 2003, 19, 5971-5974
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Enzymes as Ultrasensitive Probes for Protein Adsorption in Flow Systems Keith Lenghaus, Jeff W. Dale, J. Caroline Henderson, David C. Henry, Evelina R. Loghin, and James J. Hickman* Department of Bioengineering, Clemson University, Clemson, South Carolina 29634 Received February 19, 2003. In Final Form: May 12, 2003 We have developed an assay system that can quantitatively determine protein adsorption of different materials down to submonolayer surface coverage at low solution concentrations under flow conditions. Understanding and controlling protein adsorption under these conditions will be a key element in the performance of microdevices that interact with proteins and other biomolecules because the surface area to volume ratio of these devices is substantially larger than those of current systems. Alkaline phosphatase and glucose oxidase were evaluated as probes for quantitative protein adsorption, and loss of protein due to adsorption onto capillary surfaces under flow conditions has been determined at fractional surface coverages on a variety of microfluidic materials. It was found that polyether ether ketone (PEEK) and Teflon had high affinity for both enzymes, while unmodified fused silica, fused silica treated with poly(ethylene glycol), and polyacrylamide had much lower affinity for the two enzymes under the conditions tested. Experimental confirmation of laminar diffusion was also demonstrated with this assay system.
Introduction We have developed an assay to determine protein adsorption at submonolayer coverage in microfluidic devices and systems. Because the assay is based on enzymatic activity, it will also be able to address the functional state of the protein (active or denatured and inactive) in future embodiments. The importance of this work is that understanding protein adsorption at low bulk concentrations is likely to be necessary across a wide range of areas; the trend in chemical/biological analysis is toward the handling of ever smaller quantities of analyte at significantly lower concentrations. In addition, the surfaceto-volume ratio increases as device size decreases as epitomized in microfluidic devices or other microelectromechanical systems (MEMS).1 Finely resolved (fractions of a monolayer coverage) protein adsorption isotherms may yield important insights not only for biotechnology devices but also for understanding biocompatibility of biological systems. Many biological systems can respond to minute deviations and changes in protein interactions greatly out of proportion to the magnitude of the change (e.g., the immune response or the cascade of chemical signals leading to thrombogenesis). It is our goal that this approach, using readily available materials and requiring relatively low capital expenditure, will be a useful model system for the initial assessment of system biocompatibility, especially for microfluidic devices. It is well established in the gas adsorption literature that isotherms obtained at low bulk concentrations contain a significant amount of information about the adsorption mechanisms and the sites involved.2 Though not precisely analogous, the adsorption behavior of proteins at low bulk concentrations should also be expected to yield information about the adsorption process and thus the nature of the surface sites. We will extend this idea in this report to show that certain enzymes can be highly sensitive probes for measuring protein adsorption at low coverages. * To whom correspondence should be addressed. (1) Kovacs, G. T. A. Micromachined Transducers Sourcebook; McGrawHill: New York, 1998. (2) Gregg, S. J.; Sing, K. S. W. Adsorption, surface area and porosity; Academic Press: London, 1967.
The sensitivity of enzyme linked assays indicates that the presence or absence of particular enzymes can be ascertained with confidence and reproducibility at exceedingly small amounts of total protein.3-5 Enzymes are catalytic proteins, which makes them ideal candidates to probe the early stages of protein adsorption in terms of coverage as well as whether they are in an active or denatured state. Previous studies have shown that proteins can denature upon adsorption to a surface under static conditions3,4,6-10 and that this process can be concentration dependent.11,12 Furthermore, experiments have been designed for the use of enzyme assays to determine surface coverage and surface conformation in static protein adsorption experiments.4,6-8,10,13-15 However, little has been reported about protein adsorption under flow conditions in microfluidic devices. We have initially developed these assays to study protein adsorption in order to compare and contrast flow and static conditions in microfluidic devices and systems. Our studies on adsorp(3) Butler, J. E.; Navarro, P.; Sun, J. Adsorption-Induced Antigenic Changes and their Significance in ELISA and Immuological Disorders. Immun. Invest. 1997, 26, 39-54. (4) Butler, J. E.; et al. The Immunochemistry of Sandwich ELISAs - VI Greater than 90% of Monoclonal and 75% of Polyclonal antiFluorescyl Capture Antibodies (CAbs) are Denatured by Passive Adsorption. Mol. Immun. 1993, 30, 1165-1175. (5) Butler, J. E. In Methods in Enzymology; Academic Press: New York, 1981; pp 482-523. (6) Norde, W.; Zoungrana, T. Activity and structural stability of adsorbed enzymes. Prog. Biotechnol. 1998, 15, 495-504. (7) Norde, W.; Zoungrana, T. Surface-induced changes in the structure and activity of enzymes physically immobilized at solid/liquid interfaces. Biotechnol. Appl. Biochem. 1998, 28, 133-143. (8) Norde, W.; Giacomelli, C. E. Conformational changes in proteins at interfaces. From solution to the interface, and back. Macromol. Symp. 1999, 145, 125-136. (9) Vermeer, A. W. P.; Bremer, M. G. E. G.; Norde, W. Structural changes of IgG induced by heat treatment and by adsorption onto a hydrophobic Teflon surface studied by circular dichroism spectroscopy. Biochim. Biophys. Acta 1998, 1425, 1-12. (10) Zoungrana, T.; Findenegg, G. H.; Norde, W. Structure, stability, and activity of adsorbed enzymes. J. Colloid Interface Sci. 1997, 190, 437-448. (11) Sandwick, R. K.; Schray, K. J. The Inactivation of Enzymes upon Interaction with a Hydrophobic Latex Surface. J. Colloid Interface Sci. 1987, 115, 130-138. (12) Sandwick, R. K.; Schray, K. J. Conformational States of Enzymes Bound to Surfaces. J. Colloid Interface Sci. 1988, 121, 1-12.
10.1021/la034294o CCC: $25.00 © 2003 American Chemical Society Published on Web 06/18/2003
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tion within flow systems are outlined here. Future papers will examine the static case and denaturing versus desorption. To study protein adsorption under flow conditions at low protein concentrations (30-150 ng/mL), five different capillaries were chosen which span the range of surface properties from a broad commercial utilization or interest. These five materials represent characteristics from hydrophobic to hydrophilic and charged and uncharged systems; they also have been shown to exhibit low (i.e., poly(ethylene glycol) (PEG)16,17 and polyacrylamide) to high (Teflon and silicon18,19) protein adsorption under static conditions. This is the first comprehensive attempt to elucidate initial protein adsorption in a flow environment. Materials and Methods The capillaries studied were polyether ether ketone (PEEK) and Teflon (both from Zeus industries, 65 µm i.d.), as well as fused silica and fused silica coated with poly(ethylene glycol) and polyacrylamide (all from MicrosolvTech, 50 µm i.d.). The capillaries were cut to length (150 mm for the fused silica based capillaries and 100 mm for the Teflon and PEEK capillaries) and glued into a Luer Lock needle with epoxy. The different lengths were due to the significant back pressure observed with the hydrophobic capillaries creating flow problems at the 150 mm length. The capillaries were equilibrated with flowing buffer (500 µL/h) for at least 2 h, and preferably overnight, prior to use. Alkaline phosphatase (AP, calf intestinal) and glucose oxidase (GO, A. niger) were obtained from Sigma. GO was diluted to the working concentration with phosphate-buffered saline (PBS, Fisher). The PBS was purchased from Fisher at 10× concentration and diluted with Milli-Q water as required. Since phosphate is a listed interfering agent for alkaline phosphatase,20 AP was diluted with tris(hydroxymethyl)amine hydrochloride (Tris) buffer (50 mM tris, 138 mM NaCl, 30 mM KCl) prepared with ACS grade or better salts in Milli-Q water. All solutions which contacted AP also contained 50 mM MgCl2 and 0.1 mM ZnCl2 to preserve AP activity.21 The surface activity of Tris was considered to be insignificant, since there was not a substantial reduction in the surface energy (measured with a KSV goniometer by the pendant drop method) of the Tris solution (69.5 mN/m) compared with water (72.8 mN/m). The enzyme activity assay was based on a simple mass balance system. The ratio of the eluant activity to the control activity was taken to be proportional to the enzyme remaining in solution. Thus, the enzyme lost equals the inlet concentration multiplied by the fractional decrease in activity. Working enzyme solutions were prepared immediately prior to use. The syringe was hooked up to a syringe pump (KDS 100), the capillary/Luer Lock combination was attached, and the syringe pump started at a flow of 100 µL/h at room temperature. (13) Okubo, M.; Ahmad, H. Enzymic activity of trypsin adsorbed on temperature-sensitive composite polymer particles. J. Polym. Sci., Part A: Polym. Chem. 1998, 36, 883-888. (14) Butler, J. E. et al. The Physical and Functional Behaviour of Capture Antibodies Adsorbed on Polystyrene. J. Immunol. Methods 1992, 150, 77-90. (15) Butler, L. G. Enzyme immobilization by adsorption on hydrophobic derivatives of cellulose and other hydrophilic materials. Arch. Biochem. Biophys. 1975, 171, 645-50. (16) Halperin, A.; Leckband, D. E. From ship hulls to contact lenses: repression of protein adsorption and the puzzle of PEO. C. R. Acad. Sci., Ser. IV: Phys., Astrophys. 2000, 1, 1171-1178. (17) Lee, J. H.; Lee, H. B.; Andrade, J. D. Blood Compatibility of Poly(ethylene oxide) Surfaces. Prog. Polym. Sci. 1995, 20, 1043-1079. (18) Horbett, T. The Role of Adsorbed Proteins in Animal Cell Adhesion. Colloid Surf., B 1994, 2, 225-240. (19) Brash, J. L.; Lyman, D. J. Adsorption of Plasma Proteins in Solution to Uncharged, Hydrophobic Polymer Surfaces. J. Biomed. Mater. Res. 1969, 3, 175-189. (20) Coburn, S. P.; Mahuren, J. D.; Jain, M.; Zubovic, Y.; Wortsman, J. Alkaline phosphatase (EC 3.1.3.1) in serum is inhibited by physiological concentrations of inorganic phosphate. J. Clin. Endocrinol. Metab. 1998, 83, 3951-7. (21) Fernley, H. N. In The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1971; p 424.
Letters
Figure 1. Alkaline phosphatase adsorption onto five capillaries with different compositions. Enzyme concentration ) 30 ng/ mL, flow rate ) 0.1 mL/h. Monolayer formation (%) vs eluant volume (mL). After being drawn into the syringe (10 mL, polypropylene, BectonDickson), a sample of the enzyme solution was pushed through the capillary into a polypropylene centrifuge tube (1.5 mL, VWR). The enzyme solution was eluted into the tubes which contained a 50/50 wt % buffer/glycerine mixture to preserve the eluted enzyme, and appropriate corrections were subsequently made for dilution effects on the apparent enzyme activity. For each experiment, samples were taken every 15 min for the first 2 h, and every hour after that for 3 additional hours. A fresh control was taken from the syringe stock solution each hour, and the activity of the eluant was measured and compared with the control. Enzymes were incubated for 30 min at 37 °C, with alkaline phosphatase being assayed via p-nitrophenol phosphate substrate (Pierce) and glucose oxidase being assayed via a coupled reaction with glucose, horseradish peroxidase, and o-dianisidine (Worthington Biochem), as described by the respective vendors. Assays were performed in the linear concentration range or diluted as appropriate to reach this range. Surface coverage of the capillary is expressed as monolayer equivalents. Since it is not known exactly in what form the protein adsorbs on the surface, it is necessary to assume a size for calculation of the surface coverage. For alkaline phosphatase, the protein was treated as a sphere with a diameter of 7 nm, based on its specific volume of 0.756 mL/g, and a molecular weight of 140 000 g/mol.22 Glucose oxidase was treated as a cylinder with dimensions of 6 nm × 7 nm × 8 nm and a molecular weight of 160 000 g/mol.23 The area occupied by alkaline phosphatase on a surface is then assumed to be ∼40 nm2 and for glucose oxidase ∼56 nm2; however these dimensions are considered to be approximations only.
Results Figure 1 shows the calculated monolayer coverage data obtained for alkaline phosphatase passed through five different capillary materials. Teflon and PEEK show significant adsorption of enzyme, while PEG and polyacrylamide do not. Unexpectedly, fused silica did not appreciably adsorb any enzyme. Glass is considered to be a protein unfriendly material, and it would be expected that fused silica would behave somewhat similarly,18 but this was not observed with the enzymes under the experimental conditions employed. However, overall the assay system with AP shows appreciable dynamic range at
