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Evaluation of several methods to quantify immobilized proteins on gold and silica surfaces
Colloids and Surfaces B: Biointerfaces 10 (1998) 273–279
Evaluation of several methods to quantify immobilized proteins on gold and silica surfaces Matthias Orschel, Andreas Katerkamp *, Markus Meusel, Karl Cammann Institut fu¨r Chemosensorik und Biosensorik, Mu¨nster, Mendel str. 7, 48149 Mu¨nster, Germany Received 23 September 1997; accepted 5 January 1998
Abstract In the present study, the application of several methods for the determination of an immobilized protein are presented and the results discussed. Streptavidin was bound covalently on gold and silica surfaces. A radio assay was used as a reference method for the quantification of the immobilized protein. Two photometric, two fluorescencespectroscopic methods and an immunochemical approach, based on a sandwich-ELISA (enzyme linked immunosorbent assay) format were applied with regard to their feasibility for sensitive protein quantification. The photometric methods were not sensitive enough, but one of the fluorescence based methods and the ELISA could be applied for the detection of low protein concentrations on the surfaces. © 1998 Elsevier Science B.V. Keywords: Biotin; Gold surface; Immobilization proteins; Protein quantification; Silica surface; Streptavidin
1. Introduction Immobilized proteins are used in many biochemical applications and often the amount of proteins attached to the surface must be determined. Several methods have been developed for this purpose. These include the measurement of the difference between added protein and protein recovered after immobilization [1], amino acid analysis after acid hydrolysis [2] or analysis of elements such as nitrogen and sulfur [3,4]. All these methods require special instrumentation or may not yield very accurate results. Gravimetric methods using quartz-crystal microbalances or optical methods, such as ellipsometry, surface plasmon resonance and total internal reflection spectroscope can be applied. The latter ones however, depend on unknown physical properties of the proteins e.g. * Corresponding author. 0927-7765/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0 9 2 7 -7 7 6 5 ( 9 8 ) 0 0 00 8 - 3
the refractive index which differs among different proteins leading to a high uncertainty in protein determination. While many fast and accurate assays for protein determination in solution exist, e.g. Bradford assay [5], Lowry assay [6 ] or BCA assay (bichinolinic acid) [7], there is no easy to handle and cheap assay for immobilized proteins. This can mainly be attributed to the differences in the chemical and biological behavior of free proteins in solution and immobilized proteins. Moreover, the low concentration of immobilized proteins on surfaces requires the method to be higly sensitive. Radiochemical assays are suitable for the determination of proteins in solution and on surfaces, because radioactivity is not influenced by the structure or binding properties of the protein as these are changed by immobilization. In addition a radio assay is very sensitive. This method, however, has several limitations such as high running and material costs, problematic waste
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disposal and potential health risk. Thus, there is a requirement for alternative methods, which should have the advantages of the radio assay, but without the considerable disadvantages. In this study several methods to quantify immobilized streptavidin on gold and silica surfaces are described and evaluated. Streptavidin was chosen as a model protein in the experiments. As a first step a radio assay with tritium labeled biotin was developed. This assay was used as a reference method for the quantification of streptavidin. All other methods were subsequently compared with the results of this radio assay. As photometric methods the BCA-assay and the protein determination with Coomassie Brilliant Blue G 250 were tested. A commercially available fluorescence kit, i.e. the NanoOrange@ Protein Quantitation Kit was used. In parallel a fluorescence assay with ophthalaldehyde was applied. Finally, an enzymelinked immunosorbent-assay based on enzymelabeled anti-streptavidin antibodies was developed to determine streptavidin on the gold and silica surfaces.
2. Experimental 2.1. Materials 2.1.1. Proteins Streptavidin was obtained from Camon ( Wiesbaden, Germany) and peroxidase-labeled goat-anti-streptavidin antibody from Zymed (San Francisco, USA). Prior to immobilization a streptavidin–biotin complex was generated by mixing the compounds with a three times molar excess of biotin to guarantee quantitative binding to streptavidin. All immobilization experiments were then performed with the streptavidin–biotin complex in a 0.05 M phosphate buffer, pH 7.4. 2.1.2. Surfaces Proteins were immobilized on gold and silica surfaces. The gold surfaces were made from glass chips, which were coated with a 40 nm titan layer as an adhesive promoter and onto this a 120 nm gold layer was deposited. Oxidized silicon chips
with an oxide film thickness of 150 nm provided the silica-surfaces. 2.1.3. Chemicals Biotin was purchased from Sigma (Deisenhofen, Germany), d-[8,9(n)-3H ] biotin from Amersham (Braunschweig, Germany). The scintillation cocktail Aqua Save 500 Liquid Scintillator was from Zinsser (Frankfurt, Germany). The Micro BCA Protein Assay Reagent was obtained from Pierce (Rockford, USA) and the NanoOrange@ Protein Quantitation Kit from Molecular Probes (Lelden, The Netherlands). All other chemicals were obtained from Sigma (Deisenhofen, Germany) or Aldrich (Steinheim, Germany) in p.a. grade quality and used without further purification. 2.2. Methods 2.2.1. Immobilization of proteins In order to achieve a high reproducibility during the immobilization, a special incubation plate was developed. In this incubation plate the gold and silica surfaces formed the bottom of a cuvette, which was constructed from a Teflon bloc. The gap between the Teflon bloc and the surfaces was sealed with a 1 mm silicone membrane. The coupling of the streptavidin–biotin complex to the gold surfaces was performed according to Duan and Meyerhoff [8], using (, )-thioctic acid and 1-ethyl-3-[3-(dimethylamino)propyl ] carbodiimide ( EDC ). Covalent binding on the silica surfaces was achieved by a method described by Williamson [9], using p-toluenesulfonyl chloride ( TSC ) as a coupling agent. Subsequently, 100 ml of the strepavidin–biotin solution (various concentrations in 0.05 M phosphate buffer pH 7.4) were added to the activated surfaces mounted in the incubation plate. The protein complex was allowed to react for 18 h at room temperature. The adsorptively bound streptavidin–biotin was washed off the surfaces with 0.5% Tween 20 in 0.05 M phosphate buffer, pH 7.4, by rinsing four times. 2.2.2. Radiochemical analysis The beta radiation was measured by scintillation spectrometry with the scintillation counter Rack Beta ‘‘Spectral’’ from LKB Wallac (Sweden). To
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determine the streptavidin–biotin on the two surfaces, d-[8,9(n)-3H ]biotin instead of biotin was used to obtain the streptavidin–biotin complex, which was immobilized. After immobilization, the gold and silica chips were incubated in 3 ml scintillation cocktail and 0.3 ml water. After shaking for 2 h at room temperature, the chips were drawn out and the beta radiation of the cocktails was measured subsequently. For calibration, various volumes of a streptavidin-d-[8,9(n)-3H ] biotin stock solution were measured.
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Fig. 1. Optical set-up for the detection of a surface fluorescence signal.
2.2.3. Photometric measurements Measurements were made with the microplate reader NM 5000 from Dynatech (microplates ‘‘Maxisorp’’ from Nunc). To determine immobilization of proteins on gold and silica surfaces, the BCA assay [7,10] and an assay using Coomassie Brilliant Blue G 250 [11–13] were used. For protein determination with the BCA assay, the prepared chips were covered with 240 ml of the micro BCA protein assay reagent and shaken for 2 h at room temperature. After taking an aliquot of 100 ml of this solution, the extinction was measured in microplates at 550 nm. The reversible binding of Coomassie Brilliant Blue G 250 to proteins was reported by Gosnell [13]. In the present study we used this effect for the protein quantification on surfaces. For this purpose the chips were covered with 100 ml of the dye solution [13], shaken for 30 min at room temperature and rinsed with the washing solution to remove the dye which was not bound to the immobilized proteins. Afterwards, protein bound dye was removed with 220 ml 0.1 M sodium hydroxide solution in 80% methanol, by shaking for 5 min at room temperature. 100 ml of this solution was placed into microtiter plates and acidified with 20 ml of 4 M hydrochloric acid. The extinction was measured at 630 nm.
the diagonal, ensuring that the reflected exitation light did not enter the entry slit of the emission monochromator (Fig. 1). Two assays were tested to determine protein immobilization on gold and silica surfaces, i.e. a method employing o-phtalaldehyde (OPT ) [14–16 ] and the NanoOrange@ Protein Quantitation Kit. According to Weidemann [16 ] the reaction between proteins, OPT and b-mercaptoethanol (MERC ) can be used to determine proteins in solution, by strongly fluorescing at 445 nm (excitation wavelenght 336 nm). The chips with the immobilized proteins, were fixed in the fluorescence cuvette. Following this, 600 ml of 0.05 M phosphate buffer, pH 7.4, were added (enough to cover the whole chip in the cuvette) followed by 5 ml of MERC. After shaking for 5 min, 5 ml of 1% OPT in methanol was added and the fluorescence intensity was measured after 30 min. To quantify proteins on surfaces the NanoOrange@ Protein Quantitation Kit was used, adding 600 ml of the NanoOrange reagent was added to the chips inside the cuvette. The cuvette, including the chip was heated to 95°C for 10 min and protected from light. Afterwards, the cuvette was allowed to cool down to room temperature for at least 20 min (still protected from light). The fluorescence intensity was measured at 590 nm with an excitation wavelength of 485 nm.
2.2.4. Fluorescence-spectroscopical measurements To measure fluorescence intensity from the gold and silica surfaces, the prepared chip had to be mounted in the fluorimeter (Perkin Elmer LS 50-B, Langen, Germany) as shown in Fig. 1. In a 1 cm quartz glass cuvette (101-QS from Hellma, Mu¨hlheim-Baden, Germany) the chip was fixed on
2.2.5. Enzyme-linked immunosorbent-assay (ELISA) To determination the immobilized streptavidin– biotin complex immunochemically, a non-competitive ELISA was performed. To prevent unspecific binding of the receptor antibody, the chips with the immobilized complexes were blocked by adding
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200 ml 0.5% bovine serum albumin (BSA) in 0.05 M phoshate buffer pH 7.4 for 1 h at room temperature. After rinsing four times with 200 ml 0.5% Tween 20 in phosphate buffer, the chips were covered with 100 ml of peroxidase–labelled goatanti-streptavidin antibody in 0.05 M phosphate buffer, pH 7.4 (dilution 1:2000) and shaken for 10 min. The chips were rinsed again and 150 ml of the substrate (40 ml of a solution from 6 mg/ml tetramethyl benzidine in dimethyl sulfoxide, 1 ml 30% hydrogen peroxide and 2 ml 0.05 M acetate buffer, pH 5.5) was added. After 3 and 6 min on the gold and silica surfaces, respectively, the enzymatic reaction was stopped by adding 100 ml of 2 M sulphuric acid. Aliquots of 100 ml of this solution were pipetted into a microtiter plate followed by measuring the extinction at 450 nm.
Fig. 2. Results of the radio assay on gold surfaces. Protein concentration applied for immobilization versus immobilized protein mass. Mean values and standard deviation of quadruplicate measurements.
3. Results and discussion 3.1. Radio assay The tritium labeled streptavidin–biotin complex was immobilized on the gold and silica surfaces in various concentrations. By measuring the impulse rate, a correlation between the impulse rate and the amount of immobilized protein was obtained. With assistance from the calibration plot obtained with the tritium labeled streptavidin–biotin stock solution (impulse rate versus the amount of streptavidin–biotin), the correlation between the amount of immobilized streptavidin–biotin complex and the amount used for immobilization was obtained. Figs. 2 and 3 show this correlation for the gold and silica surfaces, respectively. Fig. 2 shows the expected progression. At low immobilization concentrations (up to 25 mg/ml ), a linear increase in the immobilized streptavidin–biotin was observed. At concentrations higher than 50 mg/ml the curve began to saturate, indicating that all EDC–activated positions on the chip were occupied by the protein complex. The obtained surface coverage densities were 15.3 ng up to a maximum of 126.4 ng streptavidin–biotin complexes per square centimeter. The ratio of immobilized streptavidin–biotin to immobilization concentration was very low. The ratio varied between 1.25% at an immobilization concentration
Fig. 3. Results of the radio assay on silica surfaces. Protein concentration applied for immobilization versus immobilized protein mass. Mean values and standard deviation of quadruplicate measurements.
of 6.12 mg/ml and 0.32% at 196.00 mg/ml. Assuming that one streptavidin molecule occupies an area on the surface of about 25 nm2 [17], only 31.7% of the geometric gold surface were covered by the protein. Using the silica surfaces ( Fig. 3), an identical curve progression as compared with the gold surfaces, was observed. The amount of immobilized protein, however, was significantly lower. Between 0.08% (at 6.12 mg/ml ) and 0.03% (at 196.00 mg/ml ) of total protein could be immobilized. This corresponds to a streptavidin–biotin surface coverage
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density of 0.95 ng/cm2 and 12.30 ng/cm2, respectively. Thus, only 3.1% of the geometric gold surface was covered by the protein. Assuming that the accessibility of the geometric surface of the chips was the same for gold and silica, the amount of protein immobilized on the gold surfaces was 10-fold higher when compared with the silica surfaces. It has to be emphasized that these results are valid only for these two immobilization methods on these special surfaces. It is most likely, that the difference observed can be attributed to the efficiencies of the immobilization methods applied. The protein–surface density on silica was not as high as expected, but was satisfactory for our purpose. Consequently, all non-radiochemical methods tested can now be compared with the results of the radio assay, providing that the reaction conditions are the same. 3.2. UV-spectroscopical measurements The commercially available BCA-assay was developed to quantify proteins in solution [7,10], however, in principle the determination of immobilized proteins on surfaces is possible [18]. The protein determination with the BCA-assay was carried out in the incubation plate as described above. For both gold and silica surfaces the extinctions of various immobilization concentrations could not be distinguished from the blank reading within the error limits. Thus, a calibration curve could not be obtained. The BCA-assay was not suitable for determining these low masses of streptavidin–biotin on the gold and silica surfaces. No improvement could be observed using the dye Coomassie Brilliant Blue G 250 [11–13] instead of the BCA reagent. Again, the extinctions of various immobilization concentrations could not be distinguished from the blank readings. From these experiments it can be concluded that the UV-spectroscopical methods tested were not sensitive enough to determine very small amounts of streptavidin–biotin on gold and silica surfaces. 3.3. Fluorescence-spectroscopical measurements Protein determination using OPT and MERC was not successful on gold or silica surfaces, because
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Fig. 4. Results of the NanoOrange@ assay on gold surfaces. Protein surface coverage density versus fluorescence signal. The protein density was calculated for the radio assay calibration plot from gold surfaces. Mean values and standard deviation of quadruplicate measurements.
the measured values showed extremely high errors. The NanoOrange@ Protein Quantitation Kit, however, determination of protein on the gold surfaces was possible. Fig. 4 shows the correlation between the fluorescence signal and the protein surface coverage density. The immobilized protein amount was calculated from the radio assay calibration plot for gold surfaces. The curve shows a sigmodial course and a detection limit of 75 ng/cm2. Parallel experiments on the silica surfaces failed because the maximum concentration of streptavidin–biotin complex was determined to be 12.3 ng/cm2. Additional experiments demonstrated that the detection limit of the NanoOrange@ Protein Quantitation Kit in solution is dependent on the individual protein. Detection limits were determined to be 1 mg/ml and 0.1 mg/ml for streptavidin– biotin and for BSA, respectively. Therefore the NanoOrange@ assay was 10 times more sensitive for BSA than for streptavidin–biotin. Based on these results the fluorescence measurements were done again with silica chips, coated with BSA instead of streptavidin–biotin (Fig. 5). In contrast to streptavidin–biotin, a BSA calibration curve was obtained, clearly indicating that protein determination with the NanoOrange@ Protein Quantitation Kit on silica surfaces is possible. A detection limit for BSA could not be given, because a radio assay calibration plot for BSA was not obtained.
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Fig. 5. Calibration plot of the NanoOrange@ assay in solution for different proteins. Mean values and standard deviation of quadruplicate measurements.
3.4. Enzyme-linked immunosorbent–assay (ELISA) The results of the ELISA on the gold and silica surfaces are shown in Figs. 6 and 7. The extinction is plotted against the surface coverage densities, which were calculated from the radio assay. The substrate reaction time was 3 min on the gold surfaces and 6 min on the silica surfaces. At low densities a nearly linear increase was observed and at higher densities the curve was saturated. On the gold surfaces streptavidin could be determined
Fig. 7. Results of the ELISA on silica surfaces. Protein surface coverage density versus extinction signal. The protein density was calculated from the radio assay calibration plot for silica surfaces. Mean values and standard deviation of quadruplicate measurements.
between 15 ng/cm2 and 80 ng/cm2 with a precision of ±3 ng/cm2 and for the silica surfaces between 0.9 ng/cm2 to 10.0 ng/cm2 with a precision of ±0.6 ng/cm2. The nonlinear curvation of Figs. 6 and 7 is probably due to steric hindrances in the binding of the labeled detector antibody. As the detector antibody was added in excess, a saturation effect can be excluded. Nevertheless, the ELISA method was able to detect streptavidin–biotin on gold and silica surfaces with very high sensitivity. However, it had to be calibrated with the results of the radio assay.
4. Conclusion
Fig. 6. Results of the ELISA on gold surfaces. Protein surface coverage density versus extinction signal. The protein density was calculated from the radio assay calibration plot for gold surfaces. Mean values and standard deviation of quadruplicate measurements.
Several methods for the determination of the concentration of immobilized proteins on gold and silica surfaces were executed and compared. The experiments were done with the protein complex streptavidin–biotin as a model. A radio assay was used as a reference method for the determination of streptavidin–biotin surface coverage densities on gold and silica surfaces. In comparison to the results of the radio assay, it became obvious that the UV-spectroscopical BSA assay and the Coomassie Brilliant Blue G 250 assay were not feasible for the determination of very low amounts of immobilized proteins.
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Among the fluorescence-spectroscopical measurements, protein determination with OPT and NERC was not successful due to extremely high errors. However it can be concluded from the experiments with the NanoOrange@ Protein Quantitation Kit, that this fluorescence assay is well suited to the quantitation of proteins on solid surfaces. The feasibility of the NanoOrange@ kit for quantitation of immobilized proteins was demonstrated for the first time. It must be emphasized that for quantitative measurements a previous calibration with a radio assay is required. For simple comparison, however, e.g. for the comparison of different immobilization methods, etc. the NanoOrange@ assay is recommended due to its high sensitivity and ease of use. A non-competitive ELISA was also developed to determine streptavidin–biotin on the two surfaces. Again, without a radio assay calibration, no quantitation is possible but the ELISA is recommended due to its high sensitivity e.g. for the optimization and development of immobilization methods. In conclusion it was shown that, besides radiochemical determination methods fluorescence-spectroscopical and immuno-chemical methods can be recommended for the detection of immobilized proteins on gold and silica surfaces. The ELISA offers the advantage of higher sensitivity, while protein determination with the NanoOrange@ Protein Quantitation Kit is more generic, as there is no need to target specific antibodies. All non-radioactive methods, however, showed the need for external calibration. Quantitative studies, however, such as chips with different amounts of immobilized proteins or investigations into immobilization procedures, are possible.
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Acknowledgment This work was supported by the German Federal Ministry of Research and Technology (BMBF, Projektrdger BEO, Grant no. 0310 840) and the Ministry of Science and Research of the State of Northrhine-Westfalia.
References [1] R. Axen, S. Ernback, Eur. J. Biochem. 18 (1971) 351–360. [2] D. Gabel, R. Exen, Meth. Enzymol. 44 (1976) 386–393. [3] Y. Levin, M. Pecht, L. Goldstein, E. Katchalski, Biochemistry 3 (1964) 1905–1913. [4] R. Axen, P. Vretblad, Acta Chem. Scand. 25 (1971) 2711–2716. [5] M. Bradfort, Anal. Biochem. 72 (1976) 248–254. [6 ] O. Lowry, N. Rosebrough, A. Farr, R. Randall, J. Biol. Chem. 193 (1951) 265–275. [7] P. Smith, R. Krohn, G. Hermanson, A. Mallia, F. Gartner, M. Provenzano, E. Fujimoto, N. Goeke, B. Olson, D. Klenk, Anal. Biochem. 150 (1985) 76–85. [8] C. Duan, M. Meyerhoff, Anal. Chem. 66 (1994) 1369–1377. [9] M. Williamson, D. Atha, D. Reeder, P. Sundaram, Anal. Lett. 22 (1989) 803. [10] M. Redinbaugh, R. Turley, Anal. Biochem. 153 (1986) 267–271. [11] H. Ahmad, M. Saleemuddin, Anal. Biochem. 148 (1985) 533–541. [12] W. Lewis, S. Schuster, J. Biochem. Biophys. Meth. 21 (1990) 129–144. [13] M. Gosnell, H. Mottola, J. Microchem. 37 (1988) 149–154. [14] J. Stobaugh, A. Repta, L. Stemson, K. Garren, Anal. Biochem. 135 (1983) 495–504. [15] M. Roth, Anal. Chem. 43 (1971) 880–882. [16 ] E. Weidekamm, D. Wallach, R. Flu¨ckiger, Anal. Biochem. 54 (1973) 102–114. [17] R. Achtnich, Biosens. Bioelectron. 7 (1992) 279–290. [18] D. Kro¨ger, Ph.D. thesis, Westfa¨lische WilhelmsUniversita¨t, Mu¨nster, Westfa¨lia, 1995.
Evaluation of several methods to quantify immobilized proteins on gold and silica surfaces Matthias Orschel, Andreas Katerkamp *, Markus Meusel, Karl Cammann Institut fu¨r Chemosensorik und Biosensorik, Mu¨nster, Mendel str. 7, 48149 Mu¨nster, Germany Received 23 September 1997; accepted 5 January 1998
Abstract In the present study, the application of several methods for the determination of an immobilized protein are presented and the results discussed. Streptavidin was bound covalently on gold and silica surfaces. A radio assay was used as a reference method for the quantification of the immobilized protein. Two photometric, two fluorescencespectroscopic methods and an immunochemical approach, based on a sandwich-ELISA (enzyme linked immunosorbent assay) format were applied with regard to their feasibility for sensitive protein quantification. The photometric methods were not sensitive enough, but one of the fluorescence based methods and the ELISA could be applied for the detection of low protein concentrations on the surfaces. © 1998 Elsevier Science B.V. Keywords: Biotin; Gold surface; Immobilization proteins; Protein quantification; Silica surface; Streptavidin
1. Introduction Immobilized proteins are used in many biochemical applications and often the amount of proteins attached to the surface must be determined. Several methods have been developed for this purpose. These include the measurement of the difference between added protein and protein recovered after immobilization [1], amino acid analysis after acid hydrolysis [2] or analysis of elements such as nitrogen and sulfur [3,4]. All these methods require special instrumentation or may not yield very accurate results. Gravimetric methods using quartz-crystal microbalances or optical methods, such as ellipsometry, surface plasmon resonance and total internal reflection spectroscope can be applied. The latter ones however, depend on unknown physical properties of the proteins e.g. * Corresponding author. 0927-7765/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0 9 2 7 -7 7 6 5 ( 9 8 ) 0 0 00 8 - 3
the refractive index which differs among different proteins leading to a high uncertainty in protein determination. While many fast and accurate assays for protein determination in solution exist, e.g. Bradford assay [5], Lowry assay [6 ] or BCA assay (bichinolinic acid) [7], there is no easy to handle and cheap assay for immobilized proteins. This can mainly be attributed to the differences in the chemical and biological behavior of free proteins in solution and immobilized proteins. Moreover, the low concentration of immobilized proteins on surfaces requires the method to be higly sensitive. Radiochemical assays are suitable for the determination of proteins in solution and on surfaces, because radioactivity is not influenced by the structure or binding properties of the protein as these are changed by immobilization. In addition a radio assay is very sensitive. This method, however, has several limitations such as high running and material costs, problematic waste
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disposal and potential health risk. Thus, there is a requirement for alternative methods, which should have the advantages of the radio assay, but without the considerable disadvantages. In this study several methods to quantify immobilized streptavidin on gold and silica surfaces are described and evaluated. Streptavidin was chosen as a model protein in the experiments. As a first step a radio assay with tritium labeled biotin was developed. This assay was used as a reference method for the quantification of streptavidin. All other methods were subsequently compared with the results of this radio assay. As photometric methods the BCA-assay and the protein determination with Coomassie Brilliant Blue G 250 were tested. A commercially available fluorescence kit, i.e. the NanoOrange@ Protein Quantitation Kit was used. In parallel a fluorescence assay with ophthalaldehyde was applied. Finally, an enzymelinked immunosorbent-assay based on enzymelabeled anti-streptavidin antibodies was developed to determine streptavidin on the gold and silica surfaces.
2. Experimental 2.1. Materials 2.1.1. Proteins Streptavidin was obtained from Camon ( Wiesbaden, Germany) and peroxidase-labeled goat-anti-streptavidin antibody from Zymed (San Francisco, USA). Prior to immobilization a streptavidin–biotin complex was generated by mixing the compounds with a three times molar excess of biotin to guarantee quantitative binding to streptavidin. All immobilization experiments were then performed with the streptavidin–biotin complex in a 0.05 M phosphate buffer, pH 7.4. 2.1.2. Surfaces Proteins were immobilized on gold and silica surfaces. The gold surfaces were made from glass chips, which were coated with a 40 nm titan layer as an adhesive promoter and onto this a 120 nm gold layer was deposited. Oxidized silicon chips
with an oxide film thickness of 150 nm provided the silica-surfaces. 2.1.3. Chemicals Biotin was purchased from Sigma (Deisenhofen, Germany), d-[8,9(n)-3H ] biotin from Amersham (Braunschweig, Germany). The scintillation cocktail Aqua Save 500 Liquid Scintillator was from Zinsser (Frankfurt, Germany). The Micro BCA Protein Assay Reagent was obtained from Pierce (Rockford, USA) and the NanoOrange@ Protein Quantitation Kit from Molecular Probes (Lelden, The Netherlands). All other chemicals were obtained from Sigma (Deisenhofen, Germany) or Aldrich (Steinheim, Germany) in p.a. grade quality and used without further purification. 2.2. Methods 2.2.1. Immobilization of proteins In order to achieve a high reproducibility during the immobilization, a special incubation plate was developed. In this incubation plate the gold and silica surfaces formed the bottom of a cuvette, which was constructed from a Teflon bloc. The gap between the Teflon bloc and the surfaces was sealed with a 1 mm silicone membrane. The coupling of the streptavidin–biotin complex to the gold surfaces was performed according to Duan and Meyerhoff [8], using (, )-thioctic acid and 1-ethyl-3-[3-(dimethylamino)propyl ] carbodiimide ( EDC ). Covalent binding on the silica surfaces was achieved by a method described by Williamson [9], using p-toluenesulfonyl chloride ( TSC ) as a coupling agent. Subsequently, 100 ml of the strepavidin–biotin solution (various concentrations in 0.05 M phosphate buffer pH 7.4) were added to the activated surfaces mounted in the incubation plate. The protein complex was allowed to react for 18 h at room temperature. The adsorptively bound streptavidin–biotin was washed off the surfaces with 0.5% Tween 20 in 0.05 M phosphate buffer, pH 7.4, by rinsing four times. 2.2.2. Radiochemical analysis The beta radiation was measured by scintillation spectrometry with the scintillation counter Rack Beta ‘‘Spectral’’ from LKB Wallac (Sweden). To
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determine the streptavidin–biotin on the two surfaces, d-[8,9(n)-3H ]biotin instead of biotin was used to obtain the streptavidin–biotin complex, which was immobilized. After immobilization, the gold and silica chips were incubated in 3 ml scintillation cocktail and 0.3 ml water. After shaking for 2 h at room temperature, the chips were drawn out and the beta radiation of the cocktails was measured subsequently. For calibration, various volumes of a streptavidin-d-[8,9(n)-3H ] biotin stock solution were measured.
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Fig. 1. Optical set-up for the detection of a surface fluorescence signal.
2.2.3. Photometric measurements Measurements were made with the microplate reader NM 5000 from Dynatech (microplates ‘‘Maxisorp’’ from Nunc). To determine immobilization of proteins on gold and silica surfaces, the BCA assay [7,10] and an assay using Coomassie Brilliant Blue G 250 [11–13] were used. For protein determination with the BCA assay, the prepared chips were covered with 240 ml of the micro BCA protein assay reagent and shaken for 2 h at room temperature. After taking an aliquot of 100 ml of this solution, the extinction was measured in microplates at 550 nm. The reversible binding of Coomassie Brilliant Blue G 250 to proteins was reported by Gosnell [13]. In the present study we used this effect for the protein quantification on surfaces. For this purpose the chips were covered with 100 ml of the dye solution [13], shaken for 30 min at room temperature and rinsed with the washing solution to remove the dye which was not bound to the immobilized proteins. Afterwards, protein bound dye was removed with 220 ml 0.1 M sodium hydroxide solution in 80% methanol, by shaking for 5 min at room temperature. 100 ml of this solution was placed into microtiter plates and acidified with 20 ml of 4 M hydrochloric acid. The extinction was measured at 630 nm.
the diagonal, ensuring that the reflected exitation light did not enter the entry slit of the emission monochromator (Fig. 1). Two assays were tested to determine protein immobilization on gold and silica surfaces, i.e. a method employing o-phtalaldehyde (OPT ) [14–16 ] and the NanoOrange@ Protein Quantitation Kit. According to Weidemann [16 ] the reaction between proteins, OPT and b-mercaptoethanol (MERC ) can be used to determine proteins in solution, by strongly fluorescing at 445 nm (excitation wavelenght 336 nm). The chips with the immobilized proteins, were fixed in the fluorescence cuvette. Following this, 600 ml of 0.05 M phosphate buffer, pH 7.4, were added (enough to cover the whole chip in the cuvette) followed by 5 ml of MERC. After shaking for 5 min, 5 ml of 1% OPT in methanol was added and the fluorescence intensity was measured after 30 min. To quantify proteins on surfaces the NanoOrange@ Protein Quantitation Kit was used, adding 600 ml of the NanoOrange reagent was added to the chips inside the cuvette. The cuvette, including the chip was heated to 95°C for 10 min and protected from light. Afterwards, the cuvette was allowed to cool down to room temperature for at least 20 min (still protected from light). The fluorescence intensity was measured at 590 nm with an excitation wavelength of 485 nm.
2.2.4. Fluorescence-spectroscopical measurements To measure fluorescence intensity from the gold and silica surfaces, the prepared chip had to be mounted in the fluorimeter (Perkin Elmer LS 50-B, Langen, Germany) as shown in Fig. 1. In a 1 cm quartz glass cuvette (101-QS from Hellma, Mu¨hlheim-Baden, Germany) the chip was fixed on
2.2.5. Enzyme-linked immunosorbent-assay (ELISA) To determination the immobilized streptavidin– biotin complex immunochemically, a non-competitive ELISA was performed. To prevent unspecific binding of the receptor antibody, the chips with the immobilized complexes were blocked by adding
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200 ml 0.5% bovine serum albumin (BSA) in 0.05 M phoshate buffer pH 7.4 for 1 h at room temperature. After rinsing four times with 200 ml 0.5% Tween 20 in phosphate buffer, the chips were covered with 100 ml of peroxidase–labelled goatanti-streptavidin antibody in 0.05 M phosphate buffer, pH 7.4 (dilution 1:2000) and shaken for 10 min. The chips were rinsed again and 150 ml of the substrate (40 ml of a solution from 6 mg/ml tetramethyl benzidine in dimethyl sulfoxide, 1 ml 30% hydrogen peroxide and 2 ml 0.05 M acetate buffer, pH 5.5) was added. After 3 and 6 min on the gold and silica surfaces, respectively, the enzymatic reaction was stopped by adding 100 ml of 2 M sulphuric acid. Aliquots of 100 ml of this solution were pipetted into a microtiter plate followed by measuring the extinction at 450 nm.
Fig. 2. Results of the radio assay on gold surfaces. Protein concentration applied for immobilization versus immobilized protein mass. Mean values and standard deviation of quadruplicate measurements.
3. Results and discussion 3.1. Radio assay The tritium labeled streptavidin–biotin complex was immobilized on the gold and silica surfaces in various concentrations. By measuring the impulse rate, a correlation between the impulse rate and the amount of immobilized protein was obtained. With assistance from the calibration plot obtained with the tritium labeled streptavidin–biotin stock solution (impulse rate versus the amount of streptavidin–biotin), the correlation between the amount of immobilized streptavidin–biotin complex and the amount used for immobilization was obtained. Figs. 2 and 3 show this correlation for the gold and silica surfaces, respectively. Fig. 2 shows the expected progression. At low immobilization concentrations (up to 25 mg/ml ), a linear increase in the immobilized streptavidin–biotin was observed. At concentrations higher than 50 mg/ml the curve began to saturate, indicating that all EDC–activated positions on the chip were occupied by the protein complex. The obtained surface coverage densities were 15.3 ng up to a maximum of 126.4 ng streptavidin–biotin complexes per square centimeter. The ratio of immobilized streptavidin–biotin to immobilization concentration was very low. The ratio varied between 1.25% at an immobilization concentration
Fig. 3. Results of the radio assay on silica surfaces. Protein concentration applied for immobilization versus immobilized protein mass. Mean values and standard deviation of quadruplicate measurements.
of 6.12 mg/ml and 0.32% at 196.00 mg/ml. Assuming that one streptavidin molecule occupies an area on the surface of about 25 nm2 [17], only 31.7% of the geometric gold surface were covered by the protein. Using the silica surfaces ( Fig. 3), an identical curve progression as compared with the gold surfaces, was observed. The amount of immobilized protein, however, was significantly lower. Between 0.08% (at 6.12 mg/ml ) and 0.03% (at 196.00 mg/ml ) of total protein could be immobilized. This corresponds to a streptavidin–biotin surface coverage
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density of 0.95 ng/cm2 and 12.30 ng/cm2, respectively. Thus, only 3.1% of the geometric gold surface was covered by the protein. Assuming that the accessibility of the geometric surface of the chips was the same for gold and silica, the amount of protein immobilized on the gold surfaces was 10-fold higher when compared with the silica surfaces. It has to be emphasized that these results are valid only for these two immobilization methods on these special surfaces. It is most likely, that the difference observed can be attributed to the efficiencies of the immobilization methods applied. The protein–surface density on silica was not as high as expected, but was satisfactory for our purpose. Consequently, all non-radiochemical methods tested can now be compared with the results of the radio assay, providing that the reaction conditions are the same. 3.2. UV-spectroscopical measurements The commercially available BCA-assay was developed to quantify proteins in solution [7,10], however, in principle the determination of immobilized proteins on surfaces is possible [18]. The protein determination with the BCA-assay was carried out in the incubation plate as described above. For both gold and silica surfaces the extinctions of various immobilization concentrations could not be distinguished from the blank reading within the error limits. Thus, a calibration curve could not be obtained. The BCA-assay was not suitable for determining these low masses of streptavidin–biotin on the gold and silica surfaces. No improvement could be observed using the dye Coomassie Brilliant Blue G 250 [11–13] instead of the BCA reagent. Again, the extinctions of various immobilization concentrations could not be distinguished from the blank readings. From these experiments it can be concluded that the UV-spectroscopical methods tested were not sensitive enough to determine very small amounts of streptavidin–biotin on gold and silica surfaces. 3.3. Fluorescence-spectroscopical measurements Protein determination using OPT and MERC was not successful on gold or silica surfaces, because
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Fig. 4. Results of the NanoOrange@ assay on gold surfaces. Protein surface coverage density versus fluorescence signal. The protein density was calculated for the radio assay calibration plot from gold surfaces. Mean values and standard deviation of quadruplicate measurements.
the measured values showed extremely high errors. The NanoOrange@ Protein Quantitation Kit, however, determination of protein on the gold surfaces was possible. Fig. 4 shows the correlation between the fluorescence signal and the protein surface coverage density. The immobilized protein amount was calculated from the radio assay calibration plot for gold surfaces. The curve shows a sigmodial course and a detection limit of 75 ng/cm2. Parallel experiments on the silica surfaces failed because the maximum concentration of streptavidin–biotin complex was determined to be 12.3 ng/cm2. Additional experiments demonstrated that the detection limit of the NanoOrange@ Protein Quantitation Kit in solution is dependent on the individual protein. Detection limits were determined to be 1 mg/ml and 0.1 mg/ml for streptavidin– biotin and for BSA, respectively. Therefore the NanoOrange@ assay was 10 times more sensitive for BSA than for streptavidin–biotin. Based on these results the fluorescence measurements were done again with silica chips, coated with BSA instead of streptavidin–biotin (Fig. 5). In contrast to streptavidin–biotin, a BSA calibration curve was obtained, clearly indicating that protein determination with the NanoOrange@ Protein Quantitation Kit on silica surfaces is possible. A detection limit for BSA could not be given, because a radio assay calibration plot for BSA was not obtained.
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Fig. 5. Calibration plot of the NanoOrange@ assay in solution for different proteins. Mean values and standard deviation of quadruplicate measurements.
3.4. Enzyme-linked immunosorbent–assay (ELISA) The results of the ELISA on the gold and silica surfaces are shown in Figs. 6 and 7. The extinction is plotted against the surface coverage densities, which were calculated from the radio assay. The substrate reaction time was 3 min on the gold surfaces and 6 min on the silica surfaces. At low densities a nearly linear increase was observed and at higher densities the curve was saturated. On the gold surfaces streptavidin could be determined
Fig. 7. Results of the ELISA on silica surfaces. Protein surface coverage density versus extinction signal. The protein density was calculated from the radio assay calibration plot for silica surfaces. Mean values and standard deviation of quadruplicate measurements.
between 15 ng/cm2 and 80 ng/cm2 with a precision of ±3 ng/cm2 and for the silica surfaces between 0.9 ng/cm2 to 10.0 ng/cm2 with a precision of ±0.6 ng/cm2. The nonlinear curvation of Figs. 6 and 7 is probably due to steric hindrances in the binding of the labeled detector antibody. As the detector antibody was added in excess, a saturation effect can be excluded. Nevertheless, the ELISA method was able to detect streptavidin–biotin on gold and silica surfaces with very high sensitivity. However, it had to be calibrated with the results of the radio assay.
4. Conclusion
Fig. 6. Results of the ELISA on gold surfaces. Protein surface coverage density versus extinction signal. The protein density was calculated from the radio assay calibration plot for gold surfaces. Mean values and standard deviation of quadruplicate measurements.
Several methods for the determination of the concentration of immobilized proteins on gold and silica surfaces were executed and compared. The experiments were done with the protein complex streptavidin–biotin as a model. A radio assay was used as a reference method for the determination of streptavidin–biotin surface coverage densities on gold and silica surfaces. In comparison to the results of the radio assay, it became obvious that the UV-spectroscopical BSA assay and the Coomassie Brilliant Blue G 250 assay were not feasible for the determination of very low amounts of immobilized proteins.
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Among the fluorescence-spectroscopical measurements, protein determination with OPT and NERC was not successful due to extremely high errors. However it can be concluded from the experiments with the NanoOrange@ Protein Quantitation Kit, that this fluorescence assay is well suited to the quantitation of proteins on solid surfaces. The feasibility of the NanoOrange@ kit for quantitation of immobilized proteins was demonstrated for the first time. It must be emphasized that for quantitative measurements a previous calibration with a radio assay is required. For simple comparison, however, e.g. for the comparison of different immobilization methods, etc. the NanoOrange@ assay is recommended due to its high sensitivity and ease of use. A non-competitive ELISA was also developed to determine streptavidin–biotin on the two surfaces. Again, without a radio assay calibration, no quantitation is possible but the ELISA is recommended due to its high sensitivity e.g. for the optimization and development of immobilization methods. In conclusion it was shown that, besides radiochemical determination methods fluorescence-spectroscopical and immuno-chemical methods can be recommended for the detection of immobilized proteins on gold and silica surfaces. The ELISA offers the advantage of higher sensitivity, while protein determination with the NanoOrange@ Protein Quantitation Kit is more generic, as there is no need to target specific antibodies. All non-radioactive methods, however, showed the need for external calibration. Quantitative studies, however, such as chips with different amounts of immobilized proteins or investigations into immobilization procedures, are possible.
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Acknowledgment This work was supported by the German Federal Ministry of Research and Technology (BMBF, Projektrdger BEO, Grant no. 0310 840) and the Ministry of Science and Research of the State of Northrhine-Westfalia.
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