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Real time sensing of specific molecular binding using surface plasmon resonance spectroscopy
Sensors and Actuators B 54 (1999) 166 – 175
Real time sensing of specific molecular binding using surface plasmon resonance spectroscopy Peter Pfeifer a,*, Ulrich Aldinger b, Gu¨nter Schwotzer a, Stephan Diekmann b, Peter Steinru¨cke b a b
Institut fu¨r Physikalische Hochtechnologie (IPHT), Helmholtzweg 4, 07743, Jena, Germany Institut fu¨r Molekulare Biotechnologie (IMB), Beutenbergstraße 10, 07745, Jena, Germany
Abstract A new planar surface plasmon resonance sensor with spectral interrogation was constructed and tested. The SPR device was directly compared with a high-end commercial BIAcore 2000 instrument. In immunological applications, we used the SPR-device for the detection of myoglobin and to measure the concentration of staphylokinase in a competitive assay. The lower limits of detection were tested when the binding of Ni2 + -ions on a matrix with nitrilotriacetic acid (NTA) was measured. The tested sensor was comparable in performance to the commercial instrument. Its detection of the plasmon resonance in a spectral mode and its fiber optical connection between sensor and detector unit offer additional advantages for future applications in on-site analysis with a portable detector. © 1999 Elsevier Science S.A. All rights reserved. Keywords: Surface plasmon resonance; Spectroscopy; Portable instrument; On-site measurements; Remote control
1. Introduction Surface plasmon resonance (SPR) is the result of the interaction between electromagnetic waves of the incident light and the free electron systems in a conducting surface layer [1]. The appearance of SPR relates to the phenomenon of internal reflection at an interface between a metal layer and a dielectricum. An electromagnetic evanescent surface wave propagates along the conducting layer of gold or silver. Within the volume of the evanescent wave biomolecular interactions are detected as alterations in the refractive index. This also changes the resonance conditions of the detection. The resonance angle is shifted at which the intensity of the reflected light becomes minimal. Alternatively the wavelength dependence of SPR can be used for detection. In this case polychromatic light is used for the excitation of the plasmons at a fixed angle of incidence. The refractive index change is measured as a shift, Dl, of the resonance wavelength (see Fig. 1).The evanescent wave decays exponentially with the distance from the interface. It effectively penetrates the lower refractive index medium to a depth of approximately one wave* Corresponding author.
length. Thus, SPR only detects changes in the refractive index very close to the surface. Surface plasmon resonance detectors do not require the labelling of the interacting components. The genuine molecular interaction can be directly observed. In 197l, Kretschmann [2] reported the determination of optical constants of thin metallic layers by measuring the resonance absorption of light by surface plasmon oscillations. In 1983, Liedberg et. al. [3] first demonstrated the exploitation of surface plasmon resonance (SPR) for chemical sensing. Since then many SPR sensor configurations based on bulk prism or optical fibers have been realised for biochemical applications [4–7]. A family of SPR-devices has been commercialised by BIAcore (Sweden) [8]. The instruments either use an angular interrogation in connection with a bulk prism, or apply a spectrometer with an optical multimode fiber as sensing element. We developed an alternative planar optical sensor with a technology based on SPR in the wavelength domain. In this paper we report on its sensitivity and on the results of a comparative study with a high-end BIAcore 2000 instrument. Both SPR-devices were tested in biological applications. Our instrument shows a comparable sensitivity but also has the potential for
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Fig. 1. Detection principle of the Jena SPR-device. Biomolecular interactions at the sensing surface layer are monitored as a shift, Dl, in the resonance wavelength.
miniaturisation and remote sensing, combined with the advantages and sensitivity of a planar bulk device as described by Robinson [9]. Our sensor configuration uses the Kretschmann geometry [2] consisting of a glass prism and a glass slide with a surface plasmon active gold layer. Collimated light from a broad-band source excites a surface plasmon wave on a prism base at the surface between the
gold layer and the sensed medium. The excitation of the surface plasmon resonance is detectable as a minimum in the intensity of the reflected light at a particular wavelength. Since the surface plasmon resonance strongly depends on the refractive index of the medium adjacent to the gold layer, changes in the optical properties of the sensed medium can be measured by determining the resonance wavelength at which SPR occurs.
Fig. 2. Calculated shift of the SPR-wavelength vs. refractive index for the layer system, BK7 glass with 50 nm gold surface layer. (A) aqueous bulk environment (open symbols), (B) hydrogel matrix with 100 nm thickness as in the BIAcore CM-5 chip (filled symbols). Angles of incidence used for the calculation: 65.8°; " 67.5°.
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Fig. 3. The Jena SPR-device. For details see text. Experimental setup with spectral interrogation.
2. Experimental Our aim was to calculate and measure the sensitivity of the Jena SPR-device and to test its suitability as an analytical tool for the investigation of biomolecular interactions. For comparisons we used the BIAcore 2000 instrument, a high-end SPR-instrument which measures the binding of molecules on the sensing surface as a shift in the resonance angle of the monochromatic reflected light. In contrast, the Jena SPR-device works at fixed angles. Binding of molecules on the sensor surface is detected as a shift of the resonance wavelength of the polychromatic reflected light. Experiments on both instruments were done with commercial sensor chips from BIAcore.
2.1. Theoretical determination of the sensiti6ity We calculated the shift in the resonance wavelength caused by changes of the refractive index of the sensed medium using the well-established matrix method for optical layers. These calculations were carried out for two configurations of the sensor: (i) by variations of the refractive index of the bulk environment; and (ii) by monitoring the changes of the refractive index which are caused by homogeneous adsorption of proteins within a 100 nm-thick aqueous hydrogel matrix adjacent to the gold layer. Our calculations are based on assuming all glass substrates being made of BK7 glass [10] with 50 nm thin gold layers [11]. Calculations were done for a spectral range from 700 to 950 nm and for angles of the incident polychromatic light of 65.8 and 67.5°. Fig. 2 displays the dependence of the SPR wave-
length on changes of the refractive index of the adjacent bulk medium. We changed the refractive index of the sensed aqueous medium on a gold surface from n= 1.33 (water) to n= 1.36. The slope of the curves increases with higher refractive indices. This indicates an increasing sensitivity of the device for media with higher refractive index. The signal dependence on the fixed angle of incidence is also illustrated, an increase in sensitivity is shown for smaller angles of incidence. Fig. 2 also indicates a decrease in sensitivity if the bulk medium is replaced by a 100 nm-thick hydrogel matrix adjacent to the gold layer. The general behaviour of the detector is as described before. The sensitivity however is smaller. The reason is that the penetration depth of the evanescent SPR-wave is larger than the thickness of the binding matrix. The sensitivity of the sensor was estimated for the case of protein adsorption to the matrix adjacent to the gold layer. Concentration changes, Dc, linearly correlate with changes in the refractive index, Dn, according to Dc = Dn × dc/dn. For protein molecules we assume a value of dc/dn = 5 g cm − 3 [4]. Angles of incidence of 65.8 and 67.5° were chosen. For a refractive index of the matrix of n= 1.33 (water) and an angle of incidence of 67.5° we obtain Dl/Dc =360 nm (g cm − 3) − 1 from Fig. 2. The spectral analysis in our instrument is performed by a polychromator with an assumed accuracy of 0.1 nm. Taking into account the matrix thickness of 100 nm this results in a detectable surface concentration of 27 pg mm − 2. Changing the parameters to 65.8° for the angle of incidence and n= 1.36 for the refractive index would decrease this level to a surface concentration of 14 pg mm − 2.
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Fig. 4. Schematic design of the flow cell. The volume of the cell was : 2.5 ml.
2.2. Instrumentation
2.3. Sensory performance
The sensor uses a broad band optical source, a sensing head and a spectrum analyser (Fig. 3). The sensing head comprises a 69°–42° – 69° prism made from BK7 glass. For temperature control it was mounted on a thermoelectric cooler (Peltier element). As a light source we used a miniature halogen lamp which was connected to a collimating lens optics by a multimode optical fiber. The reflected light was also coupled into a multimode fiber by collimating optics and analysed by a high resolution spectrum analyser. In order to detect only the TM-polarized light a foil polarizer was inserted into the collimated beam. In our experiments, a PC controlled high resolution polychromator with CCD detector was employed which had been developed at the Institute for Physical High Technology, Jena [12]. It has a dispersion of 0.18 nm per CCD pixel. Within the chosen spectral range from 730 to 910 nm this allows us to measure the shift in the SPR wavelength with a resolution of better than 0.1 nm. Resonance wavelength shifts are detected as changes in the pixel ouput of the CCD-detector. The detector pixel at which the intensity of the measured light is minimal correlates to the SPR-wavelength. In all experiments we have used commercial sensor chips from BIAcore. The chips were attached to the glass prism with a low-viscous immersion oil [n = 1.515, Novodirekt (Germany)]. A small chamber (volume 2.5 ml) was made from Teflon and a silicon ring which was pressed to the chip by a fixing screw to create a flow cell (see Fig. 4). We used this setup to measure the sensory performance of the Jena SPR-device.
We characterised the sensitivity, dynamic range and the resolution of our sensor. The chamber was filled with a buffer solution [10 mM N-2-hydroxyethylpiperazine-N%-2-ethanesulfonic acid (HEPES, from SIGMA Germany) pH 7.4, n= 1.335] and with a solution of 15% (w/v) sucrose in 10 mM HEPES, pH 7.4 (n= 1.357). As shown in Fig. 5 the SPR-wavelength changes by :792 pixel or 141 nm. With a refractive index change of Dn = 0.022 between HEPES and sucrose/HEPES buffers we can calculate an average sensitivity of 6400 nm per refractive index unit (RIU). This is in good agreement with our calculations from Fig. 2 (6900 nm RIU − 1). We also measured the resolution of the sensor. The probe chamber was filled with water. The temperature of the sensing head was increased in steps of 0.5 K over a range of 2.5 K. Changes in SPR wavelength are mainly caused by temperature dependent alterations of the water refractive index. Assuming a change of Dn/ DT = 1×10 − 4 K − 1 we obtained a sensitivity of 7120 nm RIU − 1 (theoretical 8000 nm RIU − 1). Fig. 6 shows a noise level of : 0.02 nm for this experiment. The Jena device thus can resolve changes in refractive index of about 3× 10 − 6.
2.4. Biological applications As a next step we analysed biomolecular interactions. The BIAcore 2000 instrument was used as a reference system. For both instruments sensor chips from BIA-
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Fig. 5. Shift in the SPR-response upon changing the refractive index of the bulk environment. The flow cell was filled with (a) and (c) HEPES solution (n =1.335) and (b) with solution of 15% sucrose in HEPES (n =1.357). As measured on the Jena device, 1 pixel corresponds to a resonance wavelength shift of 0.178 nm. The angle of incidence was 67.5°.
core were used thus minimizing differences in the architecture of the sensing surface. Antibodies provide an excellent tool for the specific molecular recognition of biomolecules. We monitored the recognition of myoglobin by a monoclonal antibody immobilized to the sensor chip. We further investigated the immuno-recognition of an antithrombolytic
pharmaceutical protein, staphylokinase [13], by a polyclonal rabbit antiserum in a competitive immunoassay. To determine the mass sensitivity of our device, we measured the binding of a low-molecular weight ligand to the matrix of a sensor chip. We were able to detect the binding of Ni2 + -ions to a chip covered with a metallochelating matrix (nitrilotriacetic acid, NTA).
Fig. 6. Temperature dependence of the SPR-response. The shift in SPR-response was measured by changing the temperature of the sensor in steps of 0.5 K over a range of 2.5 K. As measured by the Jena device, 1 pixel corresponds to a resonance wavelength shift of 0.178 nm. The angle of incidence was 65.8°.
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Fig. 7. Signal comparison between the BIAcore 2000 and the Jena SPR-device. Sucrose solutions of different concentrations were injected. The flow rate was 1 ml min − 1.
2.4.1. Sample application A flow-cell was formed by a Teflon body attached to the sensor chip by a silicon ring of 0.2 mm height and 4 mm diameter. The inlet and outlet were realised by two drill-holes in the Teflon body which fit to injection needles (see Fig. 4). The volume of the flow-cell was : 2.5 ml. A high precision microflux system as in the BIAcore 2000 instrument was not available for our device. Samples were manually injected into a sample loop. We used an Omnifit (Machery Nagel, Du¨ren, Germany) injection septum and a Hamilton (Darmstadt, Germany) syringe for this purpose. To avoid dispersion effects within the sample solution, the sample probe was trapped between two air bubbles. Teflon and silicon tubes connecting the detector parts were kept at a minimum length. The internal tube diameter was either 0.5 or 1 mm. Sample loop and carrier flow were separated by a 4-way valve (Hamilton HVP4-4, Darmstadt, Germany). Sample binding to the sensor chips was either carried out in a flow of carrier buffer or by direct application of the material onto the sensor field with a micropipette. In the latter case the chip was subsequently incubated in a petri dish under gentle shaking for the specified time and under water-saturated vapour conditions. A Hamilton dispensor Microlab 541 B (Hamilton, Darmstadt, Germany) was used for all flow applications. In control experiments, the signal output of the BIAcore 2000 instrument was corrected for unspecific binding according to the manufacturer’s instructions. Data obtained with the Jena device were
corrected likewise. For data processing, we used a PC (Peacock, Germany) under MS-DOS 6.2 with a Kethley DAS 801 data acquisition board (Keathley, Munich, Germany) for A/D conversion. If not stated otherwise, the sampling rate was adjusted to 0.36 Hz. Recorded spectral data of the Jena SPR-device were processed using Microcal Origin 5.0 (Microcal Software, Northampton, USA) under Microsoft Windows NT4.0 For calculations with the BIAcore 2000 data we used the BIAevaluation and BIAsimulation software of BIAcore.
2.4.2. Signal comparison The direct comparison of the BIAcore 2000 and the Jena SPR-device required an interconversion factor for the signal outputs of both SPR instruments. We used the signal output of sucrose solutions [concentrations between 20% (w/v) and 0.5% (w/v)] to calibrate the data of both instruments. A signal response of 1 pixel on the Jena detector equals 41 resonance units (RU) on the BIAcore 2000 instrument (see Fig. 7). 2.4.3. Immunological detection of myoglobin In the BIAcore 2000 instrument, myoglobin was detected using a CM-5 chip (BIAcore). All chemicals were from BIAcore. The chip carries a hydrogel of carboxymethylated dextran which was activated with 1ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Monoclonal antibodies were aminocoupled to the chip surface according to the BIAcore protocol. For biomolecular interaction with the antigen, 40 ml of
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recombinant human myoglobin (2.5 mg ml − 1) were applied at a flow rate of 10 ml min − 1. The same reagents were used in the Jena detector. Due to the larger dimensions of the detector cell, all sample volumes were doubled and the flow rate for sample application was lowered to 5 mg min − 1. The sensorgrams are given in Fig. 8. The BIAcore 2000 records a shift of 460 RU compared to a shift of about 9 pixels. This roughly agrees with the interconversion factor obtained with sucrose solutions (see Section 2.4.2).
2.4.4. Competiti6e immunoassay calibration cur6e for the detection of staphylokinase We performed a competitive immunoassay to determine the concentration of the antithrombolytic protein staphylokinase [13]. We used a staphylokinase-specific polyclonal rabbit antiserum to record calibration graphs. The calibration graph mainly reflects the specificity and binding properties of the applied antibodies. It can be used to measure the unknown staphylokinase
Fig. 8. Sensorgram for the binding of myoglobin to an immobilized monoclonal antibody. Reagents were purchased from BIAcore. Antibodies were immobilized to the sensor surface by aminocoupling as recommended by the manufacturer. (a) Baseline (b) binding of myoglobin to the immobilized antibody (c) new baseline after antigen binding (*) artefacts due to air bubbles in the detector cell. The air bubbles have been used to trap the sample volume in order to minimize sample dispersion.
concentration of samples. Its course should be independent from the used SPR-instrumentation. The antiserum was diluted with running buffer (10 mM HEPES, 150 mM NaCl, 0.005% (v/v) Tween 20, pH 7.4) to a final concentration of 750 ng ml − 1. Free staphylokinase was added in various concentrations as indicated in Fig. 9 and the mixtures were preincubated for 1 h at room temperature prior to use. The mixtures were applied to a CM-5 chip (BIAcore) which had been loaded with aminocoupled staphylokinase. Free and immobilized staphylokinase compete for the binding of antibodies. Thus, at higher concentrations of free staphylokinase only few antibodies can bind to the sensor chip and the SPR signal decreases. Measurements were carried out in a series of decreasing concentrations of staphylokinase. The chip was regenerated with injections of glycin (0.2 M, pH 2.8) and NaOH (0.1 M in 1 M NaCl) at a flow rate of 5 ml min − 1 before each measurement. The calibration graph was measured kinetically in the BIAcore 2000. The assay was carried out under mass transport-limited conditions [14]. Under such circumstances the binding rate kon of the antibodies to the immobilized staphylokinase is directly proportional to the concentration of staphylokinase. The antithrombolyticum was aminocoupled in high concentrations to a CM-5 chip. The bound protein was equivalent to an SPR-signal of 2600 RU. Samples preincubated with antibodies and known amounts of staphylokinase were applied for detection. Samples 5 ml in volume were injected at a flow rate of 1 ml min − 1. A simulation with the BIAsimulation software (version 2.1, BIAcore) showed that under these conditions the assumption of mass transport-limitation was justified. Binding rates (kon) were calculated using the BIAevaluation software package (version 2.1) and plotted against the concentration of added staphylokinase (Fig. 9). The Jena device lacks a well-designed microflux system which is a prerequirement for precise kinetic measurements. Instead of a kinetic assay a calibration graph was therefore recorded under equilibrium conditions. The preincubated antiserum/staphylokinase mixtures were directly applied to the sensor and incubated for 30 min at room temperature prior to detection. The SPR-signal output was plotted against the concentration of added staphylokinase. The calibration graph is given in Fig. 9.
2.4.5. Binding of Ni 2 + on a nitrilotriacetic acid-chip At the lower limit of detection we investigated whether the Jena SPR-device would be able to monitor the binding of nickel ions onto a metallochelating NTA-matrix. NTA is able to bind efficiently to nickel thus forming a Ni–NTA complex. Ni–NTA can efficiently be applied to bind proteins, which have been engineered to
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Fig. 9. Determination of staphylokinase in a competitive immunoassay. Staphylokinase was immobilized onto a CM-5 chip by amino-coupling. Samples of known staphylokinase concentration were preincubated with a fixed amount of staphylokinase-specific polyclonal antibodies. In the BIAcore 2000 instrument, the concentration of staphylokinase was measured kinetically under mass transport-limited conditions. In the Jena SPR-device, experiments were carried out as equilibrium experiments. In the latter case, samples were directly applied to the sensor surface and measured after equilibrium conditions had been established.
carry a stretch of histidyl residues (‘His-tag’). While His-tagged proteins are retained on a Ni – NTA chromatography column, other proteins are easily washed out. Retained His-tagged proteins are subsequently eluted with imidazol. This feature makes Ni – NTA a valuable tool for protein purification in biotechnology [18]. For non-covalent immobilization of proteins also NTA-sensor chips (BIAcore) have been introduced. These sensor chips must be loaded with nickel before use. Provided the SPR device has a sufficient sensitivity, the loading process can be monitored online. The experiment is easily carried out and is a simple test of both SPR-devices at the lower limits of detection. A 40 ml volume of 500 mM NiSO4 was applied to a NTA-sensor chip (BIAcore) at a flow rate of 5 ml min − 1. After extensive washing with carrier buffer (10 mM HEPES, 150 mM NaCl, 50 mM EDTA, 0.005% Tween 20, pH 7.4) the increase of the SPR-signal was detected. For regeneration of the chip, 40 ml of 100 mM EDTA were injected followed by washing the chip with carrier buffer. The sensorgram is given in Fig. 10.
3. Results and discussion We constructed and tested a surface plasmon resonance (SPR) biosensor device which uses polychromatic light. The binding of molecules within the volume of the evanescent wave leads to a change in the refractive
index. It is detected as a shift in the resonance wavelength of the reflected light. Since the angles of incidence and reflection are fixed, our device does not contain moveable parts. This construction principle is less sensitive to interference from mechanical vibrations. In addition, the integration of fiber optics offers the advantage to separate the detector head from the detecting unit. Therefore, our SPR-device can be considerably miniaturized and might be used for the construction of portable instruments for on-site analysis. Prior to further modifications we wanted to test the Jena SPR-device in biological applications and compare it with a high-end BIAcore 2000 instrument. The calculated sensory performance (see Section 2.3) is in good agreement with our experimental data. The calculated surface concentration of 14 pg mm − 2 is close to the 10 pg mm − 2 which is typical for the sensitivity of surface plasmon devices [1]. Surface plasmon resonance conditions change due to the binding of molecules on the sensing surface. This allows the immediate observation of biomolecular interactions. Specific molecular recognition is a prerequirement for the detection of analytes. Antibodies are well-known for their ability to specifically recognize their antigens and to bind to them. Thus, SPR is ideally suited for immunological assays [15]. In the first immunological application we monitored the recognition of myoglobin by a specific monoclonal antibody (see Section 2.4.3, Fig. 8). The antibody was covalently immoblized to the chip and the binding of
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Fig. 10. Sensorgram for the binding of Ni2 + on a NTA-chip. The flowrate was adjusted to 5 ml min − 1. (a) Baseline (b) application of 40 ml 500 mM NiSO4 for loading of the NTA-matrix with nickel (c) baseline after nickel binding (signal increase 3 pixels). Signal drops are due to refractive index changes caused by buffer changes. Larger artefacts (*) are due to air bubbles and the lack of an evolved microflux system.
myoglobin was observed. A comparison with the BIAcore sensorgram showed that the Jena SPR-device is suitable for investigating biomolecular interactions. Despite shortcomings due to the non-perfect microflux system, the Jena SPR-device allowed an immediate observation of the antigen binding to the sensor chip. The non-equilibrium measurement of molecular interactions is possible by a kinetic approach. Provided that mass transport-limited conditions are maintained, the binding rate of the analyte to the matrix is proportional to its concentration. This experiment requires a very precise microflux system which works with high reproducibility [16]. Since such a flux system is not available yet for our device, measurements with the Jena SPR-device were carried out as equilibrium experiments. In both cases the antigen was immobilized and the binding of free polyclonal antibodies to the sensor surface was monitored. Staphylokinase is an antithrombolytic pharmaprotein interrupting the blood coagulation cascade. It might be applied to the acute treatment of strokes or heart attacks [13]. For its sensoric detection we used a competitive immunosorbent assay (see Section 2.4.4) [17]. In Fig. 9 the kinetically measured calibration graph (measured by a BIAcore 2000) is directly compared with the graph that resulted from equilibrium experiments with the Jena detector. The BIAcore experiments were carried out under conditions of mass transport limitation. Therefore the observed binding rates are proportional to the analyte concentrations and can be compared with the signal
output we have measured for the equilibrium binding of our antibodies to the immobilized antigen. The polyclonal serum allows a reliable detection of staphylokinase in the nanomolar concentration range. The calibration graphs of a competitive assay should mainly reflect the properties of the antigen/antibody pair. Despite two very different approaches, the resulting graphs show the expected similarities. There are no major instrumental interferences from our Jena device that might hamper the sensitivity of the staphylokinase detection. Depending on the binding kinetics, the time demand for an equilibrium binding experiment can be considerably higher than in a kinetic measurement. Here, a time demand of 5 min for a kinetic assay compares to more than one hour for a single corresponding equilibrium experiment. The importance and time efficiency of a well-designed microflux system becomes evident. As can be seen from the quite similar course of the two graphs, both approaches are suitable for the detection of the antigen. We also developed an SPR-assay for the detection of endoproteolytic activity. It will be published elsewhere. Our immunological studies covered the main experimental approaches to monitor the antigen-antibody recognition, binding of the antigen to the immobilized antibody, binding of the antibody to the immobilized antigen, kinetic measurements and equilibrium assays. With the exception of kinetic studies the Jena SPRdevice though lacking a microflux system already proved suitable for such applications.
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Monitoring the biomolecular interaction of macromolecules gives a large SPR signal response. We wanted to determine the sensitivity of our detection system using small molecular weight compounds. Metal chelate chromatography uses the selective binding of histidinetagged proteins onto nickel ions that are chelated by a NTA matrix [18]. A chip with a NTA-surface is loaded with nickel ions to form a Ni-NTA sensor chip. In our experiment we were able to detect this binding of nickel ions to the NTA surface (see Section 2.4.5, Fig. 10). The resulting signal of :3 pixels is in good agreement with the : 100 RU observed with the BIAcore instrument. With a standard deviation of 0.5 pixel for our measurements, the signal increase was significant and reproducible. Even under a steady purging buffer flow the signal output was stable in the first 15 min. Our results demonstrate that the Jena SPR-device is a suitable instrument for measuring biomolecular interactions with high sensitivity. Even the binding of metal ions to the matrix could be detected. However, to obtain optimal performance our device still requires the integration of a well-designed microflux system. As an alternative, a stirred microcuvette might be integrated as a sensor which would allow the exact measurement of kinetic association (kass) and dissociation (kdiss) constants with considerably shortened detection times. The Jena SPR-device is measuring the shift of the resonance wavelength. It thus works as a spectrometer. In contrast to other SPR instruments no angle measurements are carried out. This offers a number of advantages: 1. Interferences due to mechanical vibrations are minimal. 2. The detector head and the instrument can be spatially separated using long distance fiber optical connections. 3. This concept enables the miniaturization of the detector head. 4. Disposable detector heads or even sensor arrays can be envisaged. 5. We can build portable systems for on-site measurements of analytes. 6. The Jena SPR-device can be manufactured with substantially less mechanical and technical expenditure.
Acknowledgements The authors gratefully acknowledge the support of the Institute of Physical High-Technology (IPHT) and the Institute for Molecular Biotechnology (IMB). Recombinant staphylokinase was a kind gift of Dr Bern.
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hard Schlott (IMB Jena). The staphylokinase specific rabbit antiserum was a kind gift of Dr Johannes Gumpert (IMB Jena). We also thank Stephan Sieben (IMB Jena) for fruitful discussions. This work was funded by the Thu¨ringer Ministerium fu¨r Wissenschaft, Forschung und Kultur (TMWFK), project number 3/ 95-31.
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Real time sensing of specific molecular binding using surface plasmon resonance spectroscopy Peter Pfeifer a,*, Ulrich Aldinger b, Gu¨nter Schwotzer a, Stephan Diekmann b, Peter Steinru¨cke b a b
Institut fu¨r Physikalische Hochtechnologie (IPHT), Helmholtzweg 4, 07743, Jena, Germany Institut fu¨r Molekulare Biotechnologie (IMB), Beutenbergstraße 10, 07745, Jena, Germany
Abstract A new planar surface plasmon resonance sensor with spectral interrogation was constructed and tested. The SPR device was directly compared with a high-end commercial BIAcore 2000 instrument. In immunological applications, we used the SPR-device for the detection of myoglobin and to measure the concentration of staphylokinase in a competitive assay. The lower limits of detection were tested when the binding of Ni2 + -ions on a matrix with nitrilotriacetic acid (NTA) was measured. The tested sensor was comparable in performance to the commercial instrument. Its detection of the plasmon resonance in a spectral mode and its fiber optical connection between sensor and detector unit offer additional advantages for future applications in on-site analysis with a portable detector. © 1999 Elsevier Science S.A. All rights reserved. Keywords: Surface plasmon resonance; Spectroscopy; Portable instrument; On-site measurements; Remote control
1. Introduction Surface plasmon resonance (SPR) is the result of the interaction between electromagnetic waves of the incident light and the free electron systems in a conducting surface layer [1]. The appearance of SPR relates to the phenomenon of internal reflection at an interface between a metal layer and a dielectricum. An electromagnetic evanescent surface wave propagates along the conducting layer of gold or silver. Within the volume of the evanescent wave biomolecular interactions are detected as alterations in the refractive index. This also changes the resonance conditions of the detection. The resonance angle is shifted at which the intensity of the reflected light becomes minimal. Alternatively the wavelength dependence of SPR can be used for detection. In this case polychromatic light is used for the excitation of the plasmons at a fixed angle of incidence. The refractive index change is measured as a shift, Dl, of the resonance wavelength (see Fig. 1).The evanescent wave decays exponentially with the distance from the interface. It effectively penetrates the lower refractive index medium to a depth of approximately one wave* Corresponding author.
length. Thus, SPR only detects changes in the refractive index very close to the surface. Surface plasmon resonance detectors do not require the labelling of the interacting components. The genuine molecular interaction can be directly observed. In 197l, Kretschmann [2] reported the determination of optical constants of thin metallic layers by measuring the resonance absorption of light by surface plasmon oscillations. In 1983, Liedberg et. al. [3] first demonstrated the exploitation of surface plasmon resonance (SPR) for chemical sensing. Since then many SPR sensor configurations based on bulk prism or optical fibers have been realised for biochemical applications [4–7]. A family of SPR-devices has been commercialised by BIAcore (Sweden) [8]. The instruments either use an angular interrogation in connection with a bulk prism, or apply a spectrometer with an optical multimode fiber as sensing element. We developed an alternative planar optical sensor with a technology based on SPR in the wavelength domain. In this paper we report on its sensitivity and on the results of a comparative study with a high-end BIAcore 2000 instrument. Both SPR-devices were tested in biological applications. Our instrument shows a comparable sensitivity but also has the potential for
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Fig. 1. Detection principle of the Jena SPR-device. Biomolecular interactions at the sensing surface layer are monitored as a shift, Dl, in the resonance wavelength.
miniaturisation and remote sensing, combined with the advantages and sensitivity of a planar bulk device as described by Robinson [9]. Our sensor configuration uses the Kretschmann geometry [2] consisting of a glass prism and a glass slide with a surface plasmon active gold layer. Collimated light from a broad-band source excites a surface plasmon wave on a prism base at the surface between the
gold layer and the sensed medium. The excitation of the surface plasmon resonance is detectable as a minimum in the intensity of the reflected light at a particular wavelength. Since the surface plasmon resonance strongly depends on the refractive index of the medium adjacent to the gold layer, changes in the optical properties of the sensed medium can be measured by determining the resonance wavelength at which SPR occurs.
Fig. 2. Calculated shift of the SPR-wavelength vs. refractive index for the layer system, BK7 glass with 50 nm gold surface layer. (A) aqueous bulk environment (open symbols), (B) hydrogel matrix with 100 nm thickness as in the BIAcore CM-5 chip (filled symbols). Angles of incidence used for the calculation: 65.8°; " 67.5°.
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Fig. 3. The Jena SPR-device. For details see text. Experimental setup with spectral interrogation.
2. Experimental Our aim was to calculate and measure the sensitivity of the Jena SPR-device and to test its suitability as an analytical tool for the investigation of biomolecular interactions. For comparisons we used the BIAcore 2000 instrument, a high-end SPR-instrument which measures the binding of molecules on the sensing surface as a shift in the resonance angle of the monochromatic reflected light. In contrast, the Jena SPR-device works at fixed angles. Binding of molecules on the sensor surface is detected as a shift of the resonance wavelength of the polychromatic reflected light. Experiments on both instruments were done with commercial sensor chips from BIAcore.
2.1. Theoretical determination of the sensiti6ity We calculated the shift in the resonance wavelength caused by changes of the refractive index of the sensed medium using the well-established matrix method for optical layers. These calculations were carried out for two configurations of the sensor: (i) by variations of the refractive index of the bulk environment; and (ii) by monitoring the changes of the refractive index which are caused by homogeneous adsorption of proteins within a 100 nm-thick aqueous hydrogel matrix adjacent to the gold layer. Our calculations are based on assuming all glass substrates being made of BK7 glass [10] with 50 nm thin gold layers [11]. Calculations were done for a spectral range from 700 to 950 nm and for angles of the incident polychromatic light of 65.8 and 67.5°. Fig. 2 displays the dependence of the SPR wave-
length on changes of the refractive index of the adjacent bulk medium. We changed the refractive index of the sensed aqueous medium on a gold surface from n= 1.33 (water) to n= 1.36. The slope of the curves increases with higher refractive indices. This indicates an increasing sensitivity of the device for media with higher refractive index. The signal dependence on the fixed angle of incidence is also illustrated, an increase in sensitivity is shown for smaller angles of incidence. Fig. 2 also indicates a decrease in sensitivity if the bulk medium is replaced by a 100 nm-thick hydrogel matrix adjacent to the gold layer. The general behaviour of the detector is as described before. The sensitivity however is smaller. The reason is that the penetration depth of the evanescent SPR-wave is larger than the thickness of the binding matrix. The sensitivity of the sensor was estimated for the case of protein adsorption to the matrix adjacent to the gold layer. Concentration changes, Dc, linearly correlate with changes in the refractive index, Dn, according to Dc = Dn × dc/dn. For protein molecules we assume a value of dc/dn = 5 g cm − 3 [4]. Angles of incidence of 65.8 and 67.5° were chosen. For a refractive index of the matrix of n= 1.33 (water) and an angle of incidence of 67.5° we obtain Dl/Dc =360 nm (g cm − 3) − 1 from Fig. 2. The spectral analysis in our instrument is performed by a polychromator with an assumed accuracy of 0.1 nm. Taking into account the matrix thickness of 100 nm this results in a detectable surface concentration of 27 pg mm − 2. Changing the parameters to 65.8° for the angle of incidence and n= 1.36 for the refractive index would decrease this level to a surface concentration of 14 pg mm − 2.
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Fig. 4. Schematic design of the flow cell. The volume of the cell was : 2.5 ml.
2.2. Instrumentation
2.3. Sensory performance
The sensor uses a broad band optical source, a sensing head and a spectrum analyser (Fig. 3). The sensing head comprises a 69°–42° – 69° prism made from BK7 glass. For temperature control it was mounted on a thermoelectric cooler (Peltier element). As a light source we used a miniature halogen lamp which was connected to a collimating lens optics by a multimode optical fiber. The reflected light was also coupled into a multimode fiber by collimating optics and analysed by a high resolution spectrum analyser. In order to detect only the TM-polarized light a foil polarizer was inserted into the collimated beam. In our experiments, a PC controlled high resolution polychromator with CCD detector was employed which had been developed at the Institute for Physical High Technology, Jena [12]. It has a dispersion of 0.18 nm per CCD pixel. Within the chosen spectral range from 730 to 910 nm this allows us to measure the shift in the SPR wavelength with a resolution of better than 0.1 nm. Resonance wavelength shifts are detected as changes in the pixel ouput of the CCD-detector. The detector pixel at which the intensity of the measured light is minimal correlates to the SPR-wavelength. In all experiments we have used commercial sensor chips from BIAcore. The chips were attached to the glass prism with a low-viscous immersion oil [n = 1.515, Novodirekt (Germany)]. A small chamber (volume 2.5 ml) was made from Teflon and a silicon ring which was pressed to the chip by a fixing screw to create a flow cell (see Fig. 4). We used this setup to measure the sensory performance of the Jena SPR-device.
We characterised the sensitivity, dynamic range and the resolution of our sensor. The chamber was filled with a buffer solution [10 mM N-2-hydroxyethylpiperazine-N%-2-ethanesulfonic acid (HEPES, from SIGMA Germany) pH 7.4, n= 1.335] and with a solution of 15% (w/v) sucrose in 10 mM HEPES, pH 7.4 (n= 1.357). As shown in Fig. 5 the SPR-wavelength changes by :792 pixel or 141 nm. With a refractive index change of Dn = 0.022 between HEPES and sucrose/HEPES buffers we can calculate an average sensitivity of 6400 nm per refractive index unit (RIU). This is in good agreement with our calculations from Fig. 2 (6900 nm RIU − 1). We also measured the resolution of the sensor. The probe chamber was filled with water. The temperature of the sensing head was increased in steps of 0.5 K over a range of 2.5 K. Changes in SPR wavelength are mainly caused by temperature dependent alterations of the water refractive index. Assuming a change of Dn/ DT = 1×10 − 4 K − 1 we obtained a sensitivity of 7120 nm RIU − 1 (theoretical 8000 nm RIU − 1). Fig. 6 shows a noise level of : 0.02 nm for this experiment. The Jena device thus can resolve changes in refractive index of about 3× 10 − 6.
2.4. Biological applications As a next step we analysed biomolecular interactions. The BIAcore 2000 instrument was used as a reference system. For both instruments sensor chips from BIA-
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Fig. 5. Shift in the SPR-response upon changing the refractive index of the bulk environment. The flow cell was filled with (a) and (c) HEPES solution (n =1.335) and (b) with solution of 15% sucrose in HEPES (n =1.357). As measured on the Jena device, 1 pixel corresponds to a resonance wavelength shift of 0.178 nm. The angle of incidence was 67.5°.
core were used thus minimizing differences in the architecture of the sensing surface. Antibodies provide an excellent tool for the specific molecular recognition of biomolecules. We monitored the recognition of myoglobin by a monoclonal antibody immobilized to the sensor chip. We further investigated the immuno-recognition of an antithrombolytic
pharmaceutical protein, staphylokinase [13], by a polyclonal rabbit antiserum in a competitive immunoassay. To determine the mass sensitivity of our device, we measured the binding of a low-molecular weight ligand to the matrix of a sensor chip. We were able to detect the binding of Ni2 + -ions to a chip covered with a metallochelating matrix (nitrilotriacetic acid, NTA).
Fig. 6. Temperature dependence of the SPR-response. The shift in SPR-response was measured by changing the temperature of the sensor in steps of 0.5 K over a range of 2.5 K. As measured by the Jena device, 1 pixel corresponds to a resonance wavelength shift of 0.178 nm. The angle of incidence was 65.8°.
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Fig. 7. Signal comparison between the BIAcore 2000 and the Jena SPR-device. Sucrose solutions of different concentrations were injected. The flow rate was 1 ml min − 1.
2.4.1. Sample application A flow-cell was formed by a Teflon body attached to the sensor chip by a silicon ring of 0.2 mm height and 4 mm diameter. The inlet and outlet were realised by two drill-holes in the Teflon body which fit to injection needles (see Fig. 4). The volume of the flow-cell was : 2.5 ml. A high precision microflux system as in the BIAcore 2000 instrument was not available for our device. Samples were manually injected into a sample loop. We used an Omnifit (Machery Nagel, Du¨ren, Germany) injection septum and a Hamilton (Darmstadt, Germany) syringe for this purpose. To avoid dispersion effects within the sample solution, the sample probe was trapped between two air bubbles. Teflon and silicon tubes connecting the detector parts were kept at a minimum length. The internal tube diameter was either 0.5 or 1 mm. Sample loop and carrier flow were separated by a 4-way valve (Hamilton HVP4-4, Darmstadt, Germany). Sample binding to the sensor chips was either carried out in a flow of carrier buffer or by direct application of the material onto the sensor field with a micropipette. In the latter case the chip was subsequently incubated in a petri dish under gentle shaking for the specified time and under water-saturated vapour conditions. A Hamilton dispensor Microlab 541 B (Hamilton, Darmstadt, Germany) was used for all flow applications. In control experiments, the signal output of the BIAcore 2000 instrument was corrected for unspecific binding according to the manufacturer’s instructions. Data obtained with the Jena device were
corrected likewise. For data processing, we used a PC (Peacock, Germany) under MS-DOS 6.2 with a Kethley DAS 801 data acquisition board (Keathley, Munich, Germany) for A/D conversion. If not stated otherwise, the sampling rate was adjusted to 0.36 Hz. Recorded spectral data of the Jena SPR-device were processed using Microcal Origin 5.0 (Microcal Software, Northampton, USA) under Microsoft Windows NT4.0 For calculations with the BIAcore 2000 data we used the BIAevaluation and BIAsimulation software of BIAcore.
2.4.2. Signal comparison The direct comparison of the BIAcore 2000 and the Jena SPR-device required an interconversion factor for the signal outputs of both SPR instruments. We used the signal output of sucrose solutions [concentrations between 20% (w/v) and 0.5% (w/v)] to calibrate the data of both instruments. A signal response of 1 pixel on the Jena detector equals 41 resonance units (RU) on the BIAcore 2000 instrument (see Fig. 7). 2.4.3. Immunological detection of myoglobin In the BIAcore 2000 instrument, myoglobin was detected using a CM-5 chip (BIAcore). All chemicals were from BIAcore. The chip carries a hydrogel of carboxymethylated dextran which was activated with 1ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Monoclonal antibodies were aminocoupled to the chip surface according to the BIAcore protocol. For biomolecular interaction with the antigen, 40 ml of
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recombinant human myoglobin (2.5 mg ml − 1) were applied at a flow rate of 10 ml min − 1. The same reagents were used in the Jena detector. Due to the larger dimensions of the detector cell, all sample volumes were doubled and the flow rate for sample application was lowered to 5 mg min − 1. The sensorgrams are given in Fig. 8. The BIAcore 2000 records a shift of 460 RU compared to a shift of about 9 pixels. This roughly agrees with the interconversion factor obtained with sucrose solutions (see Section 2.4.2).
2.4.4. Competiti6e immunoassay calibration cur6e for the detection of staphylokinase We performed a competitive immunoassay to determine the concentration of the antithrombolytic protein staphylokinase [13]. We used a staphylokinase-specific polyclonal rabbit antiserum to record calibration graphs. The calibration graph mainly reflects the specificity and binding properties of the applied antibodies. It can be used to measure the unknown staphylokinase
Fig. 8. Sensorgram for the binding of myoglobin to an immobilized monoclonal antibody. Reagents were purchased from BIAcore. Antibodies were immobilized to the sensor surface by aminocoupling as recommended by the manufacturer. (a) Baseline (b) binding of myoglobin to the immobilized antibody (c) new baseline after antigen binding (*) artefacts due to air bubbles in the detector cell. The air bubbles have been used to trap the sample volume in order to minimize sample dispersion.
concentration of samples. Its course should be independent from the used SPR-instrumentation. The antiserum was diluted with running buffer (10 mM HEPES, 150 mM NaCl, 0.005% (v/v) Tween 20, pH 7.4) to a final concentration of 750 ng ml − 1. Free staphylokinase was added in various concentrations as indicated in Fig. 9 and the mixtures were preincubated for 1 h at room temperature prior to use. The mixtures were applied to a CM-5 chip (BIAcore) which had been loaded with aminocoupled staphylokinase. Free and immobilized staphylokinase compete for the binding of antibodies. Thus, at higher concentrations of free staphylokinase only few antibodies can bind to the sensor chip and the SPR signal decreases. Measurements were carried out in a series of decreasing concentrations of staphylokinase. The chip was regenerated with injections of glycin (0.2 M, pH 2.8) and NaOH (0.1 M in 1 M NaCl) at a flow rate of 5 ml min − 1 before each measurement. The calibration graph was measured kinetically in the BIAcore 2000. The assay was carried out under mass transport-limited conditions [14]. Under such circumstances the binding rate kon of the antibodies to the immobilized staphylokinase is directly proportional to the concentration of staphylokinase. The antithrombolyticum was aminocoupled in high concentrations to a CM-5 chip. The bound protein was equivalent to an SPR-signal of 2600 RU. Samples preincubated with antibodies and known amounts of staphylokinase were applied for detection. Samples 5 ml in volume were injected at a flow rate of 1 ml min − 1. A simulation with the BIAsimulation software (version 2.1, BIAcore) showed that under these conditions the assumption of mass transport-limitation was justified. Binding rates (kon) were calculated using the BIAevaluation software package (version 2.1) and plotted against the concentration of added staphylokinase (Fig. 9). The Jena device lacks a well-designed microflux system which is a prerequirement for precise kinetic measurements. Instead of a kinetic assay a calibration graph was therefore recorded under equilibrium conditions. The preincubated antiserum/staphylokinase mixtures were directly applied to the sensor and incubated for 30 min at room temperature prior to detection. The SPR-signal output was plotted against the concentration of added staphylokinase. The calibration graph is given in Fig. 9.
2.4.5. Binding of Ni 2 + on a nitrilotriacetic acid-chip At the lower limit of detection we investigated whether the Jena SPR-device would be able to monitor the binding of nickel ions onto a metallochelating NTA-matrix. NTA is able to bind efficiently to nickel thus forming a Ni–NTA complex. Ni–NTA can efficiently be applied to bind proteins, which have been engineered to
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Fig. 9. Determination of staphylokinase in a competitive immunoassay. Staphylokinase was immobilized onto a CM-5 chip by amino-coupling. Samples of known staphylokinase concentration were preincubated with a fixed amount of staphylokinase-specific polyclonal antibodies. In the BIAcore 2000 instrument, the concentration of staphylokinase was measured kinetically under mass transport-limited conditions. In the Jena SPR-device, experiments were carried out as equilibrium experiments. In the latter case, samples were directly applied to the sensor surface and measured after equilibrium conditions had been established.
carry a stretch of histidyl residues (‘His-tag’). While His-tagged proteins are retained on a Ni – NTA chromatography column, other proteins are easily washed out. Retained His-tagged proteins are subsequently eluted with imidazol. This feature makes Ni – NTA a valuable tool for protein purification in biotechnology [18]. For non-covalent immobilization of proteins also NTA-sensor chips (BIAcore) have been introduced. These sensor chips must be loaded with nickel before use. Provided the SPR device has a sufficient sensitivity, the loading process can be monitored online. The experiment is easily carried out and is a simple test of both SPR-devices at the lower limits of detection. A 40 ml volume of 500 mM NiSO4 was applied to a NTA-sensor chip (BIAcore) at a flow rate of 5 ml min − 1. After extensive washing with carrier buffer (10 mM HEPES, 150 mM NaCl, 50 mM EDTA, 0.005% Tween 20, pH 7.4) the increase of the SPR-signal was detected. For regeneration of the chip, 40 ml of 100 mM EDTA were injected followed by washing the chip with carrier buffer. The sensorgram is given in Fig. 10.
3. Results and discussion We constructed and tested a surface plasmon resonance (SPR) biosensor device which uses polychromatic light. The binding of molecules within the volume of the evanescent wave leads to a change in the refractive
index. It is detected as a shift in the resonance wavelength of the reflected light. Since the angles of incidence and reflection are fixed, our device does not contain moveable parts. This construction principle is less sensitive to interference from mechanical vibrations. In addition, the integration of fiber optics offers the advantage to separate the detector head from the detecting unit. Therefore, our SPR-device can be considerably miniaturized and might be used for the construction of portable instruments for on-site analysis. Prior to further modifications we wanted to test the Jena SPR-device in biological applications and compare it with a high-end BIAcore 2000 instrument. The calculated sensory performance (see Section 2.3) is in good agreement with our experimental data. The calculated surface concentration of 14 pg mm − 2 is close to the 10 pg mm − 2 which is typical for the sensitivity of surface plasmon devices [1]. Surface plasmon resonance conditions change due to the binding of molecules on the sensing surface. This allows the immediate observation of biomolecular interactions. Specific molecular recognition is a prerequirement for the detection of analytes. Antibodies are well-known for their ability to specifically recognize their antigens and to bind to them. Thus, SPR is ideally suited for immunological assays [15]. In the first immunological application we monitored the recognition of myoglobin by a specific monoclonal antibody (see Section 2.4.3, Fig. 8). The antibody was covalently immoblized to the chip and the binding of
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Fig. 10. Sensorgram for the binding of Ni2 + on a NTA-chip. The flowrate was adjusted to 5 ml min − 1. (a) Baseline (b) application of 40 ml 500 mM NiSO4 for loading of the NTA-matrix with nickel (c) baseline after nickel binding (signal increase 3 pixels). Signal drops are due to refractive index changes caused by buffer changes. Larger artefacts (*) are due to air bubbles and the lack of an evolved microflux system.
myoglobin was observed. A comparison with the BIAcore sensorgram showed that the Jena SPR-device is suitable for investigating biomolecular interactions. Despite shortcomings due to the non-perfect microflux system, the Jena SPR-device allowed an immediate observation of the antigen binding to the sensor chip. The non-equilibrium measurement of molecular interactions is possible by a kinetic approach. Provided that mass transport-limited conditions are maintained, the binding rate of the analyte to the matrix is proportional to its concentration. This experiment requires a very precise microflux system which works with high reproducibility [16]. Since such a flux system is not available yet for our device, measurements with the Jena SPR-device were carried out as equilibrium experiments. In both cases the antigen was immobilized and the binding of free polyclonal antibodies to the sensor surface was monitored. Staphylokinase is an antithrombolytic pharmaprotein interrupting the blood coagulation cascade. It might be applied to the acute treatment of strokes or heart attacks [13]. For its sensoric detection we used a competitive immunosorbent assay (see Section 2.4.4) [17]. In Fig. 9 the kinetically measured calibration graph (measured by a BIAcore 2000) is directly compared with the graph that resulted from equilibrium experiments with the Jena detector. The BIAcore experiments were carried out under conditions of mass transport limitation. Therefore the observed binding rates are proportional to the analyte concentrations and can be compared with the signal
output we have measured for the equilibrium binding of our antibodies to the immobilized antigen. The polyclonal serum allows a reliable detection of staphylokinase in the nanomolar concentration range. The calibration graphs of a competitive assay should mainly reflect the properties of the antigen/antibody pair. Despite two very different approaches, the resulting graphs show the expected similarities. There are no major instrumental interferences from our Jena device that might hamper the sensitivity of the staphylokinase detection. Depending on the binding kinetics, the time demand for an equilibrium binding experiment can be considerably higher than in a kinetic measurement. Here, a time demand of 5 min for a kinetic assay compares to more than one hour for a single corresponding equilibrium experiment. The importance and time efficiency of a well-designed microflux system becomes evident. As can be seen from the quite similar course of the two graphs, both approaches are suitable for the detection of the antigen. We also developed an SPR-assay for the detection of endoproteolytic activity. It will be published elsewhere. Our immunological studies covered the main experimental approaches to monitor the antigen-antibody recognition, binding of the antigen to the immobilized antibody, binding of the antibody to the immobilized antigen, kinetic measurements and equilibrium assays. With the exception of kinetic studies the Jena SPRdevice though lacking a microflux system already proved suitable for such applications.
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Monitoring the biomolecular interaction of macromolecules gives a large SPR signal response. We wanted to determine the sensitivity of our detection system using small molecular weight compounds. Metal chelate chromatography uses the selective binding of histidinetagged proteins onto nickel ions that are chelated by a NTA matrix [18]. A chip with a NTA-surface is loaded with nickel ions to form a Ni-NTA sensor chip. In our experiment we were able to detect this binding of nickel ions to the NTA surface (see Section 2.4.5, Fig. 10). The resulting signal of :3 pixels is in good agreement with the : 100 RU observed with the BIAcore instrument. With a standard deviation of 0.5 pixel for our measurements, the signal increase was significant and reproducible. Even under a steady purging buffer flow the signal output was stable in the first 15 min. Our results demonstrate that the Jena SPR-device is a suitable instrument for measuring biomolecular interactions with high sensitivity. Even the binding of metal ions to the matrix could be detected. However, to obtain optimal performance our device still requires the integration of a well-designed microflux system. As an alternative, a stirred microcuvette might be integrated as a sensor which would allow the exact measurement of kinetic association (kass) and dissociation (kdiss) constants with considerably shortened detection times. The Jena SPR-device is measuring the shift of the resonance wavelength. It thus works as a spectrometer. In contrast to other SPR instruments no angle measurements are carried out. This offers a number of advantages: 1. Interferences due to mechanical vibrations are minimal. 2. The detector head and the instrument can be spatially separated using long distance fiber optical connections. 3. This concept enables the miniaturization of the detector head. 4. Disposable detector heads or even sensor arrays can be envisaged. 5. We can build portable systems for on-site measurements of analytes. 6. The Jena SPR-device can be manufactured with substantially less mechanical and technical expenditure.
Acknowledgements The authors gratefully acknowledge the support of the Institute of Physical High-Technology (IPHT) and the Institute for Molecular Biotechnology (IMB). Recombinant staphylokinase was a kind gift of Dr Bern.
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hard Schlott (IMB Jena). The staphylokinase specific rabbit antiserum was a kind gift of Dr Johannes Gumpert (IMB Jena). We also thank Stephan Sieben (IMB Jena) for fruitful discussions. This work was funded by the Thu¨ringer Ministerium fu¨r Wissenschaft, Forschung und Kultur (TMWFK), project number 3/ 95-31.
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