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Advancements toward magneto immunoassays
Biosensors & Bioelectronics 13 (1998) 817–823
Advancements toward magneto immunoassays Kirstin Kriz b
a,b
, Janin Gehrke a, Dario Kriz
a,b,*
a European Institute of Science, Research Park Ideon, SE-223 70 Lund, Sweden Department of Pure and Applied Biochemistry, Chemical Center, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden
Received 16 February 1998; accepted 17 April 1998
Abstract Recently, a magneto binding assay was conducted. The operational principle is based on a ‘sandwich’ mode of detection, where the target analyte (Concanavalin A) becomes bound nonselectively by protein adsorption between the solid support (silica carrier particles) and the ferromagnetic label (magneto markers). A magnetic transducer was employed for the detection. The binding assay gave a linear response in the dynamic range of 0–1.9 M and proved to be sensitive by having a limit of detection at 250 nM. Furthermore, the relative standard deviation observed was 6.2% (n = 3). The demonstration of the ‘sandwich’ approach is a step towards achieving magneto immunoassays (MIA). 1998 Elsevier Science S.A. All rights reserved. Keywords: Magneto immunoassay; MIA; Ferrofluid; Ferromagnetic markers
1. Introduction Immunological techniques, such as immunoassays, have employed antibodies for the detection and quantification of specific molecules even in the presence of compounds with similar molecular structures. These highly quantitative assays are achieved by combining the high specificity of the biological recognition occurring at the molecular level with the sensitivity of different labelling techniques. Typically, immunoassays have involved the use of antibodies or antigens which have been coupled to markers such as radioactive compounds, enzymes, fluorophores and luminescence markers (Anderson et al., 1997). Radioactive markers were employed in radioimmunoassays (RIA) in 1959 (Yalow and Berson, 1959). Even though these markers offer high sensitivities (1– 500 pM) when used in RIA, they are expensive, exhibit low stability, require high-cost equipment for their detection, and they present radioactive hazards for the personnel. The use of enzyme markers is more popular in smaller laboratories or hospital clinics due to the fact that they are relatively cheap, quite stable and they lack the radiological hazards (Tijssen, 1985). However, these
* Corresponding author. Tel.: + 46-46-182230; Fax: + 46-46182499; E-mail:[email protected]
markers generally give lower sensitivities than radioactive markers when used in immunoassays (Oellerich, 1980). Additionally, many research groups have focused on the development of fluorophores and luminescence markers (Anderson et al., 1997). These markers offer sensitivities normally associated with radioactive markers but possess similar advantages analogous to the enzyme markers. It has previously been suggested to use magneto markers in combination with biosensor and bioassay technologies (Kriz and Kriz, 1995; Rohr, 1995; Kriz et al., 1996). These markers comprise a ferromagnetic iron oxide core coated with dextran and are therefore extremely stable, non-toxic and fairly cheap. The underlying principles for magnetic determination were previously outlined by our group. The instrumentation used for the detection of the magneto markers employs a magnetic transducer which comprises a coil placed in a Maxwell bridge (Kriz et al., 1996). Consequently, the change in magnetic permeability of a compound is detected using inductance measurements and, furthermore, analyte detection has been achieved directly (the analyte was labelled ferromagnetically) or competitively (competition between ferromagnetic labelled and unlabelled analytes exists for binding sites on the transducer) using the aforementioned device. The use of magnetic transducers in combination with ferromagnetic markers has been shown to offer advantages such as, low interference, little or no background signal, no transducer fouling and no sample pretreatment.
0956-5663/98/$—see front matter 1998 Elsevier Science S.A. All rights reserved. PII: S 0 9 5 6 - 5 6 6 3 ( 9 8 ) 0 0 0 4 7 - 5
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K. Kriz et al. / Biosensors & Bioelectronics 13 (1998) 817–823
In the work reported in this paper, we have further developed one of the possible applications of the magneto markers by taking a step closer towards achieving a magneto immunoassay (MIA). Feasibility studies were conducted to determine whether a ‘sandwich’ configuration (carrier–analyte–marker) could be employed for the detection of Concanavalin A in a binding assay based on passive protein adsorption. Further characterization of the binding kinetics and assay environment was conceived.
2. Experimental 2.1. Preparation of the magneto markers The magneto markers were produced in house at the European Institute of Science (EURIS) after modification of the methods reported earlier (Molday et al., 1977; Wikstro¨m et al., 1978; Molday and Molday, 1984; Kronick and Gilpin, 1986;). Accordingly, 0.7 mmol FeCl3·6H2O (Merck, Germany) and 0.4 mmol FeSO4·7H20 (Apoteksbolaget, Sweden) were dissolved in 0.5 ml distilled H2O and 0.75 g dextran MW 70 000 (Sigma, USA) was dissolved in 3.0 ml of distilled H2O (0.25 g/ml). The iron and dextran solutions were then mixed together and heated to 333K. Subsequently, 3 ml of a 7.5% ammonia solution (Sigma, USA) was added to the iron/dextran solution and left to incubate at 333K for 15 min. Vigorous stirring occurred periodically during the incubation. Large particles and aggregates were removed by centrifuging the solution at 5 000 rpm for 30 min. Lastly, the magneto markers were dialysed against distilled H2O overnight using a Spectra/Por cellulose ester dialysis membrane MWCO 300 000 (Spectrum, USA) and filtered through a 200 nm filter (Sartorius, Germany). 2.2. Evaluation of the magneto markers From the prepared magneto marker solution, which was defined to be 100% (1000 g/l), a series of aqueous solutions containing different concentrations (0, 10, 100, 250, 500, 1000 g/l) of magneto markers were made. Their relative magnetic permeabilities (arbitrary units in mV) were measured with a home-made magnetic permeability meter produced by EURIS, which employs a Maxwell bridge. The magnetic permeability determination is based on the principle that the output signal from the Maxwell bridge is proportional to the concentration of magneto markers in solution established previously by Kriz et al., 1996. Illustrated in Fig. 1, is the current hand-held device, which is a portable variant of the original magnetic permeability meter used in this work.
Fig. 1. The current magnetic permeability meter; a portable version of the device used to measure magnetic permeability in this report.
2.3. Binding kinetics of protein adsorption (BSA) to silica carrier particles Accordingly, 1.0 g silica particles (0.2–0.5 mm, Merck, Germany) were suspended in a 5 ml, 40 mM Tris buffer, pH 7.0, solution containing 0.2 mg/ml BSA (Sigma, USA). To evaluate the binding kinetics of BSA (mg) adsorption, the particles were allowed to sediment and then the supernatant was removed and its absorbance was measured at 280 nm at 0, 1, 15 and 30 min, respectively. 2.4. Effect of buffer concentration on adsorption of protein (BSA) to the silica carrier particles Accordingly, 1.0 g silica particles (0.2–0.5 mm) were suspended in 5 ml distilled H2O and subsequently, the particles were allowed to sediment and the supernatant was removed. This washing procedure was repeated 10 times with the silica particles in order to remove fines which would disturb future absorbance measurements. Following the last washing step, the silica particles were incubated for 40 min in 5 ml solutions containing 0.2 mg/ml BSA and the following buffer concentrations: 0, 10, 40, 70, 100, 200 and 500 mM Tris buffer, pH 7.0. Subsequently, the particles were allowed to sediment and the absorbance of the supernatant was measured at 280 nm to determine the disappearance of BSA (mg) from the solution. 2.5. The magneto binding assay ‘sandwich’ approach The operational principle (shown in Fig. 2) for the magneto binding assay is based on the use of magneto markers (20–200 nm) in conjunction with larger silica carrier particles (0.2–0.5 mm). In the presence of the magneto markers and silica particles, the target protein, Concanavalin A (Sigma, USA), will adsorb to the surface of the silica particles and attached itself to the outer
K. Kriz et al. / Biosensors & Bioelectronics 13 (1998) 817–823
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2.7. The magneto binding assay detection of Concanavalin A
Fig. 2. The ‘sandwich’ approach used in the magneto binding assay, where the target analyte is bound between the silica carrier particles and the magneto markers (A). Subsequent sedimentation of the protein/particle complex (B) allows the magnetic permeability meter to measure the enrichment of magnetic markers at the bottom of the vial (C).
dextran layer of the magneto markers. Thus, a ‘sandwich’ effect is observed in solution where the target analyte is held between the silica carriers and the magneto markers. Subsequently, the weight (due to their higher density) of the silica carrier particles will cause the protein/particle complex to sediment to the bottom of the vial. Lastly, the vial is placed in the home-made magnetic permeability meter shown in Fig. 1, which measures the enrichment of the magnetic markers at the bottom of the vial. This ‘sandwich’ approach is the basis for the detection of Concanavalin A in all subsequent binding assays reported here in this work. 2.6. Influence of the magneto marker concentration on the magneto binding assay response Correspondingly, 4 ml of a 1.5 M Concanavalin A solution in 20 mM Tris buffer (10 mM MnCl2, 10 mM CaCl2, pH 7.4) was added to six different tubes each containing 0.25 g silica particles (0.2–0.5 mm). Subsequently, magneto markers were added to their respective tubes (yielding the respective final concentrations 1.25, 5, 12.5, 25, 50 and 125 g/l) and incubated overnight at room temperature. In the morning each sample was washed with 20 mM Tris buffer (10 mM MnCl2, 10 mM CaCl2, pH 7.4) by allowing the silica particles (containing adsorbed protein labelled with magneto markers) to sediment for 5 min, removing the supernatant and then resuspending the silica particles in same amount of Tris buffer which was removed. Lastly, the particles were allowed to sediment again and the magnetic permeability of the samples was measured with the magnetic permeability meter.
In several test tubes each containing 0.25 g silica particles (0.2–0.5 mm), 4 ml of a Concanavalin A solution in 20 mM Tris buffer (10 mM MnCl2, 10 mM CaCl2, pH 7.4) was added. The final Concanavalin A concentration of each tube ranged from 0 to 5.6 M. Accordingly, magneto markers were then added, to a final concentration of 12.5 g/l, to each of the test tubes and incubated for 4 h at room temperature. Shaking of the samples occurred periodically throughout the incubation. Subsequently, the samples were washed with 20 mM Tris buffer (10 mM MnCl2, 10 mM CaCl2, pH 7.4) by allowing the silica particles (containing adsorbed protein labelled with magneto markers) to first sediment for 5 min, followed by removing the supernatant and then resuspending the silica particles in the same amount of Tris buffer which was removed. Lastly, the particles were allowed to sediment again and the magnetic permeability of the samples were measured with the magnetic permeability meter.
3. Results and discussion 3.1. Preparation and evaluation of the magneto markers A brown, colloidal solution was obtained from the preparation procedure, which exhibited magnetic properties (the meniscus was pulled towards the wall of the test tube) when in close proximity to a permanent magnet. Additionally, the magneto marker preparate was filtered through a filter with a pore size of 200 nm in order to define the upper limit of the particle size. The lower limit of the particle size was determined during the preparation procedure since the preparate was dialysed with a membrane which had a molecular weight cut-off of 300 000. Therefore, everything smaller than approximately 20 nm could freely diffuse out of the membrane. Thus, we determined the magneto markers to range in size from 20 to 200 nm. Confirmation of size distribution was done by gel chromatography as earlier reported (Kriz et al., 1996). A calibration curve was established for further evaluation of the magneto marker preparate. Illustrated in Fig. 3 is the magneto marker calibration curve where the relative magnetic permeability (mV) is plotted against the magneto marker concentration (g/l). As shown in the graph a linear regression (y = − 0.56 + 0.042x, R = 0.99941) was obtained. The calibration curve was repeated and the slope of each obtained linear regression was used in the determination of the relative standard deviation (R.S.D.). The calculated R.S.D. was 10.4% for n = 4. The R.S.D. was relatively high due to the wide range (0–40 mV) of magnetic permeabilities
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BSA can adsorb per gram silica at a buffer concentration of 40 mM.
3.2.1. Binding kinetics The binding kinetics of BSA adsorption to 1.0 g silica particles are shown in Fig. 4, in which the amount of adsorbed BSA (mg) is plotted against the incubation time (min). Consequently, it was concluded that the binding kinetics was a fast process which was confirmed by the fact that 95% of the maximum BSA adsorption was achieved in approximately 4 min. Thus, it can be calculated from the maximum adsorption that 0.22 mg
3.2.2. Effect of buffer concentration on protein adsorption The relationship between buffer concentration and protein adsorption is illustrated in Fig. 5, where the amount of adsorbed BSA (mg) to 1.0 g silica particles is plotted against the concentration of Tris buffer (mM), pH 7.0. As shown in the graph, at low salt concentrations ( ⱕ 40 mM) maximum protein adsorption is observed ( ⱖ 0.28 mg). Conversely, at high salt concentrations ( ⱖ 400 mM) little or no protein adsorption ( ⬍ 0.01 mg) is observed. Additionally, silica particles which contained 0.28 mg adsorbed BSA were incubated overnight in 500 mM Tris buffer, pH 7.0. After the overnight incubation, all of the adsorbed protein had been washed from the particles and remained in the supernatant. Thus, the noncovalent interactions (hydrophobic, ionic and hydrogen interactions) between the silica and protein were, as expected, weak. Furthermore, we concluded that using a 40 mM Tris buffer, pH 7.0, would give us the most optimal protein (BSA) adsorption to the silica particles, in terms of both the amount of protein adsorbed and the strength of the adsorption. Admittedly, these results are not completely transferable to the Concanavalin A adsorption due to the fact that the adsorption is dependent on factors such as isoelectric point, glycosylation, surface activity and geometry. In addition to this, the complex Concanavalin A-magneto marker, has probably other silica binding characteristics compared to the Concanavalin A itself. Despite this fact, the BSA adsorption data were used to achieve a crude estimation of maximal binding capacity and binding time. All subsequent experiments were carried out in buffers having a salt concentration of 40 mM. Theoretically, we wanted to account for the maximum
Fig. 4. The binding kinetics of BSA adsorption is displayed here, where the amount of adsorbed BSA (mg) to 1.0 g silica particles is shown as a function of the incubation time (min).
Fig. 5. The relationship between buffer concentration and protein adsorption is illustrated above, where the amount of adsorbed BSA (mg) to 1.0 g silica particles is shown as a function of the Tris buffer concentration (mM), pH 7.0.
Fig. 3. The magneto marker calibration curve (y = − 0.56 + 0.042x, R = 0.99941, R.S.D. = 10.4%, n = 4), where the relative magnetic permeability (mV) is shown as a function of the magneto marker concentration (g/l).
measured. Lastly, we concluded that the magneto markers did not bind to silica; after an incubation with silica (0.2–0.5 mm) and subsequent washing in 20 mM Tris buffer, pH 7.0, the silica particles did not exhibit significant magnetic permeability (data not shown). 3.2. Evaluation of protein (BSA) binding to the silica carrier particles
K. Kriz et al. / Biosensors & Bioelectronics 13 (1998) 817–823
adsorption of BSA which could occur per gram silica using the geometrical surface area and volume of a silica particle. First we defined the number of protein molecules that could adsorbed to a particle’s surface (BSA/particle). The number of BSA molecules per particle was estimated by dividing the geometrical surface area of a silica particle by the cross-sectional area of the BSA protein. Thus, we found that theoretically there were 8.5 × 109 BSA molecules adsorbed per silica particle. Secondly, by using the density of silica ( = 2.2 g/cm3) and the molecular weight (MW = 66 430) of BSA we estimated that theoretically 0.019 mg BSA could adsorb per gram silica. The maximum binding of BSA to 1.0 g silica particles illustrated above in Fig. 5, was determined to be 0.28 mg BSA. Thus, there is a 15-fold difference in the actual amount of BSA adsorbed compared to our theoretical calculations. This difference can be explained by the fact that our theoretical model did not take into account the porosity of the silica particles (increasing the surface area) and the fact that proteins can be adsorbed in several layers. According to the manufacturer the surface area of the silica particles was 500 m2/g, which is five orders of magnitude larger than the geometrical area used in our calculations above. However, most of this surface area is positioned in small pores that are not accessible to the BSA or Concanavalin A protein molecules due to sterical hindrances. 3.3. A magneto binding assay of a model analyte (Concanavalin A) 3.3.1. Influence of the magneto markers on the magneto binding assay response The effect of the magneto marker concentration (0– 125 g/l) on the magneto binding assay response for 1.5 M Concanavalin A is shown in Fig. 6. In this graph
Fig. 6. The influence of the magneto marker concentration on the magneto binding assay response, where the relative magnetic permeability (mV) is shown as a function of the magneto marker concentration (g/l).
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the relative magnetic permeability (mV) of the sample is plotted against the magneto marker concentration (g/l). As illustrated in the figure, increasing the marker concentration from 0 to 5 g/l yields a higher signal. At these marker concentrations, the magneto markers are not present in excess. Therefore, most of the markers are not left free in solution, but have attached to the model analyte which is adsorbed to silica. Between 5 and 12.5 g/l a maximum and constant signal is achieved. At this concentration interval, the system becomes saturated with the magneto markers. Thus, the markers are no longer the limiting factor in the detection of Concanavalin A at concentrations below 1.5 M. Consequently, in all subsequent binding assays a magneto marker concentration of 12.5 g/l was employed. Unexpectedly, as we increased the magneto marker concentration of the system above 12.5 g/l, instead of observing a levelling of the signal, we observed a rapid decrease in the detected magnetic permeability. One explanation to this observation could be the fact that the equilibrium between silica adsorbed and free Concanavalin A becomes pushed towards the latter state due to the removal of the free Concanavalin A through binding to magneto markers present in excess. Another plausible reason lies in Concanavalin A’s multiple binding sites (4) which would likely allow a maximum of two magneto markers to interact with one given molecule, thus, sterically hindering subsequent adsorption. At high marker concentrations, the effect of this sterical hindrance is more noticeable. 3.3.2. A magneto binding assay of Concanavalin A The magneto binding assay was conducted for the detection of various concentrations (0–5.6 M) of the protein, Concanavalin A. Illustrated in Fig. 7 is the detection of Concanavalin A, in which the relative magnetic permeability (mV) is plotted against the concentration (nM). As shown in Fig. 7(a), a linear response was obtained up to 2.0 M, after which there was an observed saturation at the relative magnetic permeability 0.25 mV. The magneto marker optimization was performed on a 1.5 M Concanavalin A solution and consequently, this is approximately the upper limit of the observed linear response. However, we foresee that this linear range can be extended to cover even higher concentrations of analyte by increasing the concentration of the magneto markers used. In a control experiment, incubations were done in the presence of 200 mM glucose (which competed for the Concanavalin A binding sites). These yielded no responses suggesting that the binding was selective and that the magnetic markers were left unbound in the solution. The linear range is shown in Fig. 7(b), where the mean values for three consecutive calibration curves are plotted. The obtained linear regression yielded y = 0.032 +
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250 nM), accurate (R.S.D. = 6.2%) and linear in the dynamic range of 0–1.9 M. However, the presented model system possesses a few limitations based on the fact the target analyte is nonselectively adsorbed to the surface of the silica carrier particles. Therefore, in a complex sample such as blood, where there are many proteins present in solution, including the target analyte, detection of a specific analyte would not be possible since all proteins can adsorb to the surface of the silica carriers. In order to bring some enlightenment to the interference issue, preliminary studies were conducted to determine whether the presence of other proteins interfered with the signal normally obtained for a specific concentration of Concanavalin A. It has been illuminated that indeed the presence of proteins caused a reduction in signal. Our next objective is to increase the selectivity of the assay by chemically coupling antibodies which are specific for a given analyte or antigen, to the surface of the silica carrier particles and the magneto markers. By functionalizing the markers and the carrier particles with such natural receptors, detection of a serum protein in a complex sample such as whole blood can be conceived. Additionally, we believe that by employing smaller silica particles in the assays, and thereby increasing the surface area for protein attachment, the detection limit can be pushed down to the pico molar range. In this work, we have reported a step towards achieving magneto immunoassays (MIA): a successful sandwich approach in combination with magnetic markers. Fig. 7. The magneto binding assay of the protein Concanavalin A. The relative magnetic permeability (mV) is shown as a function of the concentration of Concanavalin A (nM) in both (a) and (b). (a) illustrates the detection of various concentrations (0–5.6 M) of the target protein (three sets of data are plotted) and (b) focuses on the linear range of the response (mean values are given).
0.00010x, R = 0.9772. We estimated (by applying the approach of 3SD) that the assay had a limit of detection at 250 nM. The binding assay was repeated three different times and the obtained slopes of the linear regressions were used to calculate the relative standard deviation (R.S.D.). The R.S.D. was determined to be 6.2% for n = 3. It should also be noted in Fig. 7(b), that the linear regression obtained intercepts the y-axis at 0.03 mV, due to the fact that there are probably traces of markers left in the pores or surrounding the silica particles.
4. Conclusions Recently, a magneto binding assay was conducted. This involved the detection of a model protein Concanavalin A using a ‘sandwich’ approach. The method has shown to be sensitive (having a limit of detection at
References Anderson, D.J., Guo, B., Xu, Y., Ng, L.M., Kricka, L.J., Skogerboe, K.J., Hage, D.S., Schoeff, L., Wang, J., Sokoll, L.J., Chan, D.W., Ward, K.M., Davis, K.A., 1997. Clinical chemistry. Analytical Chemistry 69, 165R–229R. Kriz, D., Kriz, B.C., 1995. Procedure for quantitative and qualitative determination of chemical substances, based on molecular recognition and measurement of the magnetic permeability. US patent application 08/847,125 (priority date August 20, 1995). Kriz, C.B., Rådevik, K., Kriz, D., 1996. Magnetic permeability measurements in bioanalysis and biosensors. Analytical Chemistry 68, 1966–1970. Kronick, P., Gilpin, R.W., 1986. Use of superparamagnetic particles for isolation of cells. Journal of Biochemistry and Biophysics Methods 12, 73–80. Molday, R.S., Molday, L.L., 1984. Separation of cells labelled with immunospecific iron dextran microspheres using high gradient magnetic chromatography. FEBS 170, 232–238. Molday, R.S., Yen, S.P.S., Rembaum, A., 1977. Application of magnetic microspheres in labelling and separation of cells. Nature 268, 437–438. Oellerich, M., 1980. Enzyme immunoassays in clinical chemistry: present status and trends. Journal of Clinical Chemistry and Clinical Biochemistry 18, 197–208. Rohr, T., 1995. Magnetically assisted binding assays using magnetically labelled binding members. US patent 5,445,970 (priority date August 29, 1995).
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Tijssen, P., 1985. In: Burdon, R.H. and van Knippenberg, P.H. (Eds), Practice and Theory of Enzyme Immunoassays. Elsevier, Amsterdam. Wikstro¨m, P., Flygare, S., Gro¨ndalen, A., Larsson, P.-O., 1978. Magnetic aqueous two-phase separation: a new technique to increase
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rate of phase-separation, using dextran–ferrofluid or larger iron oxide particles. Analytical Biochemistry 167, 331–339. Yalow, R.S., Berson, S.A., 1959. Assay of plasma insulin in human subjects by immunological methods. Nature 184, 1648–1649.
Advancements toward magneto immunoassays Kirstin Kriz b
a,b
, Janin Gehrke a, Dario Kriz
a,b,*
a European Institute of Science, Research Park Ideon, SE-223 70 Lund, Sweden Department of Pure and Applied Biochemistry, Chemical Center, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden
Received 16 February 1998; accepted 17 April 1998
Abstract Recently, a magneto binding assay was conducted. The operational principle is based on a ‘sandwich’ mode of detection, where the target analyte (Concanavalin A) becomes bound nonselectively by protein adsorption between the solid support (silica carrier particles) and the ferromagnetic label (magneto markers). A magnetic transducer was employed for the detection. The binding assay gave a linear response in the dynamic range of 0–1.9 M and proved to be sensitive by having a limit of detection at 250 nM. Furthermore, the relative standard deviation observed was 6.2% (n = 3). The demonstration of the ‘sandwich’ approach is a step towards achieving magneto immunoassays (MIA). 1998 Elsevier Science S.A. All rights reserved. Keywords: Magneto immunoassay; MIA; Ferrofluid; Ferromagnetic markers
1. Introduction Immunological techniques, such as immunoassays, have employed antibodies for the detection and quantification of specific molecules even in the presence of compounds with similar molecular structures. These highly quantitative assays are achieved by combining the high specificity of the biological recognition occurring at the molecular level with the sensitivity of different labelling techniques. Typically, immunoassays have involved the use of antibodies or antigens which have been coupled to markers such as radioactive compounds, enzymes, fluorophores and luminescence markers (Anderson et al., 1997). Radioactive markers were employed in radioimmunoassays (RIA) in 1959 (Yalow and Berson, 1959). Even though these markers offer high sensitivities (1– 500 pM) when used in RIA, they are expensive, exhibit low stability, require high-cost equipment for their detection, and they present radioactive hazards for the personnel. The use of enzyme markers is more popular in smaller laboratories or hospital clinics due to the fact that they are relatively cheap, quite stable and they lack the radiological hazards (Tijssen, 1985). However, these
* Corresponding author. Tel.: + 46-46-182230; Fax: + 46-46182499; E-mail:[email protected]
markers generally give lower sensitivities than radioactive markers when used in immunoassays (Oellerich, 1980). Additionally, many research groups have focused on the development of fluorophores and luminescence markers (Anderson et al., 1997). These markers offer sensitivities normally associated with radioactive markers but possess similar advantages analogous to the enzyme markers. It has previously been suggested to use magneto markers in combination with biosensor and bioassay technologies (Kriz and Kriz, 1995; Rohr, 1995; Kriz et al., 1996). These markers comprise a ferromagnetic iron oxide core coated with dextran and are therefore extremely stable, non-toxic and fairly cheap. The underlying principles for magnetic determination were previously outlined by our group. The instrumentation used for the detection of the magneto markers employs a magnetic transducer which comprises a coil placed in a Maxwell bridge (Kriz et al., 1996). Consequently, the change in magnetic permeability of a compound is detected using inductance measurements and, furthermore, analyte detection has been achieved directly (the analyte was labelled ferromagnetically) or competitively (competition between ferromagnetic labelled and unlabelled analytes exists for binding sites on the transducer) using the aforementioned device. The use of magnetic transducers in combination with ferromagnetic markers has been shown to offer advantages such as, low interference, little or no background signal, no transducer fouling and no sample pretreatment.
0956-5663/98/$—see front matter 1998 Elsevier Science S.A. All rights reserved. PII: S 0 9 5 6 - 5 6 6 3 ( 9 8 ) 0 0 0 4 7 - 5
818
K. Kriz et al. / Biosensors & Bioelectronics 13 (1998) 817–823
In the work reported in this paper, we have further developed one of the possible applications of the magneto markers by taking a step closer towards achieving a magneto immunoassay (MIA). Feasibility studies were conducted to determine whether a ‘sandwich’ configuration (carrier–analyte–marker) could be employed for the detection of Concanavalin A in a binding assay based on passive protein adsorption. Further characterization of the binding kinetics and assay environment was conceived.
2. Experimental 2.1. Preparation of the magneto markers The magneto markers were produced in house at the European Institute of Science (EURIS) after modification of the methods reported earlier (Molday et al., 1977; Wikstro¨m et al., 1978; Molday and Molday, 1984; Kronick and Gilpin, 1986;). Accordingly, 0.7 mmol FeCl3·6H2O (Merck, Germany) and 0.4 mmol FeSO4·7H20 (Apoteksbolaget, Sweden) were dissolved in 0.5 ml distilled H2O and 0.75 g dextran MW 70 000 (Sigma, USA) was dissolved in 3.0 ml of distilled H2O (0.25 g/ml). The iron and dextran solutions were then mixed together and heated to 333K. Subsequently, 3 ml of a 7.5% ammonia solution (Sigma, USA) was added to the iron/dextran solution and left to incubate at 333K for 15 min. Vigorous stirring occurred periodically during the incubation. Large particles and aggregates were removed by centrifuging the solution at 5 000 rpm for 30 min. Lastly, the magneto markers were dialysed against distilled H2O overnight using a Spectra/Por cellulose ester dialysis membrane MWCO 300 000 (Spectrum, USA) and filtered through a 200 nm filter (Sartorius, Germany). 2.2. Evaluation of the magneto markers From the prepared magneto marker solution, which was defined to be 100% (1000 g/l), a series of aqueous solutions containing different concentrations (0, 10, 100, 250, 500, 1000 g/l) of magneto markers were made. Their relative magnetic permeabilities (arbitrary units in mV) were measured with a home-made magnetic permeability meter produced by EURIS, which employs a Maxwell bridge. The magnetic permeability determination is based on the principle that the output signal from the Maxwell bridge is proportional to the concentration of magneto markers in solution established previously by Kriz et al., 1996. Illustrated in Fig. 1, is the current hand-held device, which is a portable variant of the original magnetic permeability meter used in this work.
Fig. 1. The current magnetic permeability meter; a portable version of the device used to measure magnetic permeability in this report.
2.3. Binding kinetics of protein adsorption (BSA) to silica carrier particles Accordingly, 1.0 g silica particles (0.2–0.5 mm, Merck, Germany) were suspended in a 5 ml, 40 mM Tris buffer, pH 7.0, solution containing 0.2 mg/ml BSA (Sigma, USA). To evaluate the binding kinetics of BSA (mg) adsorption, the particles were allowed to sediment and then the supernatant was removed and its absorbance was measured at 280 nm at 0, 1, 15 and 30 min, respectively. 2.4. Effect of buffer concentration on adsorption of protein (BSA) to the silica carrier particles Accordingly, 1.0 g silica particles (0.2–0.5 mm) were suspended in 5 ml distilled H2O and subsequently, the particles were allowed to sediment and the supernatant was removed. This washing procedure was repeated 10 times with the silica particles in order to remove fines which would disturb future absorbance measurements. Following the last washing step, the silica particles were incubated for 40 min in 5 ml solutions containing 0.2 mg/ml BSA and the following buffer concentrations: 0, 10, 40, 70, 100, 200 and 500 mM Tris buffer, pH 7.0. Subsequently, the particles were allowed to sediment and the absorbance of the supernatant was measured at 280 nm to determine the disappearance of BSA (mg) from the solution. 2.5. The magneto binding assay ‘sandwich’ approach The operational principle (shown in Fig. 2) for the magneto binding assay is based on the use of magneto markers (20–200 nm) in conjunction with larger silica carrier particles (0.2–0.5 mm). In the presence of the magneto markers and silica particles, the target protein, Concanavalin A (Sigma, USA), will adsorb to the surface of the silica particles and attached itself to the outer
K. Kriz et al. / Biosensors & Bioelectronics 13 (1998) 817–823
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2.7. The magneto binding assay detection of Concanavalin A
Fig. 2. The ‘sandwich’ approach used in the magneto binding assay, where the target analyte is bound between the silica carrier particles and the magneto markers (A). Subsequent sedimentation of the protein/particle complex (B) allows the magnetic permeability meter to measure the enrichment of magnetic markers at the bottom of the vial (C).
dextran layer of the magneto markers. Thus, a ‘sandwich’ effect is observed in solution where the target analyte is held between the silica carriers and the magneto markers. Subsequently, the weight (due to their higher density) of the silica carrier particles will cause the protein/particle complex to sediment to the bottom of the vial. Lastly, the vial is placed in the home-made magnetic permeability meter shown in Fig. 1, which measures the enrichment of the magnetic markers at the bottom of the vial. This ‘sandwich’ approach is the basis for the detection of Concanavalin A in all subsequent binding assays reported here in this work. 2.6. Influence of the magneto marker concentration on the magneto binding assay response Correspondingly, 4 ml of a 1.5 M Concanavalin A solution in 20 mM Tris buffer (10 mM MnCl2, 10 mM CaCl2, pH 7.4) was added to six different tubes each containing 0.25 g silica particles (0.2–0.5 mm). Subsequently, magneto markers were added to their respective tubes (yielding the respective final concentrations 1.25, 5, 12.5, 25, 50 and 125 g/l) and incubated overnight at room temperature. In the morning each sample was washed with 20 mM Tris buffer (10 mM MnCl2, 10 mM CaCl2, pH 7.4) by allowing the silica particles (containing adsorbed protein labelled with magneto markers) to sediment for 5 min, removing the supernatant and then resuspending the silica particles in same amount of Tris buffer which was removed. Lastly, the particles were allowed to sediment again and the magnetic permeability of the samples was measured with the magnetic permeability meter.
In several test tubes each containing 0.25 g silica particles (0.2–0.5 mm), 4 ml of a Concanavalin A solution in 20 mM Tris buffer (10 mM MnCl2, 10 mM CaCl2, pH 7.4) was added. The final Concanavalin A concentration of each tube ranged from 0 to 5.6 M. Accordingly, magneto markers were then added, to a final concentration of 12.5 g/l, to each of the test tubes and incubated for 4 h at room temperature. Shaking of the samples occurred periodically throughout the incubation. Subsequently, the samples were washed with 20 mM Tris buffer (10 mM MnCl2, 10 mM CaCl2, pH 7.4) by allowing the silica particles (containing adsorbed protein labelled with magneto markers) to first sediment for 5 min, followed by removing the supernatant and then resuspending the silica particles in the same amount of Tris buffer which was removed. Lastly, the particles were allowed to sediment again and the magnetic permeability of the samples were measured with the magnetic permeability meter.
3. Results and discussion 3.1. Preparation and evaluation of the magneto markers A brown, colloidal solution was obtained from the preparation procedure, which exhibited magnetic properties (the meniscus was pulled towards the wall of the test tube) when in close proximity to a permanent magnet. Additionally, the magneto marker preparate was filtered through a filter with a pore size of 200 nm in order to define the upper limit of the particle size. The lower limit of the particle size was determined during the preparation procedure since the preparate was dialysed with a membrane which had a molecular weight cut-off of 300 000. Therefore, everything smaller than approximately 20 nm could freely diffuse out of the membrane. Thus, we determined the magneto markers to range in size from 20 to 200 nm. Confirmation of size distribution was done by gel chromatography as earlier reported (Kriz et al., 1996). A calibration curve was established for further evaluation of the magneto marker preparate. Illustrated in Fig. 3 is the magneto marker calibration curve where the relative magnetic permeability (mV) is plotted against the magneto marker concentration (g/l). As shown in the graph a linear regression (y = − 0.56 + 0.042x, R = 0.99941) was obtained. The calibration curve was repeated and the slope of each obtained linear regression was used in the determination of the relative standard deviation (R.S.D.). The calculated R.S.D. was 10.4% for n = 4. The R.S.D. was relatively high due to the wide range (0–40 mV) of magnetic permeabilities
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BSA can adsorb per gram silica at a buffer concentration of 40 mM.
3.2.1. Binding kinetics The binding kinetics of BSA adsorption to 1.0 g silica particles are shown in Fig. 4, in which the amount of adsorbed BSA (mg) is plotted against the incubation time (min). Consequently, it was concluded that the binding kinetics was a fast process which was confirmed by the fact that 95% of the maximum BSA adsorption was achieved in approximately 4 min. Thus, it can be calculated from the maximum adsorption that 0.22 mg
3.2.2. Effect of buffer concentration on protein adsorption The relationship between buffer concentration and protein adsorption is illustrated in Fig. 5, where the amount of adsorbed BSA (mg) to 1.0 g silica particles is plotted against the concentration of Tris buffer (mM), pH 7.0. As shown in the graph, at low salt concentrations ( ⱕ 40 mM) maximum protein adsorption is observed ( ⱖ 0.28 mg). Conversely, at high salt concentrations ( ⱖ 400 mM) little or no protein adsorption ( ⬍ 0.01 mg) is observed. Additionally, silica particles which contained 0.28 mg adsorbed BSA were incubated overnight in 500 mM Tris buffer, pH 7.0. After the overnight incubation, all of the adsorbed protein had been washed from the particles and remained in the supernatant. Thus, the noncovalent interactions (hydrophobic, ionic and hydrogen interactions) between the silica and protein were, as expected, weak. Furthermore, we concluded that using a 40 mM Tris buffer, pH 7.0, would give us the most optimal protein (BSA) adsorption to the silica particles, in terms of both the amount of protein adsorbed and the strength of the adsorption. Admittedly, these results are not completely transferable to the Concanavalin A adsorption due to the fact that the adsorption is dependent on factors such as isoelectric point, glycosylation, surface activity and geometry. In addition to this, the complex Concanavalin A-magneto marker, has probably other silica binding characteristics compared to the Concanavalin A itself. Despite this fact, the BSA adsorption data were used to achieve a crude estimation of maximal binding capacity and binding time. All subsequent experiments were carried out in buffers having a salt concentration of 40 mM. Theoretically, we wanted to account for the maximum
Fig. 4. The binding kinetics of BSA adsorption is displayed here, where the amount of adsorbed BSA (mg) to 1.0 g silica particles is shown as a function of the incubation time (min).
Fig. 5. The relationship between buffer concentration and protein adsorption is illustrated above, where the amount of adsorbed BSA (mg) to 1.0 g silica particles is shown as a function of the Tris buffer concentration (mM), pH 7.0.
Fig. 3. The magneto marker calibration curve (y = − 0.56 + 0.042x, R = 0.99941, R.S.D. = 10.4%, n = 4), where the relative magnetic permeability (mV) is shown as a function of the magneto marker concentration (g/l).
measured. Lastly, we concluded that the magneto markers did not bind to silica; after an incubation with silica (0.2–0.5 mm) and subsequent washing in 20 mM Tris buffer, pH 7.0, the silica particles did not exhibit significant magnetic permeability (data not shown). 3.2. Evaluation of protein (BSA) binding to the silica carrier particles
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adsorption of BSA which could occur per gram silica using the geometrical surface area and volume of a silica particle. First we defined the number of protein molecules that could adsorbed to a particle’s surface (BSA/particle). The number of BSA molecules per particle was estimated by dividing the geometrical surface area of a silica particle by the cross-sectional area of the BSA protein. Thus, we found that theoretically there were 8.5 × 109 BSA molecules adsorbed per silica particle. Secondly, by using the density of silica ( = 2.2 g/cm3) and the molecular weight (MW = 66 430) of BSA we estimated that theoretically 0.019 mg BSA could adsorb per gram silica. The maximum binding of BSA to 1.0 g silica particles illustrated above in Fig. 5, was determined to be 0.28 mg BSA. Thus, there is a 15-fold difference in the actual amount of BSA adsorbed compared to our theoretical calculations. This difference can be explained by the fact that our theoretical model did not take into account the porosity of the silica particles (increasing the surface area) and the fact that proteins can be adsorbed in several layers. According to the manufacturer the surface area of the silica particles was 500 m2/g, which is five orders of magnitude larger than the geometrical area used in our calculations above. However, most of this surface area is positioned in small pores that are not accessible to the BSA or Concanavalin A protein molecules due to sterical hindrances. 3.3. A magneto binding assay of a model analyte (Concanavalin A) 3.3.1. Influence of the magneto markers on the magneto binding assay response The effect of the magneto marker concentration (0– 125 g/l) on the magneto binding assay response for 1.5 M Concanavalin A is shown in Fig. 6. In this graph
Fig. 6. The influence of the magneto marker concentration on the magneto binding assay response, where the relative magnetic permeability (mV) is shown as a function of the magneto marker concentration (g/l).
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the relative magnetic permeability (mV) of the sample is plotted against the magneto marker concentration (g/l). As illustrated in the figure, increasing the marker concentration from 0 to 5 g/l yields a higher signal. At these marker concentrations, the magneto markers are not present in excess. Therefore, most of the markers are not left free in solution, but have attached to the model analyte which is adsorbed to silica. Between 5 and 12.5 g/l a maximum and constant signal is achieved. At this concentration interval, the system becomes saturated with the magneto markers. Thus, the markers are no longer the limiting factor in the detection of Concanavalin A at concentrations below 1.5 M. Consequently, in all subsequent binding assays a magneto marker concentration of 12.5 g/l was employed. Unexpectedly, as we increased the magneto marker concentration of the system above 12.5 g/l, instead of observing a levelling of the signal, we observed a rapid decrease in the detected magnetic permeability. One explanation to this observation could be the fact that the equilibrium between silica adsorbed and free Concanavalin A becomes pushed towards the latter state due to the removal of the free Concanavalin A through binding to magneto markers present in excess. Another plausible reason lies in Concanavalin A’s multiple binding sites (4) which would likely allow a maximum of two magneto markers to interact with one given molecule, thus, sterically hindering subsequent adsorption. At high marker concentrations, the effect of this sterical hindrance is more noticeable. 3.3.2. A magneto binding assay of Concanavalin A The magneto binding assay was conducted for the detection of various concentrations (0–5.6 M) of the protein, Concanavalin A. Illustrated in Fig. 7 is the detection of Concanavalin A, in which the relative magnetic permeability (mV) is plotted against the concentration (nM). As shown in Fig. 7(a), a linear response was obtained up to 2.0 M, after which there was an observed saturation at the relative magnetic permeability 0.25 mV. The magneto marker optimization was performed on a 1.5 M Concanavalin A solution and consequently, this is approximately the upper limit of the observed linear response. However, we foresee that this linear range can be extended to cover even higher concentrations of analyte by increasing the concentration of the magneto markers used. In a control experiment, incubations were done in the presence of 200 mM glucose (which competed for the Concanavalin A binding sites). These yielded no responses suggesting that the binding was selective and that the magnetic markers were left unbound in the solution. The linear range is shown in Fig. 7(b), where the mean values for three consecutive calibration curves are plotted. The obtained linear regression yielded y = 0.032 +
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250 nM), accurate (R.S.D. = 6.2%) and linear in the dynamic range of 0–1.9 M. However, the presented model system possesses a few limitations based on the fact the target analyte is nonselectively adsorbed to the surface of the silica carrier particles. Therefore, in a complex sample such as blood, where there are many proteins present in solution, including the target analyte, detection of a specific analyte would not be possible since all proteins can adsorb to the surface of the silica carriers. In order to bring some enlightenment to the interference issue, preliminary studies were conducted to determine whether the presence of other proteins interfered with the signal normally obtained for a specific concentration of Concanavalin A. It has been illuminated that indeed the presence of proteins caused a reduction in signal. Our next objective is to increase the selectivity of the assay by chemically coupling antibodies which are specific for a given analyte or antigen, to the surface of the silica carrier particles and the magneto markers. By functionalizing the markers and the carrier particles with such natural receptors, detection of a serum protein in a complex sample such as whole blood can be conceived. Additionally, we believe that by employing smaller silica particles in the assays, and thereby increasing the surface area for protein attachment, the detection limit can be pushed down to the pico molar range. In this work, we have reported a step towards achieving magneto immunoassays (MIA): a successful sandwich approach in combination with magnetic markers. Fig. 7. The magneto binding assay of the protein Concanavalin A. The relative magnetic permeability (mV) is shown as a function of the concentration of Concanavalin A (nM) in both (a) and (b). (a) illustrates the detection of various concentrations (0–5.6 M) of the target protein (three sets of data are plotted) and (b) focuses on the linear range of the response (mean values are given).
0.00010x, R = 0.9772. We estimated (by applying the approach of 3SD) that the assay had a limit of detection at 250 nM. The binding assay was repeated three different times and the obtained slopes of the linear regressions were used to calculate the relative standard deviation (R.S.D.). The R.S.D. was determined to be 6.2% for n = 3. It should also be noted in Fig. 7(b), that the linear regression obtained intercepts the y-axis at 0.03 mV, due to the fact that there are probably traces of markers left in the pores or surrounding the silica particles.
4. Conclusions Recently, a magneto binding assay was conducted. This involved the detection of a model protein Concanavalin A using a ‘sandwich’ approach. The method has shown to be sensitive (having a limit of detection at
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