Magnetic Bead Sensing Platform for the Detection of Proteins

Anal. Chem. 2007, 79, 8669-8677

Magnetic Bead Sensing Platform for the Detection of Proteins Randy De Palma,†,‡ Gunter Reekmans,† Chengxun Liu,‡ Roel Wirix-Speetjens,*,‡ Wim Laureyn,‡ Olle Nilsson,§ and Liesbet Lagae‡

IMEC, NEXT, Kapeldreef 75, B-3001 Leuven, Belgium, Physical and Quantum Chemistry, Catholic University Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium, and Fujirebio Diagnostics, Majnabbe Terminal, SE-414 55 Gothenburg, Sweden

Over the past 5 years, the on-chip detection and manipulation of magnetic beads via magnetoelectronics has emerged as a promising new biosensor platform. Magnetic bead sensing (MBS) provides a highly sensitive and specific technique, enabling these sensors to meet the diagnostic needs that are currently not met by existing technologies. Although many studies have proven the high physical sensitivity of magnetic sensors, the establishment of dose-response curves using MBS is unexplored and their capability to sensitively detect low concentrations of target molecules for diagnostic applications has remained unproven. In this study, we have exploited an alternative MBS concept based on the repositioning of the magnetic beads toward the most sensitive location on the spin valve sensors to allow for highly sensitive immunosensing over a wide range of target concentrations. Furthermore, we present the optimization of the magnetoimmuno assay, i.e., the surface chemistry, the blocking procedure, and the type of magnetic particle, for the highly sensitive and specific detection of S100ββ, a diagnostic marker for stroke and minor head injury. Finally, a dose-response curve was established that illustrates that our MBS platform can specifically detect S100ββ down to 27 pg/ mL, while maintaining a broad dynamic detection range of ∼2 decades. Biosensors have the potential to revolutionize the field of in vitro diagnostics for human, veterinary, and food applications. Point-of-care (POC) diagnostics is considered as the most promising application field of biosensors, because of their intrinsic capability to allow for easy to perform, reliable, and cost-effective testing, without major human intervention.1 Despite great efforts, most biosensors for the label-free detection of proteins and nucleic acids currently have not met the required sensitivity and specificity to allow for their successful implementation in the various * To whom correspondence should be addressed. cphone: +32-16-281935. Fax: +32-16-281097. E-mail: [email protected]. † IMEC, NEXT. ‡ Catholic University Leuven. § Fujirebio Diagnostics. (1) Rasooley, A. Biosens. Bioelectron. 2006, 21, 1847. 10.1021/ac070821n CCC: $37.00 Published on Web 10/11/2007

© 2007 American Chemical Society

application fields envisaged.2,3 The incorporation of labels allows increasing the biosensor sensitivity and specificity, which are important parameters when attempting to detect low concentrations of target molecules in raw sample matrixes, such as whole blood.4 Furthermore, the use of superparamagnetic particles as an alternative for the commonly used fluorescent labels is very appealing considering their successful application for the isolation of cells, proteins, and nucleic acids. Baselt et al. demonstrated the use of magnetoresistive sensors for the detection of such magnetic particles.5 Since then, a variety of similar sensing platforms was proposed, as described in many excellent reviews.6-8 While the detection of DNA hybridization is being put forward as the main application, more recent reports mainly described the detection of single magnetic particles9,10 or studied the relationship between the sensor signal and the number of particles deposited on the sensor surface.11-13 Although these studies have extensively proven the intrinsically high physical sensitivity of magnetic sensors, the establishment of dose-response curves using magnetic biosensors is largely unexplored and their capability to sensitively detect low concentrations of target molecules in body fluids for diagnostic applications has remained largely unproven.7 Most publications typically describe a model system such as (2) Soper, S. A.; Brown, K.; Ellington, A.; Frazier, B.; Garcia-Manero, G.; Gau, V.; Gutman, S. I.; Hayes, D. F.; Korte, B.; Landers, J. L.; Larson, D.; Ligler, F.; Majumdar, A.; Mascini, M.; Nolte, D.; Rosenzweig, Z.; Wang, J.; Wilson, D. Biosens. Bioelectron. 2006, 21, 1932. (3) Gooding, J. J. Anal. Chim. Acta.2006, 559, 137. (4) Bange, A.; Halsall, H. B.; Heineman, W. R. Biosens. Bioelectron. 2005, 20, 2005. (5) Baselt, D. R.; Lee, G. U.; Natesan, M.; Metzger, S. W.; Sheehan, P. E.; Colton, R. J. Biosens. Bioelectron. 1998, 13, 731. (6) Graham, D. L.; Ferreira, H. A.; Freitas, P. P. Trends Biotechnol. 2004, 22, 455. (7) Megens, M.; Prins, M. J. Magn. Magn. Mater. 2005, 293, 702. (8) Wirix-Speetjens, R.; De Boeck, In, J. Encyclopedia of Sensors; Grimes, C. A., Dickey, E. C., Pishko, M. V., Eds.; American Scientific Publishers: Stevenson Ranch, CA, 2006; Vol. 5, pp 403-414. (9) Wirix-Speetjens, R.; Fyen, W.; De Boeck, J.; Borghs, G. J. Appl. Phys. 2006, 99, 103903. (10) Rife, J. C.; Miller, M. M.; Sheehan, P. E.; Tamanaha, C. R.; Tondra, M.; Whitman, L. J. Sens. Actuators, A: Phys. 2003, 107, 209. (11) Millen, R. L.; Kawaguchi, T.; Granger, M. C.; Porter, M. D. Anal. Chem. 2005, 77, 6581. (12) Li, G.; Sun, S.; Wilson, R. J.; White, R. L.; Pourmand, N.; Wang, S. X. Sens, Actuators, A: Phys. 2006, 126, 98. (13) De Boer, B. M.; Kahlman, J. A. H. M.; Jansen, T. P. G. H.; Duric, H.; Veen, J. Biosens. Bioelectron. 2007, 22, 2366.

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biotin/streptavidin14,39 or use a single concentration of analyte,15 rather then measuring over a wide concentration range.7 Moreover, in those cases where dose-response curves were reported, the probe concentration on the surface was varied rather than the target concentration in solution.16 To our knowledge, real dose-response curves, in which the target concentration is varied, have been presented only in a limited number of reports up to now,17,18 and this for obvious reasons. Setting up such a study would require the design and fabrication of a device with a sufficiently large sensing area, the development of a proper surface functionalization strategy, and the design of a magnetoimmuno assay with a sufficiently high sensitivity and specificity. In this paper, we focus on the very important and difficult task of successfully optimizing and integrating all these different aspects. (14) Ferreira, H. A.; Graham, D. L.; Freitas, P. P.; Cabral, J. M. S. J. Appl. Phys. 2003, 93, 7281. (15) Miller, M. M.; Sheehan, P. E.; Edelstein, R. L.; Tamanaha, C. R.; Zhong, L.; Bounnak, S.; Whitman, L. J.; Colton, R. J. J. Magn. Magn. Mater. 2001, 225, 138-144. (16) Schotter, J.; Kamp, P. B.; Becker, A.; Pu ¨hler, A.; Reiss, G.; Bru ¨ckl, H. Biosens. Bioelectron. 2004, 19, 1149. (17) Meyer, M. H. F.; Hartmann, M.; Krause, H. J.; Blankenstein, G.; MuellerChorus, B.; Oster, J.; Miethe, P.; Keusgen, M. Biosens. Bioelectron. 2007, 22 (6), 973., (18) Nikitin, P.I.; Vetoshko, P.M.; Ksenevich, T.I. J. Magn. Magn Mater. 2007, 311 (1), 445. (19) Heart Attack, Stroke and Cardiac Arrest Warning Signs; http://www.americanheart.org. (20) Reynolds, M. A.; Kirchick, H. J.; Dahlen, J. R.; Anderberg, J. M.; McPherson, P. H.; Nakamura, K. K.; Laskowitz, D. T.; Valkirs, G. E.; Buechler, K. F. Clin. Chem. 2003, 49, 1733. (21) Poli-de-Figueiredo, L. F.; Biberthaler, P.; Simao, Filho, C.; Hauser, C.; Mutschler, W.; Jochum, M. Clinics 2006, 61, 41. (22) http://www.canag.com/home.htm. (23) Wirix-Speetjens, R.; Liu, C.; Reekmans, G.; De Palma, R.; Laureyn, W.; Borghs, G. Sens. Actuators, B: Chem. DOI: 10.1016/J.snb.2007.05.023. (24) Luxton, R.; Badesha, J.; Kiely, J.; Hawkins, P. Anal. Chem. 2004, 76, 17151719. (25) The magnetic susceptibility of Ademtech 300-nm particles at 48 Oe is 0.05 emu/g, as measured with alternating gradient field magnetometry. (26) Huang, L.; Reekmans, G.; Saerens, D.; Friedt, J.-M.; Frederix, F.; Francis, L.; Muyldermans, S.; Campitelli, A.; Van, Hoof, C. Biosens. Bioelectron. 2005, 21, 483. (27) Graham, D. L.; Ferreira, H.; Bernardo, J.; Freitas, P. P.; Cabral, J. M. S. J. Appl. Phys. 2002, 91, 7786. (28) Lagae, L.; Wirix-Speetjens, R.; Das, J.; Graham, D.; Ferreira, H.; Freitas, P. P. F.; Borghs, G.; De Boeck, J. J. Appl. Phys. 2002, 91, 7445. (29) Edelstein, R. L.; Tamanaha, C. R.; Sheehan, P. E.; Miller, M. M.; Baselt, D. R.; Whitman, L. J.; Colton, R. J. Biosens. Bioelectron. 2000, 14, 805. (30) More information on the correction for the thermal drift on the baseline signal can be found in Supporting Information. (31) Wilson, W. D. Science 2002, 295, 2103. (32) Cooper, M. A. Nat. Rev. Drug Discovery 2002, 1, 515. (33) Four different antibodies (S21, S23, S36, S53) were tested in epitope-mapping experiments. We found that S53 as the primary antibody and S36 as the secondary antibody was the pair that gave the most sensitive and specific S100ββ detection (data not shown). (34) It has to be noted that we could couple more then 10 000 RU, but we stopped the coupling manually around 8500 RU to avoid oversaturation of the surface, which could result in hindered S100ββ binding and a drifting baseline. (35) Johnsson, B.; Lo ¨fås, S;, Lindquist, G. Anal. Biochem. 1991, 198, 268. (36) Lo ¨fås, S. Pure Appl. Chem. 1995, 67, 829. (37) Immunochemical Assays and Biosensor Technologies for the 1990s; Nakamura, R., Kasahara, Y., Rechnitz, G. A., Eds.; American Society for Microbiology: Washington, DC, 1992. (38) The noise level (N) is calculated as the standard deviation of 10 blank injections. The intercept (I) and the sensitivity (S) are determined from the first linear part of the dose-response curve. I is given as the value where the linear curve crosses the Y axis, while S is reported as the slope of the linear curve. (39) Nidumolu, B. G.; Urbina, M. C.; Hormes, J.; Kumar, C. S. S. R.; Monroe, W. T. Biotechnol. Prog. 2006, 22, 91-95.

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The diagnosis of stroke is becoming increasingly important, as stroke is the third most frequent cause of death in the Western world, next to other cardiovascular diseases and cancer.19 To be able to discriminate between ischemic and hemorrhagic stroke and timely apply the correct therapy, the measurement of the marker protein S100ββ has been proposed.20 In addition, it has been demonstrated recently that the diagnosis of S100ββ could play an important role for ruling out the need for computed tomography scans following minor head injury.21 Currently, the successful diagnosis of S100ββ at the point-of-care is hampered by its low concentration in whole blood, i.e., 1-100 pg/mL. Currently, ELISA-based assays allow the detection of S100ββ in the concentration range of 50 pg/mL-3.5 ng/mL,22 but since they require a lot of hands-on work, their applicability in, for example, POC settings, is rather limited. Considering their intrinsically high sensitivity, magnetoresistive-based biosensors could be ideal for the detection of S100ββ at such low concentration levels and in such settings. In this study, we have designed and fabricated an alternative magnetic bead sensor (MBS) platform that allows immunosensing over a wide range of target concentrations by increasing its active area as compared to the magnetic sensors designed for single magnetic bead detection. In the proposed MBS concept, we have exploited the strong dependence of the MBS signal on the specific location of the magnetic particles relative to the spin valves. By repositioning the magnetic particles to the most sensitive location on the spin valves, the MBS signal can be increased to its theoretical maximum.23 Although many papers described the successful application of magnetic particles as physical labels,16,24 none of them elucidated on the optimization of the magnetosandwich assay performance. Following the optimization of the surface chemistry, the blocking procedure, and the type of magnetic particle, we could establish a highly sensitive and specific doseresponse curve for the detection of S100ββ, over a broad dynamic detection range and down to diagnostically relevant concentrations. To our knowledge, this is the first report that elucidates the optimization of the different aspects of magnetic bead sensing and their combination into an integrated MBS platform to establish a real dose-response curve. EXPERIMENTAL SECTION Materials. All materials and reagents were used as commercially received. Mouse monoclonal anti-S100ββ antibodies (clones S21, S23, S36, and S53) and mouse monoclonal antiprostate specific antigen antibodies (PSA66) were obtained from Fujirebio Diagnostics. S100ββ antigen was purchased from Affiniti Research Products Ltd. Bovine serum albumine (BSA), sodium chloride, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 11-mercapto-1-undecanol (11-MUOH), 16-mercapto-1hexadecanoic acid (16-MHA), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were obtained from Sigma. 2-{2-[2-(11-Mercaptoundecyloxy)ethoxy]ethoxy]ethoxy}ethanol (C11-PEO3-OH), 2-(2-{2-[2-(2-[2-(11mercaptoundecyloxy)ethoxy]ethoxy)ethoxy]ethoxy}ethoxy)ethoxyacetic acid (C11-PEO6-COOH) were from Sensopath. Riedelde Hae¨n supplied calcium chloride. Tween-20 was purchased from Fluka and acetic acid from Merck. Tri(ethylene glycol) monoamine was obtained from Molecular Biosciences. Hydrogen peroxide, sulfuric acid and acetone were from Honeywell. Air Products

supplied ultrapure ethanol. Dynabeads Myone streptavidin 1-µm particles were purchased from Invitrogen, and streptavidin 300nm particles were from Ademtech. CM5 chips (carboxymethyl dextran-coated gold) were from Biacore. These particles have a magnetic content of 37 and 70%, respectively. Sensor Fabrication and Readout Instrumentation. Figure S1 (see Supporting Information) gives a schematic overview of the MBS chip. Every chip consists of 2 rows of 12 sensors, and each sensor is made of 9 parallel spin valves that function as both the detection and the alignment elements. The spin valves were sputtered as Ta 1.5 nm/Ni80Fe20 4.5 nm/Co90Fe10 0.5 nm/Cu 1.9 nm/Co90Fe10 2.5 nm/Ir80Mn20 7 nm/Ta 2.0 nm/TiW 5 nm. In a next step, the bulk spin valve was patterned as nine parallel lines by ion milling after standard photolithography. Each spin valve has a size of 100 × 2 µm (length × width). Au contacts were then thermally evaporated onto the spin valve sensors and patterned by liftoff. Afterward, 450-nm Si3N4 was deposited on the device by plasma-enhanced chemical vapor deposition, patterned and etched by CF4 plasma. In order to cover the defects in the Si3N4 passivation layer over the Au conductors, a 2.4-µm SU8 layer was spun and patterned while the sensing area remains open. Finally, the sensing area was covered with 50-nm Au on which the surface chemistry and the magnetosandwich assay was applied. The MBS chip was bonded onto a ceramic package, which was later mounted to a printed circuit board. The bonding wires were protected with epoxy. The sensor readout instrument is mainly composed of an ATMEL microprocessor (AT89C52) with peripheral driving integrated circuits. The ATMEL microprocessor was commanded by a LabVIEW program on a computer, and the sensor signal was transmitted to the same program after analogdigital conversion. In order to externally magnetize the magnetic particles, a pair of homemade Helmholtz coils was carefully mounted to the PCB of the sensor chip so that a uniform perpendicular out-of-plane magnetic field (Hext) of 48 Oe could be applied to the device.25 The coils were fed by an external dc current source. Thiol Deposition. The gold films for the surface plasmon resonance (SPR) experiments were deposited by electron beam evaporation of 2-nm Ti and 50-nm Au on 2 M NaOH cleaned glass substrates. These SPR samples were cleaned for 15 min using a homemade UV/O3 device with an ozone producing mercury grid lamp.26 The gold films for the magnetosandwich assays were deposited by electron beam evaporation of 10-nm Ti and 100-nm Au on a polished Si wafer with 1.2-µm thermally grown SiO2. These samples were cleaned by a 5-min sonication step in a 1:1 mixture of H2O and acetone followed by two 15-min UV/O3 cleanings. The packaged MBS chips were treated with the same cleaning procedure. Immediately after UV/O3 cleaning, the samples were immersed in a solution of 5% (v/v) 16-MHA and 95% (v/v) 11MUOH or 5% (v/v) C11-PEO6-COOH and 95% (v/v) C11-PEO3OH (both 1 mM in ultrapure ethanol). After deposition for 3 h, the samples were thoroughly rinsed with ethanol and dried under a stream of nitrogen. Label-Free and Sandwich Assay Detection of S100ββ. The label-free and sandwich assay detection of S100ββ was studied using a Biacore2000 SPR instrument. A continuous flow of modified HEPES-buffered saline (HBS; 10 mM HEPES pH 7.4,

150 mM NaCl, 1 mM CaCl2, and 0.005% Tween-20) was used as running buffer. All experiments were carried out at 20 °C. First, the COOH groups of the coupling layer were activated by injection of a 50 µL of a 1:1 (v/v) mixture of 0.4 M EDC and 0.1 M NHS, both dissolved in H2O. Next, 132 µL of the primary antibody solution (500 µg/mL) in 10 mM acetate buffer pH 5.0 (AB) was injected, followed by the injection of 50 µL of 1 M ethanolamine to block the remaining NHS ester groups and by two pulses of 10 mM glycine (pH 2.2). A flow rate of 5 µL/min was maintained during the immobilization period. S100ββ antigens were diluted into HBS. A total of 130 µL of the S100ββ solution was perfused over the antibody-modified surface, followed by a HBS rinse. Next, 100 µL of 10 µg/mL secondary antibody was perfused over the S100ββ-modified surface. Between each antigen concentration, regeneration of the surfaces was performed by two 10-µL pulses of 10 mM glycine (pH 2.2). A flow rate of 20 µL/min was maintained during this period. The binding levels of both S100ββ and the secondary antibody were measured as the change of the buffer baseline before and after the binding event. Both binding levels were corrected for nonspecific binding by subtraction of the binding levels on the control surface. Magnetosandwich Assay. The magnetosandwich assay was optimized using optical microscopy, and the samples were prepared as follows. The thiol-modified gold samples were statically incubated for 10 min with a 1:1 (v/v) mixture of 0.4 M EDC and 0.1 M NHS both dissolved in water. The activated samples were then statically incubated with 100 µg/mL primary antibody diluted into AB for 1 h followed by a blocking step of 30 min with 1% BSA diluted into AB (procedure 1) or 5% BSA diluted into HBS (procedure 2). The samples were subsequently blocked with 1 M tri(ethylene glycol) monoamine dissolved into water for 30 min. In between the different incubations steps, the samples were washed with HBS. Next, the antibody-modified samples were incubated with different S100ββ concentrations for 90 min followed by incubation with 1 µg/mL biotinylated secondary antibody for 90 min. Finally, the samples were incubated with 1 mg/mL streptavidin magnetic particles for 30 min. The S100ββ, the secondary antibody, and the particle binding were performed in HBS for procedure 1 and in HBS supplemented with 5% BSA and 0.1% Tween-20 for procedure 2. Following particle incubation, the samples were carefully washed with HBS. Five independent microscope images were taken of the substrate and evaluated with CorelPHOTO-PAINT. Brightness, contrast, and intensity of the gray scale-transformed images were adjusted so that the particles became completely black and the background white. The percentage black color was quantified and was correlated to the particle coverage on the substrate. In the next step, the optimized magnetosandwich assay was transferred to the MBS. The packaged MBS chips were modified in the same way as discussed above, and all incubations were performed in a static mode. After the final washing step, the device was mounted on the PCB and the sensing experiment was immediately started. THEORY Magnetic Bead Sensing: Measurement Principle. The general principle of magnetic bead sensing is to magnetize the magnetic particles and detect their stray field using giant magnetoresistive sensors. In prior art, both in-plane and out-of-plane Analytical Chemistry, Vol. 79, No. 22, November 15, 2007

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Figure 1. Schematic diagram illustrating the (A) magnetosandwich assay applied in this work for the detection of S100ββ and (B) the 3 steps performed during the MBS measurement. First (1) the MPs are specifically bound (i.e., randomly distributed) to the sensor surface, followed by (2) the regeneration of the biomolecular bonds and alignment of the MPs to the most sensitive part of the spin valves. (3) Finally, all particles are washed away from the sensor surface to perform a blank measurement.

magnetization was utilized for magnetic biosensing.5,16,27,28 Because spin valves are only sensitive to a magnetic field in the in-plane direction, we chose to apply an out-of-plane magnetization (Hext) to maximize the particle magnetization without affecting the spin valve performance. In contrast to prior art,16,29 we did not sense the signal immediately after the magnetosandwich assay was built up. Instead, the MBS measurement was performed in three subsequent steps (Figure 1B).23 First, the signal (Vrandom) was recorded with all the particles randomly spread over the sensing area. Second, 10 mM NaOH was slowly and carefully injected on top of the sensors, thereby breaking the biomolecular bonds in the sandwich assay and locally releasing all the magnetic particles from the sensor surface. During this process, Hext was on in order to magnetize the magnetic particles. In response to the magnetic field gradient generated by the sensing current sent through the spin valves, the released magnetic particles were immediately aligned to one of the edges of the spin valves, where the magnetic field is the maximum and the stray field of the magnetized particles causes a maximal change in the sensor signal. The second signal (Valigned) was then recorded after the alignment of the magnetic particles to the sensor edge. Third, Hext was switched off, followed by a stringent washing with NaOH. As a result, all the magnetic particles were washed away from the sensor surface. Then, the third signal (Vblank) was collected. In order to filter out the contribution of the thermal drift from the three signals, we used magnetic field (Hext) pulses to magnetize the particles.30 The MBS measurement principle described above is summarized in Figure S2 (see Supporting Information). The overall time to complete this procedure, including the measurement of Vrandom, Valigned, and Vblank), is ∼20 min. It has to be noted that, for a perfectly perpendicular Helmholtz coil configuration, the in-plane component Hext,x equals zero and the only contribution to the MBS signal comes from the magnetic particles’ stray field Hp. In reality, however, there is always a small in-plane component Hext,x contributing to the MBS signal (Figure 1B). Since only Hp reflects the amount of particles specifically bound to the sensor surface and correlates to the concentration 8672

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of S100ββ, we should correct for the contribution of Hext,x. Since there are no particles present during the measurement of Vblank, this signal solely represents the contribution of the Hext,x and can be used as a reference signal. The corrected MBS signals for the random and the aligned sensing scheme could thus be retrieved by respectively subtracting Vrandom and Valigned from Vblank. Furthermore, the MBS signal was normalized by taking into account the sensitivity of each individual sensor. The corrected and normalized MBS signals, i.e., “random” and “aligned”, were calculated using the following formula:

MBS signalrandom/aligned )

Vblank - Vrandom/aligned IR0HextS

(1)

where Vblank and Valigned/random are in millivolts, I is the sensing current (18 mA), R0 is the resistance in Ω at zero external field, Hext is the applied external field (48 Oe), and S is the sensitivity of the spin valve sensor in MR%/Oe. RESULTS AND DISCUSSION Influence of the Surface Chemistry on the Label-Free and Sandwich Assay Detection of S100ββ. A common bottleneck in the development of biosensors is the lack of a stable, reproducible, specific, and sensitive chemical coupling layer modified with biological receptors. Here, we compare the performance of two mixed SAMs of thiols, i.e., 16-MHA and 11-MUOH (alkanethiol) or 11-PEO6-COOH and 11-PEO3-OH (PEO-thiol) versus a commercial carboxymethyl dextran layer (CM5). The different coupling chemistries were compared using a Biacore2000 SPR instrument.31,32 The immobilization degree of proteins in a Biacore system is given in refractive units (RU) and 1 RU corresponds to 1 pg/mm2. The impact of the surface chemistry was investigated on the level of both label-free sensing and sandwich assay sensing, as ultimately a magnetosandwich assay will be used to specifically bind magnetic particles to the MBS (Figure 1A). The sandwich assay described in this paper consists of two anti-S100ββ antibod-

Figure 2. Dose-response curves determined via SPR for the labelfree (a) and sandwich assay (b) detection of S100ββ. Both detection schemes were applied on the three different coupling layers, i.e., CM5, PEO-thiol, and alkanethiol.

ies, i.e., a primary antibody covalently immobilized on the mixed thiol SAM and a secondary antibody, which can bind to the S100ββ/antibody complex.33 To obtain a sensitive S100ββ binding, a high degree of primary antibody needs to be immobilized on the surface. After activation of the surface COOH-groups with EDC/NHS, S53 can be covalently coupled via its amino groups. The CM5 surface resulted in the highest degree of coupling (8531 RU), which is due to the three-dimensional structure of the dextran layer.34-36 The alkanethiol surface resulted in the lowest degree of coupling (1081 ( 270 RU), while on the PEO-thiol we obtained a much higher coupling degree (4297 ( 122 RU). This is thought to be due to the flexibility and more three-dimensional character of the PEO-thiol as compared to the rigid, twodimensional alkanethiol. Furthermore, the PEO-thiol also allows a more reproducible immobilization with 3% standard deviation, compared to 25% for the alkanethiol. The anti-PSA antibody, used as a control for nonspecific binding, showed a coupling behavior similar to S53. First, we tested the label-free S100ββ assay on the different coupling layers and compared the stability, sensitivity, detection limit, and nonspecific binding. A dose-response curve between 0.3 and 1280 ng/mL S100ββ was generated in order to calculate the sensitivity of the label-free S100ββ detection (Figure 2a). Saturation is reached much faster on the mixed thiol SAMs while the saturation level is much higher on the CM5 surface. The latter is a result of the higher coupling degree and the higher activity of the coupled antibodies. More specifically, 46% of the available binding sites on the CM5 surface have bound S100ββ in comparison with 26 and 23% on the alkanethiol and the PEO-thiol, respectively. This observation can again be explained by the threedimensional structure of the CM5 surface, which is expected to result in less steric hindrance than a two-dimensional layer.37 To calculate the sensitivity of the assay on the different layers, the first part of the dose-response curve was fit linearly. The slope of the fitted curve is known as the sensitivity of the performed assay. The PEO-thiol and the CM5 surface have comparable sensitivities while the sensitivity of the alkanethiol is lower (Table 1). From the sensitivity, we calculated the detection limit (DL) in nanograms per milliliter using the following formula:

DL )

|(3N) - I| S

(2)

where N is the noise level (RU), I is the intercept of the linear fit (RU), and S is the sensitivity (RU mL/ng).38 The noise level N, which is an indication of the stability of the coupling layer, was calculated as the standard deviation on 10 blank buffer injections. This resulted in a noise level of 2.9, 1.3, and 4.7 RU for the alkanethiol, the PEO-thiol, and the CM5 surface, respectively. Apparently, mixed thiols exhibit a lower noise level than the CM5 surface. This resulted in comparable detection limits on the alkanethiol and the CM5 surface although the latter was much more sensitive (Table 1). The best detection limit could be obtained on the PEO-thiol. In a next step, we tested the performance of the S100ββ sandwich assay on the different surface chemistries using the same methodology as described above. The resulting doseresponse curves between 0.3 and 160 ng/mL S100ββ are presented in Figure 2b. None of the layers reached saturation in the applied concentration range. The PEO-thiol was found to be two times more sensitive than the CM5 surface, which is also two times more sensitive then the alkanethiol (Table 1). When taking into account the stability of the different layers, we obtained comparable detection limits for the sandwich assay on the alkanethiol and the CM5 surface although the latter was much more sensitive. The lowest detection limit for the sandwich assay was obtained on the PEO-thiol, similar to that observed for the label-free detection. Finally, we evaluated the specificity of the three surface chemistries by measuring the nonspecific binding of S100ββ and the secondary antibody S36 on a control antibody and by calculating the nonspecific binding of human serum dilutions on the specific antibody. Both the nonspecific binding of S100ββ and the secondary antibody were found to be negligible on all three layers. On the other hand, the nonspecific binding of human serum was much higher on the CM5 surface, indicating that the latter is much more prone to nonspecific binding (data not shown). In conclusion, we have shown that mixed thiols exhibit improved performances compared to the commercial CM5 surface chemistry. Therefore, mixed thiols were used for the magnetosandwich assay. Optimization of the Magnetosandwich Assay and the Influence of the Surface Chemistry. To allow detection with the MBS, the analyte captured by the surface-immobilized antibody is being labeled with a secondary antibody and with magnetic particles, to form a magnetosandwich assay (Figure 1A). In this study, the magnetic labeling is accomplished by the specific interaction between streptavidin magnetic particles and the biotinylated secondary antibody. Many techniques have been applied in the literature to characterize the binding of particles on surfaces, e.g., atomic force microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, etc.11,39,40 However, in our case, where relatively big magnetic particles are used (300 nm and 1 µm), optical microscopy is the easiest and most straightforward detection method.41 Magnetic particles are commercially available in a range of sizes, functionalities, and magnetic properties. To evaluate which commercial streptavidin-modified magnetic particles are (40) Csa´ki, A.; Mo¨ller, R.; Straube, W.; Ko¨hler, J. M.; Fritzsche, W. Nucleic Acids Res. 2001, 29, e81. (41) Diao, J.; Ren, D.; Engstrom, J. R.; Lee, K. H. Anal. Biochem. 2005, 343, 322-328.

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Table 1. Influence of Surface Chemistry, Type of Magnetic Particle, and Blocking Procedure on the Total Assay Performancea detection platform SPR

detection of S100ββ secondary antibody

microscope

magnetic particle (1 µm) magnetic particle (300 nm)

MBS

magnetic particle (300 nm)

surface chemistry

sensitivity

detection limit (ng/mL)

dynamic range (ng/mL)

specificity

CM5 PEO-thiol alkanethiol CM5 PEO-thiol alkanethiol

1.7 RU mL/ng 1.2 RU mL/ng 0.45 RU mL/ng 6.5 RU mL/ng 13 RU mL/ng 3.7 RU mL/ng

18 2.9 15 4.5 1.5 3.7

18-1000 2.9-300 15-300 4.5-150b 1.5-150b 3.7-150b

+ + + + + +

alkanethiol PEO-thiol alkanethiol alkanethiol + BSA blocking PEO-thiol PEO-thiol + BSA blocking

5.5 % mL/ng 6.8 % mL/ng 6.1 % mL/ng 59 % mL/ng

20 18 1.9 0.047

20-35 18-35 1.9-5b 0.047-2

+ + +

43 % mL/ng 265 % mL/ng

0.31 0.011

0.31-3 0.011-1.5

+

alkanethiol + BSA blocking

0.59 × 10-3 mL/ng

0.027

0.027-1

+

a The different detection platforms allowed us to separately evaluate the different building blocks of the magnetosandwich assay. b No measurements were performed at higher concentrations; therefore, the upper limit could not be determined.

appropriate for building a sandwich assay, we functionalized a gold substrate with thiol SAMs, covalently immobilized biotin, and directly bound the streptavidin particles. The binding was monitored using SPR and optical microscopy. The results of this study have been reported elsewhere.42 Two types of magnetic particles gave excellent binding signals in these experiments, i.e., Ademtech 300 nm and Dynal 1 µm, and were used further in this study. Both particles show a good uniformity in size and shape (see Figures S3 and S4 in Supporting Information) and are reported to have excellent magnetic properties.43 We opted for two different sizes, i.e., 1 µm and 300 nm, since we believe that the particle size has a dramatic effect on the assay performance. Figure 3 shows the percent coverage of 1-µm Dynal particles on the substrate. Both alkane- and PEO-thiols were compared to study the influence of the surface chemistry on the magnetosandwich assay characteristics. A concentration of 100 ng/mL S100ββ resulted in a high degree of particle binding on the surface, with coverages ranging up to 90% (Figure 3). In the case of 0 ng/mL S100ββ, no particle binding (