J. Phys. Chem. C 2007, 111, 12227-12235
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Magnetic Particles as Labels in Bioassays: Interactions between a Biotinylated Gold Substrate and Streptavidin Magnetic Particles Randy De Palma,*,†,‡ Chengxun Liu,† Francesca Barbagini,§ Gunter Reekmans,† Kristien Bonroy,† Wim Laureyn,† Gustaaf Borghs,† and Guido Maes‡ NEXT and UCP-ACMD, IMEC, Kapeldreef 75, B-3001 LeuVen, Belgium, and Physical and Quantum Chemistry, Katholieke UniVersiteit LeuVen, Celestijnenlaan 200F, B-3001 LeuVen, Belgium ReceiVed: April 2, 2007; In Final Form: June 22, 2007
Magnetic particles (MPs) have been attracting much interest as a labeling material for advanced biological and medical applications, such as biomagnetic separation, drug delivery, magnetic resonance imaging, and hyperthermia. In most of these applications, the MPs have been designed to specifically interact with a target, such as cells or proteins, moving freely in a solution. However, for surface-based applications, such as magnetic biosensing, these MPs must bind specifically with a target that is immobilized onto a planar substrate. Consequently, new interaction phenomena, which influence the binding of the MPs to the substrate, have to be taken into account. To achieve adequate binding characteristics and to optimize the MPs toward substrate labeling, these physicochemical interactions should be properly identified. In this paper, the interactions between 16 commercially available streptavidin MPs and a biotinylated gold substrate were monitored in real time by surface plasmon resonance technology and the particle surface coverage was calculated by optical microscopy. On the basis of the type of interactions, the MPs studied in this paper could be classified into three different cases: (I) MPs that bind to the biotinylated substrate via the specific streptavidin-biotin interactions, without showing any nonspecific interactions; (II) MPs that do not bind to the substrate; and (III) MPs that bind to the biotinylated substrate via nonspecific interactions rather than via specific streptavidin-biotin interactions. The three cases were understood by determining the surface charges of both the particle and the substrate in ζ potential measurements. It was found that binding of MPs to the substrate was strongly dependent on the amount and the sign of the charges on both surfaces. The strong influence of electrostatic interactions was validated by simulating the total interaction force between a streptavidin MP and a biotinylated substrate by use of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, while the gravitational force and the streptavidin-biotin force were accounted for. Finally, we conclude that apart from a well-controlled streptavidin coating, the surface charge of the particle and the substrate plays a pivotal role in the construction of MP assays on surfaces.
Introduction Over the past few decades, the study of magnetizable objects on the nano- and micrometer scale has generated considerable interest. These magnetic particles (MPs) have triggered numerous promising applications in the fields of advanced biological and medical sciences.1,2 They have revolutionized imaging, therapeutic, and purification processes, such as magnetic resonance imaging,3,4 site-specific drug delivery,5, cancer treatment via hyperthermia5,6 and molecular/cellular separation.7-9 These applications can be classified as solution-based applications in which the biologically functionalized MPs typically bind to a target that is moving freely in a (biological) solution. Due to the increasing interest in MPs, many commercial companies have focused their efforts on the specific design of MPs for solution-based applications, especially aiming at biological isolation and purification.10 Examples are the removal of tumor cells from bone marrow8,11 or the isolation of antibodies from * Corresponding author: MCP-ART, IMEC, Kapeldreef 75, B-3001 Leuven, Belgium. Phone +32-16-281083, fax +32-16-281097; e-mail
[email protected]. † NEXT, IMEC. ‡ KU Leuven § UCP-ACMD, IMEC.
serum.7,12 Following their increasing success, many requirements have been reported for proper use of MPs in solution-based applications.4,10,13 More recently, MPs are being increasingly used as labels in surface-based applications, such as magnetic biosensing,14,15 where they show advantages over the more commonly used labeling materials, for example, enzymes, fluorescent dyes, chemiluminescent molecules, or radioisotopes.16,17 In these surface-based applications, the target is most frequently immobilized on a planar substrate. A recent example is the detection of C-reactive protein, a marker for inflammatory processes, via magnetic biosensors.18 The presence of the substrate introduces new physical and chemical phenomena that determine the interactions between a MP and its target immobilized onto the substrate. Consequently, commercially available MPs, which are specifically optimized for solutionbased applications, are often not suitable for surface-based applications. Many papers elucidated on the use of MPs as labels for biofunctionalized substrates, especially in the field of magnetic biosensors.19,20 For example, Ferreira et al.21 and Schotter et al.22 performed an experimental comparison between various commercially available streptavidin MPs and proposed a number
10.1021/jp0725681 CCC: $37.00 © 2007 American Chemical Society Published on Web 07/31/2007
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TABLE 1: Overview of Important Physicochemical Characteristics of Streptavidin Magnetic Particles diameter (nm) supplied by company
measured via DLS
biotin binding capacity (pmol/mg)
company
product name
Dynal Ademtech Ademtech Ademtech Ademtech Miltenyi Roche
Dynabeads MyOne MasterBeads Bio-Adembeads Bio-Adembeads Bio-Adembeads Microbeads magnetic particles
1050 525 293 197 121 ∼50 1000
Case I: Specific Binding 1006 ( 249 470 ( 63 282 ( 12 216 ( 14 125 ( 8 46 ( 13 1232 ( 258
Bangs Indicia Indicia Seradyn Micromod Micromod Micromod Kisker
ProActive microspheres magnetic particles magnetic particles Sera-Mag Nanomag-silica Nanomag-D Nanomag-D dextran magnetic particles
860 350 1050 783 250 250 130 250
Case II: No Binding 786 ( 207 500 ( 182 651 ( 279 850 ( 252 211 ( 49 258 ( 137 173 ( 79 203 ( 92
Immunicon
captivate ferrofluid
∼200
Case III: Nonspecific Binding 70 ( 33
a
force barrier heightb (pN)
pI
ζa (mV)
3500 1206 387 809 3757 c 350
6.5 7.2 6.8 7.1 6.9 5.2 6.5
-7.3 -6.7 -8.4 -7.5 -7.5 -11.0 -6.9
9 1 8 3 2 4 4
878 c c 4188 70-100 70-100 70-100 70-100
4.4 4.9 4.6 4.5 2.9 2.9 3.4 4.2
-38.8 -30.6 -32.5 -29.5 -31.9 -24.5 -23.1 -24.2
941 438 622 702 196 157 101 121
∼10 000
7.7
+11.8
d
ζ potential at pH 7.4. From DLVO calculations. Not provided by the supplier. No barrier ) always attractive. b
c
of requirements for their use in magnetic biosensor applications, such as high magnetization, specific binding character, and good size/shape uniformity. However, all these reports neglected the requirements imposed by the presence of a substrate. Furthermore, only the results with “optimal” MPs (i.e., MPs that bind specifically to the immobilized target) were reported, and the underlying physical and chemical phenomena that determine the MP binding characteristics were typically ignored. For example, it is well-known that when two surfaces (e.g., particle and planar substrate) approach each other, their interaction is often dominated by van der Waals and electrostatic forces,23 which is not necessarily the case when a particle interacts with a target moving freely in solution. The variety of commercial MPs applied in magnetic biosensing is enormous,15,24-26 although streptavidin-modified MPs are among the most commonly used. In a typical magnetic biosensing experiment, these streptavidin MPs are applied in a sandwich assay format. In this type of assay, the target of interest is specifically bound to biological receptors immobilized on the sensor surface. Next, the captured analyte is labeled with a biotinylated second receptor. On the basis of the high affinity of the streptavidin-biotin system, the MPs are finally bound to the sensor surface, where their signature can be monitored by the magnetic sensor. To avoid the more complicated and time-consuming procedure of constructing an entire sandwich assay, we opted for the use of a biotinylated substrate as a model system to bind streptavidin MPs. Biotin-streptavidin is an ideal model system due to its high affinity (Ka ) 1015 M-1), its high specificity, and its generic nature (i.e., the same MP can be used to label different biotinylated analytes). According to Megens and Prins,16 one of the key challenges for future development of magnetic biosensors (or surface-based applications in general) is the implementation of suitable magnetic particles. However, the question arises, “What makes a particle suitable for a certain application?” In general, one could say that a particle is suitable for a certain application when it is specifically designed and optimized toward that particular application. But in order to perform a proper optimization, one needs to understand the physical and chemical phenomena that drive the interactions between the particle and its target. Therefore, in this paper, the interactions between 16 com-
d
mercially available streptavidin MPs and a biotinylated gold substrate were evaluated by surface plasmon resonance (SPR) and optical microscopy in order to identify which MPs are suitable for the labeling of substrates. To better understand the differences in the behavior of the different streptavidin MPs and to identify the underlying physical and chemical phenomena that drive these interactions, the surface of both the particle and the substrate were thoroughly characterized. Supported by interaction force calculations, we found that surface charges and electrostatic interactions play a dominant role in determining the MP binding to a substrate. To our knowledge, this is the first systematic report that addresses design issues for magnetic particles in surface-based labeling applications. Furthermore, this paper highlights basic concepts for the future implementation of particles in surface-based applications. Experimental Section Materials. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), 11-mercapto-1-undecanol (11-MUOH) (>97%), 16-mercapto-1-hexadecanoic acid (16MHA) (>90%), and glycine hydrochloride (99%) were obtained from Sigma. Calcium chloride was purchased from Riedel-de Hae¨n. Tween-20 and N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) (sodium salt, buffer grade) were obtained from Fluka and Applichem respectively. Tri(ethylene glycol) monoamine (NH2-PEO3-OH) was purchased from Molecular Biosciences, and EZ-link biotin-LC-PEO-amine (NH2-PEO3biotin) was purchased from Pierce. Ultrapure ethanol (Puranal) was purchased from Honeywell. NaOH (pro-analyze) and maleic acid (99%) were from Merck. Acetone (Cleanroom) and HCl (37%) were purchased from Air Products. NaCl (molecular biology grade) was from Calbiochem, and the IBIS gold sensor chips were from Metrohm. The commercial information on the MPs studied in this paper, such as supplier, product name, and diameter, are given in Table 1. It has to be noted that in the remainder of this paper the MPs are given a name based on their supplier and the hydrodynamic diameter measured in our lab by dynamic light scattering. All materials and reagents were used as commercially received. Thiol Deposition on Gold Substrates. Thiols were deposited on gold substrates following a standard procedure reported
Magnetic Particles as Labels in Bioassays elsewhere.27,28 Briefly, the IBIS sensor chips were thoroughly rinsed with acetone and cleaned for 30 min in a homemade UV/O3 device with an ozone-producing mercury grid lamp (BHK Inc.).29 Immediately after UV/O3 cleaning, the IBIS sensor chips were immersed in a mixed thiol solution of 5% (v/v) 16-MHA and 95% (v/v) 11-MUOH (1 mM in ultrapure ethanol). After a deposition time of 3 h, the samples were thoroughly rinsed with ethanol and dried under a stream of nitrogen. Biotinylation of Gold Substrate. Immediately after thiol deposition, the samples were placed into the IBIS SPR instrument (details described further). A measurement cell, containing two separate chambers, was mounted on top of the sensor chip and the measurement was immediately started. All experiments were carried out at 21 °C. The sensor surface in the first chamber was functionalized with a biotin-capped PEO3 linker (NH2PEO3-biotin) in order to investigate the specific binding of streptavidin MPs to the biotinylated gold substrate. The sensor surface in the second chamber was functionalized with a similar PEO3 linker, with an OH end group (NH2-PEO3-OH), and was used to study nonspecific interactions with the functionalized gold substrate. Both measurements were carried out in parallel on the same sensor chip, in order to guarantee a reliable comparison, and were repeated at least twice. In this first part of the IBIS experiment, maleate buffer (5 mM, pH 6.0) was used as the running buffer. To functionalize the surface with both linkers (NH2-PEO3-OH and NH2-PEO3-biotin), the following procedure was used. First, the sensor surface was pretreated with 50 µL of glycine (10 mM, pH 2.2) for 10 min. Subsequently, the COOH groups of the thiol coupling layer were activated by a 10 min injection of a 50 µL 1:1 (v/v) aqueous mixture of 0.4 M EDC and 0.1 M NHS. A freshly prepared solution of NH2-PEO3-biotin (50 µL, 500 µg/mL) in maleate buffer was then incubated on the specific substrate for 30 min. During the same time period, the nonspecific substrate was functionalized with 50 µL of NH2-PEO3-OH solution (1 M) in maleate buffer. Afterward, both substrates were incubated with 50 µL NH2-PEO3-OH (1 M in maleate buffer) for 10 min to deactivate the remaining NHS ester groups. At this stage, the sensor surface was ready to interact with the streptavidin MPs. The gold films for ζ potential measurements were deposited by electron beam evaporation of 10 nm Ti and 100 nm Au on a polished 6 in. Si wafer with 1.2 µm thermally grown SiO2. The same functionalization procedure as described above was adopted to prepare the gold substrates for ζ potential measurements. However, in this case, higher volumes of reagents were used (5 mL) and the functionalization was performed outside the IBIS instrument. To avoid contamination of the functionalized gold substrates, ζ potential measurements were carried out immediately after functionalization. Binding of Streptavidin Magnetic Particles to Biotinylated Gold Substrate. The binding of streptavidin MPs onto the functionalized gold substrate was monitored in real time during the same experiment as the one in which the sensor chip surface was functionalized. In this second part of the SPR experiment, HEPES-buffered saline (HBS) (10 mM HEPES at pH 7.4, 0.15 M NaCl, and 0.005% Tween-20) was used as the running buffer. Prior to use, the MPs were washed three times with deionized water and were finally dissolved in HBS at a concentration of 1 mg/mL. The MPs were simultaneously incubated on both functionalized sensor surfaces for 20 min to study their specific and nonspecific binding characteristics. All experiments were performed in a stagnant solution, resulting in diffusion-controlled binding events. Finally, the surface was rinsed two times with
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Figure 1. Illustration of the dominant forces acting on a streptavidin MP in the vicinity of a biotinylated substrate.
100 µL of HBS. The first rinse was performed at a speed of 10 µL/s and the second rinse at a speed of 50 µL/s. Afterward, the sensor chip was demounted from the IBIS instrument and the binding of the streptavidin MPs to the functionalized substrates was analyzed on the same sensor chips by optical microscopy in order to calculate the surface coverage. Characterization Methods. Surface plasmon resonance (SPR) measurements were performed on an IBIS II instrument (IBIS Technologies B.V., currently Ecochemie B.V.). The IBIS II is equipped with a double laser beam, allowing simultaneous evaluation of two separate areas on a single sensor chip. The measurements were performed in a stagnant environment and the solutions were replaced by an automated syringe setup that can handle volumes up to 100 µL. The IBIS sensor chips (Metrohm) were made of optical glass, coated with a thin Ti adhesive layer and ∼50 nm Au. Optical micrographs were taken on a Nikon microscope equipped with an Ikegami digital camera while a 50× magnification long-distance lens (Nikon) was used. At least three independent images were recorded and evaluated with photoprocessing software (Corel Photo-Paint). Brightness, contrast, and intensity of the grayscale-transformed images were adjusted so that the particles became completely black and the background white. The percentage of black color was quantified and used as the percentage of particle coverage on the substrate. The coverages determined via optical microscopy consistently lie within ∼10% from the real coverages. Dynamic light scattering (DLS) was used to determine the ζ potential and hydrodynamic diameter of the streptavidin MPs. DLS measurements were performed on a Zetasizer Nano ZS (Malvern), equipped with an automated titration unit. All ζ potentials were recorded in an aqueous solution with a pH varying between 2 and 12. The pH of the solution was adjusted with HCl and NaOH. Streaming potential measurements were performed on an Anton Paar EKA system and were used to determine the ζ potential of the biotinylated gold substrate. All ζ potentials were recorded in an aqueous solution with the pH adjusted between 2 and 12 with HCl and NaOH. Theory Figure 1 gives a schematic illustration of the total interaction force between a streptavidin MP and a biotinylated substrate as studied in this work. In this system, a spherical particle with radius R is positioned at a distance z from the substrate in solution under the influence of several surface forces. For clarity reasons, we describe only the force components that are aligned perpendicular to the substrate. Furthermore, other force components with minor contributions, such as the Langevin force
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due to Brownian motion, are not taken into account in this work. The total interaction force between a streptavidin MP and a biotinylated substrate consists of specific and nonspecific surface forces. Below we describe these forces in more detail. Specific Surface Force: StreptaVidin-Biotin Force (FS-B). The streptavidin-biotin system exhibits the strongest known noncovalent biological bond, which arises from a unique combination of van der Waals, hydrogen-bond, ionic, and hydrophobic interactions. A multitude of papers investigated its bonding strength via atomic force microscopy or the surfaceforce apparatus and measured a rupture force of 100-300 pN, depending on the applied loading rate, the type of instrument, and the type of streptavidin.30,31 The attractive force, giving rise to the strong and specific adhesive binding between streptavidin and biotin, is known to manifest itself at very short ranges, typically at a distance smaller than 1-2 nm.32 Nonspecific Surface Forces: Van der Waals Force (Fvdw). The van der Waals force acting between two atoms is a shortrange attractive force that originates from electromagnetic interactions between dipoles and/or induced dipoles. By use of the Derjaguin approximation, Fvdw between a sphere and a plane can be calculated as follows:33
Fvdw ) -
H132R
(1)
6z2
where z is the separation distance between the particle and the substrate, R is the radius of the particle, and H132 is the combined Hamaker constant for a particle in a medium interacting with a substrate. This constant depends on the dielectric frequency response of all three materials over the whole electromagnetic spectrum and was assumed to be 3.4 × 10-21 J.34 Fvdw is known to give an important attractive contribution, especially at short separation distances (
