Surface Plasmon Resonance Spectroscopy and Quartz Crystal

Dec 1, 2004 - Surface plasmon resonance (SPR) spectroscopy is employed for the study of biotinylated DNA assembly on streptavidin modified gold surfac...
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Langmuir 2005, 21, 348-353

Surface Plasmon Resonance Spectroscopy and Quartz Crystal Microbalance Study of Streptavidin Film Structure Effects on Biotinylated DNA Assembly and Target DNA Hybridization Xiaodi Su,*,† Ying-Ju Wu,† Rudolf Robelek,† and Wolfgang Knoll*,†,‡ Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602, and Max-Planck-Institut fu¨ r Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany Received August 10, 2004. In Final Form: October 12, 2004 Surface plasmon resonance (SPR) spectroscopy is employed for the study of biotinylated DNA assembly on streptavidin modified gold surfaces for target DNA hybridization. Two immobilization strategies are involved for constructing streptavidin films, namely, (1) physical adsorption on biotin-containing thiol treated surfaces through biotin-streptavidin links and (2) covalent attachment to 11-mercaptoundecanoic acid (MUA) treated surfaces through amine coupling. To understand the structural properties of the streptavidin films, a quartz crystal microbalance with energy dissipation monitoring (QCM-D) is used to monitor the streptavidin immobilization procedures. The simultaneously measured frequency (∆f) and dissipation factor (∆D) changes, together with the SPR angle shifts (∆θ), suggest that the streptavidin film assembled on the biotin-containing surface is highly rigid with a well-ordered structure while the streptavidin film formed through amine coupling is highly dissipative and less structured. The subsequent biotinylated DNA (biotin-DNA) assembly and target hybridization results show that the streptavidin film structure has distinct effects on the biotin-DNA binding amount. On the streptavidin matrix, not only the probe DNA density but also the strand orientation mediated by the streptavidin films has distinct effects on hybridization efficiency. Particularly, the molecularly ordered streptavidin films formed on the biotincontaining surfaces ensure a well-ordered DNA assembly, which in turn allows for a higher efficiency in target DNA capture and for a higher sensitivity in the hybridization analysis when compared to the biotin-DNA assembled on the less structured streptavidin films formed through amine coupling.

Introduction The efficient and reproducible hybridization of oligonucleotides with surface-immobilized DNA has become increasingly important in disease diagnosis and biomedical research. Sensitive DNA hybridization assays rely on the construction of immobilized DNA probes with sufficient stability, activity, and well-controlled packing density.1,2 For the thiol-gold-interaction-based covalent DNA immobilization, for example, great efforts have been invested to optimize the self-assembly of the SH-DNA probe for controlled probe density and controlled hybridization efficiency as well as duplex formation kinetics.2-5 Streptavidin is a basic tetrameric protein isolated from Streptomyces avidinii. It binds tightly to biotin, a small vitamin ligand. A combination of high affinity (Kd ∼ 10-15 M for unlabeled biotin and streptavidin),6 binding capacity, reproducibility, and chemical resistance makes the biotinstreptavidin system extensively used for DNA immobi* Corresponding authors. Phone: (65) 68748420 (X.S.); 49 (0) 6131 379 160 (W.K.). Fax: (65) 68720785 (X.S.); 49 (0) 6131 379 360 (W.K.). E-mail: [email protected] (X.S.); [email protected] or [email protected] (W.K.). † Institute of Materials Research and Engineering. ‡ Max-Planck-Institut fu ¨ r Polymerforschung. (1) Georgiadis, R. M.; Peterlinz, K. P.; Peterson, A. W. J. Am. Chem. Soc. 2000, 122, 3166-3173. (2) Peterson, A. W.; Richard, J. H.; Georgiadis, R. M. Nucleic Acids Res. 2001, 29, 5163-5168. (3) Georgiadis, R.; Peterlinz, K. P.; Peterson, A. W. J. Am. Chem. Soc. 2000, 122, 3166-3173. (4) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1998, 120, 97879792. (5) Satjapipat, M.; Sanedrin, R.; Zhou, F. M. Langmuir 2001, 17, 7637-7644. (6) Wilchek, M.; Bayer, E. A. Anal. Biochem. 1998, 171, 1-6.

lization in chip- and sensor-based bioassays, upon preconstruction of streptavidin films on the substrates.7-14 Among many protein immobilization methods, amine coupling is most frequently used for covalent attachment of streptavidin on carboxyl-group-containing surfaces.13,15 The primary amines of the surface exposed lysine of streptavidin are involved in the amide bond formation via a nucleophilic displacement of N-hydroxysuccinimide (NHS) esters formed during N-(3-dimethylaminopropyl)N′-ethylcarbodiimide hydrochloride/NHS activation. This covalent surface attachment does not influence most of the biotin binding sites, thus leaving them available for binding with biotinylated ligands. Alternatively, in relatively new attempts, various chemistries have been developed to introduce biotin residues on solid substrates, onto which streptavidin molecules can be directly adsorbed via one or two biotin links.8-12,14 The dyad symmetry and (7) Caruso, F.; Rodda, E.; Furlong, D. F.; Niikura, K.; Okahata, Y. Anal. Chem. 1997, 69, 2043-2049. (8) Knoll, W.; Zizlsperger, M.; Liebermann, T.; Arnold, S.; Badia, A.; Liley, M.; Piscevic, D.; Schmitt, F.-J. Colloids Surf., A 2000, 161, 115137. (9) Nelson, K. E.; Gamble, L.; Jung, L. S.; Boeckl, M. S.; Naeemi, E.; Golledge, S. L.; Sasaki, T.; Castner, D. G.; Campbell, C. T.; Stayton, P. S. Langmuir 2001, 17, 2807-2816. (10) Jung, L. S.; Nelson, K. E.; Stayton, P. S.; Campbell, C. T. Langmuir 2000, 16, 9421-9432. (11) Shumaker-Parry, J. S.; Zarele, M. N.; Aebersold, R.; Campbell, C. T. Anal. Chem. 2004, 76, 918-929. (12) Ho¨o¨k, F.; Ray, A.; Norden, B.; Kasemo, B. Langmuir 2001, 17, 8305-8312. (13) Nilsson, P.; Persson, B.; Uhlen, M.; Nygren, P. A. Laboratory Methods for the Detection of Mutations and Polymorphisms in DNA; CRC Press: 1997; pp 253-261. (14) Su, X. Biochem. Biophys. Res. Commun. 2002, 290, 962-966. (15) Fa¨gerstam, L. Techniques in Protein Chemistry II; Academic Press: San Diego, CA, 1991; pp 71-95.

10.1021/la047997u CCC: $30.25 © 2005 American Chemical Society Published on Web 12/01/2004

SA Film Structure Effects on Biotin-DNA Assembly

the multiple biotin binding sites of the streptavidin molecule allow for a further binding of biotinylated DNA (biotin-DNA) via a biotin-streptavidin-biotin bridge chemistry. To this end, studies have been conducted to optimize the surface biotin contents in order to achieve streptavidin films with highly controlled surface physical properties.8-10,16,17 However, to the best of our knowledge, there are no understandings of the effects of streptavidin immobilization strategies on subsequent biotin-DNA binding density and orientation. Both aspects are known to be determining factors for the efficiency of target capture, which, in turn, determine the sensitivity of hybridization assays. In this paper, surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance with dissipation monitoring (QCM-D) techniques are used to study streptavidin immobilization on gold surfaces through two strategies, namely, (1) physical adsorption on biotin-containing thiol treated surfaces and (2) covalent attachment on 11mercaptoundecanoic acid treated surfaces via amine coupling. Using a 30 mer biotinylated, single-stranded oligonucleotide as the probe DNA and a 30 mer complementary oligonucleotide as the target DNA, we study the effects of streptavidin packing density and film structure on the DNA binding amount and strand orientation, and the consequent effects on target DNA hybridization efficiency. The simultaneously recorded frequency (∆f) and dissipation (∆D) changes in the QCM-D measurements, together with the SPR angle shifts (∆θ), are used to reveal the structural properties of the streptavidin films formed under different immobilization schemes. Experimental Section Materials. 11-Mercaptoundecanoic acid (MUA), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and streptavidin (SA) were purchased from Sigma-Aldrich (St. Louis, MO). N-Hydroxysuccinimide (NHS) was obtained from Acros Organics (Fisher Scientific, U.K.). The biotin- and ethylene glycol-terminated thiols were synthesized in our laboratory at the Max-Planck-Institute for Polymer Research (Mainz, Germany). Thirty base pair (bp) oligonucleotides were obtained from MWG Biotech (Germany). The probe DNA was tagged with a biotin label at the 5′ end (5′-biotin-GCACCTGACTCC TGTGGAGAAGTCTGCCGT-3′), and the target DNA contains a fully complementary sequence to the probe DNA (3′-CGTGGACTGAGGACACCTCTTCAGACGGCA-5′). Phosphate buffered saline (PBS) composed of 10 mM phosphate buffer, 137 mM NaCl, 2.7 mM KCl (pH 7.4), and sodium acetate buffer (pH 4.5) was used for streptavidin immobilization. DNA immobilization and hybridization were conducted in PBS buffer. SPR Measurement. SPR measurements were conducted using a double-channel Autolab ESPR instrument (Eco Chemie, The Netherlands). The configuration of this equipment is described elsewhere.18,19 Briefly, via a hemicylindrical lens, p-polarized laser light (λ ) 670 nm) is directed to the bottom side of the sensor disk and the reflected light is detected by a photodiode. The angle of incidence is varied using a vibrating mirror with a frequency of 44 Hz. In each cycle, SPR curves were scanned on the forward and backward movement of the mirror. In an adjustable interval time, the minimums in reflectance are determined and averaged. Binding curves are obtained by processing this minimum in reflectance in real time. The instrument is equipped with a cuvette. The gold sensor disks (diameter 17 mm) mounted on the lens (through index-matching (16) Spinke, J.; Liley, M.; Guder, H. J.; Augerwaier, L.; Knoll, W. Langmuir 1993, 9, 1821-1825. (17) Spinke, J.; Liley, M.; Schwitt, F. J.; Guder, H. J.; Augerwaier, L.; Knoll, W. J. Chem. Phys. 1993, 99, 7012-7019. (18) Wink, T.; Van Zuilen, S. J.; Bult, A.; van Bennekom, W. P. Anal. Chem. 1998, 70, 827-832. (19) Bart. M.; van Os, P. J. H. J.; Kamp, B.; Bult, A.; van Bennekom, W. P. Sens. Actuators, A 2002, 84, 129-135.

Langmuir, Vol. 21, No. 1, 2005 349 oil) form the base of the cuvette. An autosampler (Eco Chemie, The Netherlands) is used to inject or remove tested solutions. The measurements are conducted at room temperature, and the noise level is 1 mdeg. Quartz Crystal Microbalance with Energy Dissipation (QCM-D) Measurement. The QCM-D (Q-Sense, Go¨teborg, Sweden) technique, described in detail elsewhere,20 is an extension of a traditional QCM technique. It measures the frequency change (∆f) and the energy dissipation change (∆D) by periodically switching off the driving power over the crystal and recording the decay of the damped oscillation. The time constant of the decay is inversely proportional to D, and the period of the decaying signal gives f. Five megahertz, AT-cut quartz crystals (Q-Sense AB, Go¨teborg, Sweden) were used as the reaction carriers. The Sauerbrey sensitivity of these crystals is 1 Hz ) 17.7 ng/cm2. The QCM-D setup allows for subsequent measurements of up to four harmonics (fundamental frequency and 15, 25, and 35 MHz, corresponding to the overtones n ) 3, 5, and 7, respectively) of the 5 MHz crystal. For better clarity, only the normalized frequency shift (∆fnormalized ) ∆fn/n) and the dissipation shift (∆D) for the third overtone is presented. During the measurements, the crystal was mounted in a thermal static liquid chamber, designed to provide a rapid, nonperturbing exchange of the liquid over one side of the sensor. The measurements were conducted at room temperature, and the noise of f and D with the liquid load is 0.3 Hz and 0.2 × 10-6, respectively. Assay Procedures. The SPR gold disks and QCM disks were first cleaned with hot piranha solution (a 3:1 mixture of H2SO4 and H2O2). The freshly cleaned disks were then immersed in either the binary biotin-containing thiol mixture (10% biotinthiol and 90% ethylene glycol-thiol at a net concentration of 1 mM in ethanol) or a 1 mM MUA ethanol solution overnight. After the disks were rinsed with ethanol followed by being dried using nitrogen, they were ready to use for the measurements. For covalent streptavidin immobilization on an MUA treated surface, the surface was first activated by an exposure of 7 min to a 1:1 mixture of 0.4 M EDC and 0.1 M NHS aqueous solution. At the end of the activation, the surfaces were calibrated with sodium acetate buffer (pH 4.5). Streptavidin (0.05-0.2 mg/mL) in the same buffer solution was then applied for immobilization. At saturation, the surfaces were rinsed, and the remaining active esters were deactivated by ethanolamine hydrochloride at pH 8.5. For SA adsorption through biotin-streptavidin links, the biotin-thiol treated sensor surface was directly exposed to streptavidin (0.05-0.2 mg/mL) in PBS buffer for incubation. In both the MUA and biotin-thiol systems, biotin-DNA assembly (0.2-1 µM) and target DNA hybridization (0.01-1 µM) were conducted in PBS buffer.

Results and Discussion SPR and QCM-D Measurement of Streptavidin Immobilization. Figure 1 shows a schematic illustration of the surface binding procedures involved in this study, including streptavidin immobilization through either noncovalent adsorption on a biotin-thiol modified surface (named the “biotin-thiol method”, Figure 1A) or covalent attachment to an MUA treated surface (named the “MUA method” or “amine coupling method”, Figure 1B), biotinDNA assembly, and target DNA hybridization. As indicated in previous studies,8,9,12,16,17 streptavidin molecules adsorbed on biotin-containing surfaces are in an ordered arrangement with two of the biotin binding pockets occupied and the other two facing the solution. For the covalently attached streptavidin, however, the molecules are supposed to be randomly orientated, as illustrated in Figure 1B. According to the several NH2 binding anchors exhibited by surface exposed lysine residues, the protein molecules may be anchored through one or more of their Lys groups. Figures 2 and 3 show the SPR measurements of the sequential bindings of SA (0.2 mg/mL), biotin-DNA (1 (20) Rogahl, M.; Ho¨o¨k, F.; Krozer, A.; Brzezinski, P.; Kasemo, B. Rev. Sci. Instrum. 1995, 66, 3924-3930.

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Figure 3. SPR responses to the sequential reaction steps of streptavidin immobilization (0.2 mg/mL in pH 4.5 acetate acid buffer) on an MUA treated surface, biotin-DNA assembly (1 µM in PBS buffer), and target hybridization (1 µM in PBS buffer). The inset shows the normalized streptavidin adsorption curves in the biotin-thiol system (data from Figure 2) and MUA system.

Figure 1. Schematic illustration of the binding reactions involved in the study, including streptavidin immobilization, biotin-DNA assembly, and target DNA hybridization. The streptavidin is immobilized through either (A) biotin-streptavidin interaction on a biotin-containing thiol modified surface or (B) covalent attachment on a carboxyl-containing surface using amine coupling. In part B, the unoccupied residues are deactivated using ethanolamine-HCl buffer solution to form passive -CO-NH-R groups.

Figure 2. SPR responses to the sequential reaction steps of streptavidin immobilization (0.2 mg/mL) on a biotin-thiol treated surface, biotin-DNA assembly (1 µM), and target hybridization (1 µM). The V arrows indicate the time of rinsing the surface.

µM), and target DNA (1 µM) in the biotin-thiol and MUA systems, respectively. In the MUA system (Figure 3), the on-site EDC/NHS activation before streptavidin application and the ethanolamine hydrochloride deactivation after streptavidin immobilization are not presented, as they are detected as drastic SPR angle shifts (changes in refractive index of the liquid media) that affect the observation of the protein and DNA binding reactions. It can be seen from Figures 2 and 3 that the binding reactions are detected as stepwise SPR angle increases, with different kinetics and equilibrium amounts, depending on the streptavidin immobilization mechanisms. Using an Autolab SPR sensitivity of 120 mdeg ) 100 ng/cm2 for

protein and DNA and molecular weights of MWSA ) 60 kDa, MWbiotin-DNA ) 9.6 kDa, and MWtarget DNA ) 9.2 kDa, we convert the saturated SPR angle shift of each step (averaged over five to seven experiments) to the saturated molecular binding capacities in units of molecules per square centimeter (Table 1). These calculations assumed an equivalent SPR response per unit coverage for protein, single-stranded DNA, and double-stranded DNA. This assumption is feasible,21,22 as the incremental changes in refractive index with concentration (dn/dC), the determining factor of the SPR sensitivity, for protein and DNA are very similar (0.18 for protein and 0.19 for DNA).22 Using the saturated binding capacities, the biotin-DNA/ SA binding ratio and target hybridization efficiency (HE, %) are also estimated. In this section, we focus on the understanding of the streptavidin surface binding behaviors associated with the two immobilization mechanisms. The subsequent effects on probe assembly and target hybridization will be discussed in the following sections. First of all, from the repeated experiments, we found that streptavidin immobilization through physical adsorption on biotin-thiol treated surfaces is a robust procedure, leading to reproducible streptavidin binding (relative standard deviation (RSD) ≈4%) and subsequent biotin-DNA binding (RSD < 8%). However, in the MUA system, streptavidin immobilization is subjected to preactivation of the carboxyl groups with the EDC/NHS reagents, of which the role is to activate the carboxyl groups to form O-acylurea intermediates and NHS esters that promote the formation of amide bonds with the amines on the protein.14 This chemistry is, however, sensitive to the temperature and time of exposure and thus makes the quality of the activation a concern and bigger variations in streptavidin binding (RSD ≈ 8%) and successive DNA binding (RSD > 10%) possible. To compare the SA binding kinetics under the two schemes, the adsorption curves are normalized as compared in the inset of Figure 3. On the biotin-thiol treated surface, streptavidin adsorption shows a rapid initial rate, reaching saturation within a few minutes. We attribute this to the extremely strong affinity of the biotin/ streptavidin reaction (even though the biotin-DNA/SA binding can be a few magnitudes lower in affinity compared to the biotin/SA binding). However, in the (21) Larsson, C.; Rodahl, M.; Ho¨o¨k, F. Anal. Chem. 2003, 75, 50805087. (22) Peterson, A. W.; Wolf, L. K.; Georgiadis, R. M. J. Am. Chem. Soc. 2002, 124, 14601-14607.

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Table 1. Summary of the Molecular Binding Capacities Calculated from the Measured SPR Angle Shifts and the Biotin-DNA/SA Binding Ratio and Hybridization Efficiencya biotin-thiol method

MUA (amine coupling) method

step

angle shift (mDeg)

surface coverageb (×1012 molecules/cm2)

angle shift (mDeg)

surface coverageb (×1012 molecules/cm2)

streptavidin biotin-DNA target DNA biotin-DNA/SA ratio hybridization efficiency (%)c

457.5 ( 19.2 111.2 ( 10.2 62.0 ( 3.1 1.5 61

3.8 ( 0.1 5.8 ( 0.5 3.4 ( 0.2

406.1 ( 36.1 81.7 ( 14.3 45.2 ( 8.0 1.2 60

3.4 ( 0.3 4.3 ( 0.5 2.5 ( 0.4

a Average from five to seven experiments. b Surface coverage is calculated using an AutoLab SPR sensitivity of 120 mDeg )100 ng/cm2 and molecular weights of MWSA ) 60 kDa, MWbiotin-DNA ) 9.6 kDa, and MWtarget DNA ) 9.2 kDa. c Hybridization efficiencies are calculated as the fraction of hybridized target coverage divided by the immobilized probe coverage at saturation.

Figure 4. QCM-D measurement (∆f and ∆D) of streptavidin adsorption (0.2 mg/mL in PBS buffer) on a biotin-thiol treated surface.

Figure 5. QCM-D measurement (∆f and ∆D) of streptavidin immobilization (0.2 mg/mL in pH 4.5 sodium acetate buffer) on an MUA treated surface, after EDC/NHS activation of the surface.

covalent attachment, it takes a longer time for the protein to reach the equilibrium binding. The relatively slow molecular accumulation on the surface may be determined by the nucleophilic displacement of the NHS esters during the amide bond formation. From the SPR response speed alone, we may not be able to deduct the structural properties of the streptavidin films. However, the simultaneously recorded frequency (∆f) and dissipation (∆D) from the QCM-D measurements (Figures 4 and 5), together with the SPR signals, reveal the viscoelastic properties and the hydration rates of the streptavidin films formed, which are related to the protein conformation and film structure. As can be seen from Figures 4 and 5, similar kinetic trends for the streptavidin assembly are observed as compared to the SPR curves, that is, a rapid adsorption through biotin-streptavidin interaction and a relatively slow molecular accumulation through amine coupling. Using the Sauerbrey sensitivity of the 5 MHz QCM (17.7 ng/cm2), the measured ∆f value at saturation (25.2 ( 1.5 Hz in the biotin-thiol system and 32.5 ( 3.5 Hz in the amine coupling method) can be

converted to streptavidin mass uptakes (∆massQCM) of 446.0 ( 26.5 and 575.3 ( 62.0 ng/cm2, respectively, which are 1.2 and 1.7 times their corresponding SPR masses (∆massSPR), respectively. Considering the fact that SPR spectroscopy measures the “dry” mass (or molar mass) of the adsorbed protein film23 (this is because SPR spectroscopy senses the refractive index changes during the replacement of water by probed molecules), the QCM measures the protein mass (molar mass) together with water trapped between the macromolecules,23,24 and furthermore, the amount of trapped water varies significantly depending on the structure of the protein film.21 We conclude that the bound streptavidin molecules through amine coupling are oriented in such a way that more water entrapment occurs when compared to those immobilized on the biotin-thiol treated surface. At the same time, the induced ∆D values provide an assessment of the viscoelasticity of the protein films. For the streptavidin film assembled in the biotin-thiol system (∆f ) 25.2 Hz), only a slight dissipation increase of ∼0.3 × 10-6 is detectable, leading to a ∆D/∆f ratio (induced energy dissipation per coupled unit mass) of 12 × 10-9 Hz -1. However, using amine coupling, the frequency change, that is, ∆f ) 32.5 Hz, is accompanied with a significant ∆D increase of 4.0 × 10-6. The resulting ∆D/∆f value (123 × 10-9 Hz -1) is significantly larger than that in the biotinthiol system. These ∆D/∆f values suggest that the streptavidin film assembled on the biotin-thiol surface is highly rigid and compact, while the covalently bound streptavidin molecules form a water-rich film, being flexible and dissipative. The observed ∆massQCM/∆massSPR (1.2 for the SA in the biotin-thiol system and 1.7 for the SA in the MUA system) and ∆D/∆f values are in agreement with the emerging understanding of the QCM and SPR behaviors of films composed of solvated macromolecules, that is, the larger the ∆D/∆f ratio, the larger the difference in mass uptake measured by QCM analysis and SPR spectroscopy.24 From the subsequent biotin-DNA immobilization and target DNA hybridization measured by SPR spectroscopy, we found that the streptavidin film structure has distinct effects on the biotin-DNA binding amounts and strand orientation, which in turn determine the target hybridization efficiencies. We will be discussing these effects sequentially below. Effects of Streptavidin Packing Density/Film Structure on Biotin-DNA Binding Amounts. As given in Table 1, the saturated streptavidin binding amount in the biotin-thiol system is (3.8 ( 0.1) × 1012 molecules/cm2. The subsequent biotin-DNA coverage is (5.8 ( 0.5) × 1012 molecules/cm2, leading to a biotin(23) Fant, C.; Elwing, H.; Ho¨o¨k, F. Biomacromolecules 2002, 3, 732741. (24) Ho¨o¨k, F.; Voros, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B.; Ray, A.; Norden, B.; Kasemo, B. Colloids Surf., B 2002, 24, 155-170.

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DNA/SA molecular binding ratio of ∼1.5. This value, in agreement with the one we obtained using the surface plasma diffraction technique,25 suggests that on average one molecule of bound streptavidin binds 1.5 molecules of the biotin-DNA. It is not surprising that the two available biotin binding sites on the well-ordered streptavidin do not simply result in a biotin-DNA/SA ratio of 2. This is because the distance between the two biotin binding sites on the well-ordered streptavidin films is close to the hydrodynamic diameter of DNA. The combination of electrostatic repulsion and steric hindrance may prevent efficient coupling of two biotin-DNA molecules per streptavidin.21 On the MUA surface, a slightly lower surface coverage ((3.4 ( 0.3) × 1012 molecules/cm2) is obtained for the randomly oriented streptavidin (see Table 1). The successive biotin-DNA binding amount is (4.2 ( 0.5) × 1012 molecules/cm2, corresponding to a smaller biotin-DNA/ SA ratio of ∼1.2. In fact, the covalently coupled streptavidin molecules have most of the biotin binding residues unaffected, while the streptavidin molecules adsorbed on the biotin-thiol surfaces have only partially available biotin binding sites. The relatively smaller biotin-DNA/ SA ratio observed for the covalently bound streptavidin (which has more available biotin binding sites) may arise from the inaccessibility of the binding residues if the streptavidin molecules are randomly oriented. In other words, although the affinity of the biotin-streptavidin interaction is very strong and is considered irreversible, the success of biotin-DNA attachment is obviously subjected to the thermodynamic equilibrium conditions and the accessibility of the biotin binding pockets of the surface-bound streptavidin. Up to now, the discussions have been based on saturated streptavidin films achieved at a high streptavidin immobilization concentration (0.2 mg/mL). Figure 6 shows the streptavidin surface coverage effects on biotin-DNA binding amounts over a wide range of streptavidin coverage controlled by varying the concentrations (0.050.2 mg/mL) used for immobilization. The biotin-DNA/ SA ratios at different streptavidin coverages are calculated. It can be seen, in an overall trend, that the biotinDNA amounts decrease with a decrease of the streptavidin amounts in both the biotin-thiol (Figure 6A) and MUA (Figure 6B) systems. Over the tested streptavidin surface coverage in the biotin-thiol system ((4.0-1.7) × 1012 SA/ cm2), a consistent biotin-DNA/SA ratio of ∼1.5 is observed, while, for the covalently coupled SA, the reduction of the biotin-DNA amount is less sensitive to the decrease of the streptavidin surface coverage. At lower coverage, the biotin-DNA/SA ratio can reach up to 2 or even 2.5 in a few cases. We attribute this to the steric hindrance effects; that is, when the streptavidin surface coverage is low, the spatially scattered streptavidin molecules leave most of the available biotin binding sites accessible. On the other hand, the streptavidin density independent biotin-DNA/ SA ratio in the biotin-thiol system reflects that it is the biotin-streptavidin-biotin bridge chemistry that is responsible for the binding of the biotin-DNA. Effects of DNA Probe Density and Orientation on Target DNA Hybridization Efficiencies. For the selfassembled SH-DNA probe, previous studies have reported a strong dependence of hybridization efficiencies and kinetics on probe density.2-5 At varied SH-DNA probe densities of (2-12) × 1012 molecules/cm2, for a 25 bp probetarget DNA system, for example, Peterson et al.2 observed a decrease of the hybridization efficiencies (HEs) from 100 to 10%. At higher probe densities, the closely packaged (25) Yu, F.; Yao, D. F.; Knoll, W. Nucleic Acids Res. 2004, 32, e75.

Su et al.

Figure 6. Streptavidin surface coverage dependent biotinDNA binding amount in (A) the biotin-thiol system and (B) the MUA system. The biotin-DNA/SA ratios are calculated. Varied streptavidin surface coverage is achieved by varying the concentration (0.05-0.2 mg/mL) used for immobilization. The probe DNA concentration is fixed at 1 µM.

DNA probes are sterically less accessible. In this current study, we found that, for the biotin-DNA assembled on a streptavidin matrix, not only the probe density but also the probe orientation influenced by the streptavidin films affects the efficiency in target capture. As shown in Table 1, the hybridization efficiencies (HEs, %, calculated as the fraction of hybridized target coverage divided by the immobilized probe coverage at saturation) are 61 and 60% for the biotin-thiol system at a maximum probe density of (5.8 ( 0.5) × 1012 molecules/cm2 and the MUA system at a maximum probe density of (4.2 ( 0.5) × 1012 molecules/cm2, respectively. The lower probe density in the MUA system does not result in a higher hybridization efficiency, as expected. We attribute this to the orientation barrier of the randomly assembled DNA strands; that is, although the probe density is relatively low, the randomly oriented probe DNA strands mediated by the randomly oriented streptavidin molecules make the probe sequences less accessible. The molecularly ordered streptavidin film, however, leads to an ordered arrangement of the biotin-DNA strands, which facilitates the target capture. The above is a comparison of “between methods” and “between different probe densities”. In the following experiments, on saturated streptavidin surfaces, varied biotin-DNA densities are obtained by using varied biotinDNA concentrations (from 0.2 to 1 µM) for assembly. Target DNA (1 µM) is then applied for hybridization. Figure 7 gives a summary of the probe density dependent hybridization efficiencies measured for the two systems. The overall trend shows the following: (i) As expected, and in agreement with the SH-DNA system,2-4 the HE increases with an decrease of the probe density within

SA Film Structure Effects on Biotin-DNA Assembly

Figure 7. Biotin-DNA probe density dependent hybridization efficiencies in the biotin-thiol system (filled circles) and the MUA system (open circles).

Figure 8. Effects of biotin-DNA density (biotin-DNA per streptavidin molecule) on hybridization efficiency. The streptavidin film density (curve a, 345 mdeg; curve b, 270 mdeg; curve c, 154 mdeg) is controlled by varying the SA immobilization concentration. The applications of biotin-DNA (1 µM) on these streptavidin surfaces result in a similar DNA binding amount (∼70 mdeg). The subsequent target hybridization signals are different, resulting in hybridization efficiencies of 77, 60, and 31%, respectively.

each individual system. Specifically, in the tested biotinDNA density ranges ((2.5-6.2) × 1012 molecules/cm2 in the biotin-thiol system and (1.5-5.1) × 1012 molecules/ cm2 in the MUA system), the HE ranges from 96 to 61% and 97 to 55%, respectively. (ii) At a similar probe density, the well-ordered DNA strands on the orientation controlled streptavidin films in the biotin-thiol system always have higher efficiencies in target capture, compared to those in the MUA system. As discussed earlier (Figure 6B), the covalently bound streptavidin films allow different biotin-DNA binding density per bound streptavidin molecule (i.e., biotin-DNA/ SA ratio of 1.3-2.5), depending on the density of the streptavidin films. Through a careful analysis of the probe density dependent hybridization efficiency for this system, we found that not only the biotin-DNA density in terms of molecules per square centimeter of the substrate but also the biotin-DNA density in terms of molecules per streptavidin molecule has distinct effects on the hybridization efficiency. Figure 8 shows the examples where similar amounts of biotin-DNA (∼70 mdeg of SPR angle shift) are built up on streptavidin films of different surface coverages (345, 270, and 154 mdeg of SPR angle shifts). The resulting biotin-DNA/SA ratios are 1.2, 1.6, and 2.5, respectively. The hybridization of 1 µM target DNA gives rise to SPR angle shifts of 51, 40, and 21 mdeg, leading to HEs of 77, 60, and 31%, respectively. Again, we attribute the decrease of the HE with the increase of the biotinDNA amount per SA molecule to the steric hindrance

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Figure 9. SPR response to biotin-DNA immobilization on a biotin-thiol treated surface and to the sequential applications of target DNA at concentrations of 10, 50, 100, 200, 500, and 1000 nM.

effects; that is, the closely packed DNA probes on one streptavidin molecule prevent the sequences from being reached by the target DNA. Sensitivity of the DNA Hybridization Analysis. It can be seen from the above discussions that streptavidin films constructed on biotin-thiol treated gold surfaces are an ideal platform for a well-ordered biotin-DNA assembly that facilitates target capture. Using this platform, we tested the sensitivity of the DNA hybridization analysis. Figure 9 shows the SPR response to biotinDNA immobilization on the biotin-thiol surface and the sequential exposure of bound DNA probes to target DNA samples at increased concentrations (10, 50, 100, 200, 500, and 1000 nM). A plot of the SPR angel shift (∆θ) in millidegrees versus target concentrations shows a linear response of ∆θ ) 0.2293C + 0.2257, R2 ) 0.987, in the range 10-150 nM. Considering a noise of 2 mdeg of the AutoLab SPR instrument, the detection limit is 20 nM. This detection limit is comparable with most of the labelfree techniques, for example, quartz crystal microbalance measurements (unpublished data in our lab). Conclusion We have demonstrated that, for biotin-DNA assembly on streptavidin modified surfaces, not only the probe density but also the probe orientation influenced by the streptavidin films has distinct effects on the hybridization efficiencies, and in turn the sensitivity of the hybridization analysis. On a planar surface, an evenly distributed, wellordered streptavidin film is expected to provide a wellcontrolled biotin-DNA assembly. As an example, streptavidin film constructed on a biotin-containing surface is proven to be an ideal platform for biotin-DNA assembly and hybridization assays. In contrast, the randomly assembled streptavidin molecules through amine coupling do not favor the ordered DNA assembly for a higher efficiency in target capturing. It ought to be mentioned that in this study a comparison of the SA film structure is made between two immobilization strategies on a planner gold surface. The question as to how the planar biotin-thiol/SA platform is comparable to a 3-D streptavidin matrix, for example, that constructed on a brush structured dextran hydrogel of the BIAcore SPR sensor disks, is the subject of further investigations. In addition, the QCM-D analysis of the biotin-DNA structure mediated by different streptavidin films is also an ongoing study in our lab. LA047997U