Article pubs.acs.org/JPCB
Estimation of Molecular Interaction Force Using Atomic Force Microscopy for Bioapplication Hweiyan Tsai,†,§ Zihkai Chen,‡ Huiwen Deng,‡ Sinmei Tsai,‡ and C. Bor Fuh*,‡ †
School of Medical Applied Chemistry, Chung Shan Medical University, Taichung 402 Taiwan Department of Applied Chemistry, National Chi Nan University, Puli, Nantou 545 Taiwan § Department of Medical Education, Chung Shan Medical University, Taichung 402 Taiwan ‡
ABSTRACT: We report a method that involves using atomic force microscopy to estimate molecular interaction forces for bioapplications. Experimental parameters, comprising the labeling concentrations of tips and substrates and the loading rates of tips, were optimized for estimating molecular interaction forces for three pairs of model molecules (IgG/anti-IgG, BSA/anti-BSA, streptavidin/biotin). The estimated molecular interaction forces of IgG/anti-IgG, BSA/anti-BSA, and streptavidin/biotin were 121 ± 3, 185 ± 4, and 241 ± 4 pN, respectively. The measured values were consistent and within the range of values reported in the literature. Estimation of molecular interaction forces in force−distance curves for bioapplication is still challenging. There are many potential bioapplications with further investigations. Providing additional screening reference for microsensing applications is one example. This method demonstrates favorable potential for effectively estimating molecular interaction forces for various applications of protein−ligand, antibody−antigen, ligand−receptor complexes, and other bioreactions. This method is also useful for studies of the structures and properties of molecular, cellular, and bacterial surfaces.
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INTRODUCTION Interaction forces between protein−protein and protein−ligand play crucial roles in many biological applications such as protein function, protein folding, drug design, and separation technology.1−4 Therefore, evaluating molecular interaction forces is essential for these applications. Conventional methods entail measuring the properties of large ensembles of molecules. However, the ability to probe structure and functional heterogeneity of tested molecules is also essential. The methods commonly used for examining the structure and functional heterogeneity are laser tweezer, magnetic tweezer, and atomic force microscopy (AFM).5−8 Laser tweezers can cause photo damage to biological samples, and magnetic tweezers have the disadvantage of force hysteresis. AFM demonstrates favorable potential for this application and has the advantages of sensitivity of displacement, a small tip−sample contact area (∼10 nm2), and operation under physiological conditions. The intermolecular force between antibodies and antigens could be measured to identify bound molecules and provide their distributions on a sample when one of interacting molecules (antibody or antigen) is known and under control. Therefore, studying intermolecular interaction forces by AFM deserves further investigation. AFM measurements of intermolecular forces were reported in the literature, but the experimental parameters were scattered and not well-documented, especially for widely used immunocomplexes.9 Effective methods for modification and characterization of micro- and nanosurface are essential for successful measurement of intermolecular forces. We studied © 2016 American Chemical Society
intermolecular forces by using AFM with a simple approach on experimental setup, tip modification, and optimization involving two test molecules. The main steps in estimating intermolecular forces in this study can be summarized as follows. One molecule (probe) of the complex was fixed, modified, and attached to an AFM tip. The other molecule (test) in the complex was bound to the surface of a glass plate (substrate) by conjugation, as shown in Figure 1A. The AFM tip with the attached molecule approached the other molecule on the surface of the substrate to form a specific complex. The AFM tip retracted from the surface of the substrate after the tip made contact with the substrate to form a complex at a controlled loading rate. The force−distance curve measured from the approach−retract cycle of the tip showed sharp jumps of the cantilever that provided the intermolecular force, as shown in Figure 1B. The blue and red arrows show the moving effects of the tip approach and retraction on the intermolecular force, respectively. Three pairs of model complexes (IgG/antiIgG, BSA/anti-BSA, streptavidin/biotin) were used to test this approach.
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EXPERIMENTAL SECTION Chemicals and Materials. Phosphate buffered saline (PBS), 3-aminopropyl-triethoxysilane, 1-ethyl-(dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), Received: July 12, 2016 Revised: September 8, 2016 Published: October 18, 2016 10932
DOI: 10.1021/acs.jpcb.6b06985 J. Phys. Chem. B 2016, 120, 10932−10935
Article
The Journal of Physical Chemistry B
curve was measured in triplicate. The histograms of force distributions were fitted into two Gaussians with the first Gaussian being a nonspecific reaction and the second Gaussian being a specific reaction using a software Origin. The molecules on substrate surfaces were treated with an eight-fold concentration of their counter-paired molecules before force measurements in the blocking experiments.
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RESULTS AND DISCUSSION Optimization of Surface Modification on Tips and Substrates. Modifying the tips and substrates is essential to the success of this method for estimating interaction forces for bioapplication. Covalent bonding was used to immobilize reacting molecular pairs on the AFM tips and substrate surfaces to prevent the detachment of biomolecules during experiments. Biotin, anti-IgG, and anti-BSA molecules were used to modify the AFM tips. The molecular concentrations used to modify the tips were tested from 10−6 to 10−10 M. Fluorescein-conjugated BSA (0.5 mL of 10−6 M BSA-FITC) was used to monitor and minimize the immobilization of anti-BSA on the AFM tips under a fluorescent microscope. The labeled concentrations of anti-BSA were reduced to ∼10−8 M by minimizing the complex fluorescence on the AFM tips. Force curve measurement of specific reaction with ≥10% of total reactions was then used to ensure that there was still anti-BSA attached to the tip. The optimal concentration for immobilizing molecules on the tip was determined to be 1.0 × 10−9 M. Streptavidin, IgG, and BSA were used to modify the substrate surfaces. The molecular concentrations used to modify the substrates were tested from 10−5 to 10−7 M. Fluoresceinconjugated anti-BSA (1 mL of 10−6 M anti-BSA FITC) was used to monitor and adjust the number of BSA immobilized on the substrate surfaces with minimal clustering. The optimal concentration for immobilizing molecules on the substrate surfaces was determined to be 3.0 × 10−7 M. These optimal concentrations were used for all reactants that modified the tips and substrates for all the model molecules and were used in rest of experiments. Effect of Loading Rates of Tips on Force Measurement. The loading rate of a tip can affect its force measurement. The loading rate is equal to retracting velocity multiplied by force constant. Figure 2A shows the effects of the loading rate on force measurement with BSA/anti-BSA as an example. The force measurements increase linearly as the loading rate increases from 5.6 to 440 nN/s. However, the forces vary slightly with a mean of 200 ± 12 pN for loading rates from 5.6 to 11 nN/s. After these calibration trials, a loading rate of 11 nN/s was used for the experimental force measurements. Determination of Intermolecular Force. Figure 2B shows the force curve of the IgG/anti-IgG molecule pair. The force curve of a specific reaction is quite different from those of nonspecific and control reactions. The nonspecific reaction shows a small negative force, and the control reaction shows a considerably smaller negative force in immediate proximity to the 0 position of piezo movement. The control conditions were measured when either the tips or the substrates were not modified with reacting molecules. Figure 3 shows the histograms of force distributions for three pairs of model molecules. The most probable forces were determined by fitting Gaussians to the histograms of force distributions. Each force histograms can be fitted into two Gaussians with the first being nonspecific and the second
Figure 1. (A) Schematic view of interaction of AFM tip with substrate surface. (B) Schematic view of a specific force measurement curve.
bovine serum albumin (BSA), BSA-fluorescein isothiocyanate conjugate (FITC), anti-BSA, human IgG, antihuman IgG, biotin, and streptavidin were purchased from Sigma-Aldrich (St. Louis, MO). Preparation of Functional Tips and Sample Surfaces. Glass coverslips were cleaned in Piranha solution (H2O2/ H2SO4 3:7, Handle with Caution!) to remove contaminants, rinsed thoroughly with distilled water, and dried under N2 gas before use as substrate supports. AFM tips and glass slips were functionalized with amine groups by incubating them in a solution of 10% (v/v) 3-aminopropyl-triethosilan in methanol for 2 h. The immobilization of all reacting molecules on AFM tips or glass surfaces used the method of EDC and NHS coupling at room temperature for 4 h to form covalent bonds. In brief, 2 mg/mL of NHS and 15 mg/mL of EDC in PBS solution were reacted with functional molecules and then reacted with immobilized tips or substrates. Biotin, anti-IgG, and anti-BSA were linked to the AFM tips. Streptavidin, IgG, and BSA were linked to the glass surfaces. Instrumentation and Measurement of Intermolecular Force. Atomic force microscope (Innova, Bruker) equipped with a liquid cell was used for force measurement. Silicon nitride AFM probes from a Budget sensor were used in the experiments. Spring constants of cantilevers were measured using thermal fluctuation analysis from the AFM supplier. The spring constants of modified tips were determined to be 0.056 ± 0.004 N/m. All force measurements were performed in phosphate-buffered solution at room temperature. Each force 10933
DOI: 10.1021/acs.jpcb.6b06985 J. Phys. Chem. B 2016, 120, 10932−10935
Article
The Journal of Physical Chemistry B
Figure 2. (A) Effects of retracting rates on force measurement. (B) Typical force−distance curves of AFM from measurement of IgG/anti-IgG pair; nonspecific reaction, specific reaction, and control are shown.
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representing specific interactions. Figure 3A shows the force histogram of IgG/anti-IgG with a most probable force of 121 ± 3 pN. The reference forces from the literature are 65.8 ± 3.0, 108.1 ± 4.1, 131.1 ± 11.2, 149.5 ± 4.7, and 239.5 ± 3.1 pN.10 Figure 3A also shows the histogram from the blocking experiment of the IgG/anti-IgG pair as a controlled experiment. Most of the forces were from nonspecific interactions, and very little (∼6%) of the total force was from specific interactions of residuals. Figure 3B shows a force histogram of BSA/anti-BSA with a most probable forces of 185 ± 4 pN. The reference forces from the literature are 142 ± 8 and 250 ± 16 pN.11 Figure 3C shows a force histogram of streptavidin/biotin with a most probable force of 241 ± 4 pN. The reference forces from the literature are 257 ± 15 and 326 ± 19 pN.12,13 All measured forces of molecular pairs from this systematic approach were consistent and within the ranges reported in the literature. This method has the potential to provide additional screening reference for microsensing applications.
CONCLUSIONS
The method for modifying and optimizing tips and substrates in this approach is effective and useful. All measured forces of molecular pairs were consistent and within the ranges reported in the literature. Estimation of molecular interaction forces in force−distance curves for bioapplication is still challenging. There are many potential bioapplications with further investigations. Providing additional screening reference for microsensing applications is just one example. This method could provide references regarding intermolecular forces for various interactions of immunocomplexes and ligand−receptors in bioapplications. This method is also useful for studying the structures and properties of molecular, cellular, and bacterial surfaces. 10934
DOI: 10.1021/acs.jpcb.6b06985 J. Phys. Chem. B 2016, 120, 10932−10935
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The Journal of Physical Chemistry B
MY2). We thank Mr. S. S. Chuang, Ms. Y. S. Lu, and Ms. B. L. Kuo for technical assistance.
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Figure 3. Histograms of force measurements. (A) IgG/anti-IgG, (B) BSA/anti-BSA, and (C) streptavidin/biotin.
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AUTHOR INFORMATION
Corresponding Author
*Phone: 886-49-2919-779. Fax: 886-49-2917-956. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology of Taiwan (Grant MOST-102-2113-M-260-00510935
DOI: 10.1021/acs.jpcb.6b06985 J. Phys. Chem. B 2016, 120, 10932−10935
