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BIO-AFM Design and Development of Active Force Probes for Characterizing
Biomolecular Interactions |
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Of late, Atomic force microscopy (AFM) has become a powerful tool to typify biological interactions at the single molecule level. In a conventional AFM system, a passive microcantilever is used as the force-sensing element to measure the interaction forces between a microcantilever tip and an apposing surface, both decorated with biomolecules of interest. The microcantilever is brought into and out of contact with the apposing surface using a piezo actuator. By employing an optical lever detection mechanism to monitor cantilever deflection, various biophysical parameters like bond force, bond lifetimes and molecular elasticities can be extracted. Although microcantilever-based AFM has proven itself to be a powerful tool to study these interactions, extending this approach for multi-probe parallel measurements has its limitations. Moreover high-speed imaging in fluid is limited by the dynamics of both microcantilevers and piezo-actuators. To address these issues, a membrane-based active probe structure has recently been developed. The micromachined probe has self-sensing capability as it uses a dielectric membrane on a transparent substrate as the force sensing mechanical structure and has an integrated diffraction-based micro-interferometer for membrane displacement measurement. The probe membranes can also be electrostatically actuated, which removes the need for a piezo-actuator and giving self-actuation capability to the probe. Figure 1 schematically shows a membrane coupled with a microcantilever in a typical experiment.
Fig 1: Schematic of a
force spectroscopy probe membrane with self-sensing and self-actuation
capabilities coupled with an AFM cantilever in a typical force spectroscopy
experiment setup. To demonstrate the self-actuation capabilities of the membranes for single-molecular force spectroscopy measurements, relatively stiff silicon nitride/dioxide membranes were fabricated as actuators while softer AFM cantilevers were used as force sensors. We used them to measure unbinding forces between L-selectin reconstituted into membrane-supported lipid bilayers and an antibody. The stiff silicon nitride/dioxide probes are good for actuation as the characteristic of the actuator is limited by the membrane dynamic. However, if one were to use softer membrane probes that could be actuated and also act as force sensors, the need for a cantilever would be completely eliminated. We have recently fabricated soft polymer-based (parylene) membrane probes, with spring constants on the order of few tens of N/m for experimentation in liquid. These membranes are capable of detecting displacement changes with a resolution of <10 fm/√Hz for frequencies as low as 3 Hz using a differential readout scheme. This provides a force sensitivity of 0.3 – 3 pN with 1 kHz bandwidth using the membranes with spring constants of 1 – 10 N/m, which seems feasible to fabricate.
Fig 2: Displacement
noise spectrum of the 200 μm diameter membrane
probe. Due to its low elastic modulus parylene, a biocompatible polymer makes the fabrication of softer membranes possible. Moreover the hydrophilicity of parylene can be altered by oxygen plasma treatment, a key feature for their functionalization with biomolecules. These probes, functionalized with biotinylayted BSA and tested against streptavidin-coated cantilevers. While biotin could interact with streptavidin, BSA helped reduce nonspecific binding by blocking all exposed surfaces. Figure 3 shows the simultaneous data captured by both sensors.
Fig 3: Comparison of
force data simultaneously captured by a coupled AFM cantilever and a membrane
probe functionalized by biotin/streptavidin. The stiffness of the probes can be tuned using the parallel-plate type integrated electrostatic actuator which moves the membrane. An important implication of adjustable spring constant for force spectroscopy is that, a small, relatively stiff membrane with smaller coefficient of viscous damping can be used for low force noise experiments. The large spring constant of such a membrane can be reduced electrically to obtain a larger signal for unit force, i.e. higher detection sensitivity, while still being limited by thermal mechanical noise dominated by the interaction of the probe structure with the surrounding liquid. A proof of principle experiment showing sensitivity improvement was carried out by taking force curves with a single probe at two different bias points as shown in figure 4.
Fig 4 (a) Measured
and predicted variation of the spring constant of the membrane with
increasing DC bias voltage. The inset shows the measured and predicted
membrane displacement as a function of DC bias voltage. (b) Force curves
obtained by bringing the AFM tip in and out of contact with the probe
membrane biased at 35V. The top curve recorded from the AFM cantilever shows
a peak force of 400 nN. The bottom curve is recorded
from the membrane with a spring constant of 22 N/m. (c) The peak force
applied by the cantilever is kept constant at 400 nN
as shown in top trace. The bottom trace is from the same membrane with an
electrically reduced spring constant of 11 N/m. The membrane architecture allows array formation for parallel operation. The parallel AFM setup we have developed uses the array of membranes that can be coupled to an array of cantilevers to perform parallel experiments. In this setup, the membranes are used to actuate the cantilevers that serve as force sensors. It is possible to control the force on the individual cantilevers precisely since they are surface actuated in this scheme. Figure 5 shows simultaneous reading we obtained from two different cantilevers to demonstrate the feasibility of the method.
Fig 5. Cantilever 1
and 8 are engaged to the membrane probe and the substrate, respectively. The
drive signals applied to the membrane probe and the piezo actuator that
carries the microcantilevers are shown. The readout
signals obtained from cantilever-8 shows that this cantilever follows the
piezo movement whereas cantilever-1 movement is a combination of piezo and
membrane movements. Biological force studies using AFM typically involve coating microcantilevers and an apposing surface with different biomolecules of interest and retracting the cantilevers at set speeds that span a wide range. At very high speeds of pulling, the cantilevers are subjected to viscous drag forces, whose magnitudes often match or exceed the unbinding forces between the biomolecules. This introduces ambiguity in the forces being measured. We are currently modeling the hydrodynamic effects on cantilevers and membrane probes using FLUENT (CFD software) under conditions that simulate high speed pulling. Preliminary investigations have suggested that membrane-based probes are much less susceptible to drag forces than cantilevers for the same speed of pulling. A method for athermalization in atomic force microscope based force spectroscopy applications using microstructures that thermomechanically match the AFM probes has been introduced. The method uses a setup where the AFM probe is coupled with the matched structure and the displacements of both structures are read out simultaneously as shown in Fig.6. The matched structure displaces with the AFM probe as temperature changes, thus the force applied to the sample can be kept constant without the need for a separate feedback loop for thermal drift compensation, and the differential signal can be used to cancel the shift in zero-force level of the AFM.
Fig 6. Schematic of a micromachined membrane with integrated diffraction grating interferometer coupled with AFM cantilever for athermalization of the cantilever in a biomolecular experiment. Profiles of the structures before and after thermal deflection are schematically shown. Collaborators: Dr. Cheng Zhu (GT) Funding:
National Institute of Health Related
Publications: H Torun, O Finkler,
F L Degertekin, "Athermalization in atomic
force microscope based force spectroscopy using matched microstructure
coupling", Review of Scientific Instruments, 80, 076103, 2009 (pdf) H Torun, K K Sarangapani, F L Degertekin, "Spring constant tuning of active atomic force microscope probes using electrostatic spring softening effect", Appl. Phys. Lett., 91 (2007) 253113 (Also selected by the Virtual Journal of Nanoscale Science and Technology, http://www.vjnano.org/nano/ ) (pdf) H Torun, J Sutanto,
K K Sarangapani, P
Joseph, F L Degertekin, C Zhu, "Micromachined
membrane-based active probe for biomolecular mechanics measurement ", Nanotechnology,
18 (2007) 165303 ((The paper is awarded with second prize in the
Georgia Tech Student Paper Competition sponsored by SAIC.) (pdf) H Torun, K K Sarangapani, F L Degertekin, C Zhu, “Parallel active polymer probes with integrated interferometer for single molecule force spectroscopy”, International Meeting on AFM in Life Sciences and Medicine, Monterey, CA, Oct 15-18 2008 H Torun, K K Sarangapani, C Zhu, F L Degertekin, “Single molecule force spectroscopy using active polymer membrane probes with integrated interferometer”, International Congress of Nanotechnology (ICN+T 2008), Keystone, Co, July 20-25 2008 H F L Degertekin, G Onaran, M Balantekin, H Torun, "Novel AFM probes for fast imaging and quantitative material characterization", MRS Fall Meeting, Boston, MA, Nov 26-30, 2007 H Torun, K K
Sarangapani, C Zhu, F L Degertekin, "Micromachined membrane-based active probe for
biomolecular mechanics measurement", Seeing at Nanoscale
V, F L Degertekin, A G Onaran, H Torun, M Balantekin, K Sarangapani, C Zhu, "AFM probe structures with integrated interferometric sensing and electrostatic actuation", Kanazawa Workshop on Atomic Force Microscopy, Kanazawa, Japan, January 12-18th, 2007 H Torun, J B Sutanto, K K Sarangapani, C Zhu, F L Degertekin, “Membrane type displacement and force sensing transducer with sub-nano sensitivity for biological applications”, International Congress of Nanotechnology (ICNT 2006), Basel, 30 July - 4 August 2006 |
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