Final Report Summary - UTMOST (Ultra-Stable Molecular Force Spectroscopy with Micromachined Transducers)
Mechanical stimuli regulate many intra- and inter-molecular adhesion events such as immune cell tethering, antigen binding to antibody, and protein folding/unfolding through which weak biological bonds are formed. Single-molecule experiments employing an AFM can measure mechanical properties of bonds formed by biomolecules (e.g. integrins, selectins and immunoglobulins) directly.
A conventional AFM system uses a micro-scale cantilever as a force sensor. The cantilever, coated with biomolecules is actuated against a sample surface using a piezo actuator. The interaction force between the biomolecules on the cantilever and the sample surface are recorded by measuring the deflection of the cantilever. In this method, mechanical properties of the biomolecular bonds are measured directly.
Long-time scale biomechanical experiments require stability and control of drift to minimize the detrimental effects of changes in ambient conditions. Thermal drift of the cantilever due to ambient temperature fluctuation is a significant source of drift in AFM along with mechanical vibrations, material creep, and surface stress changes. This is a significant bottleneck in current AFM technology that restrains the measurement window from minute-long experiments. Yet, there is a strong need for novel instruments with low instrumental drift to expand the capabilities of AFM. Long-time scale measurements with ultra-high stability will allow conducting experiments at physiological rates in low-force regime.
The goal of the proposed research is to systematically analyze drift problem in AFM and to develop micromachined transducers that will cancel the drift when used in conjunction with microcantilevers. Thermal drift in cantilever is inevitable due to thermal bimorphic structure of the cantilever and the large mechanical loop in AFM. The proposed approach is to design the transducer that thermo-mechanically matches the cantilever. The transducer comprises a micro-stage anchored to its substrate using bimaterial and isolation legs. Bimaterial legs are made of two different materials with different values of coefficient of thermal expansion (CTE). Due to CTE mismatch, these legs deflect under thermal fluctuations. It is guaranteed by design that the micro-stage deflects identically with the AFM cantilever. This provides constant tip-to-transducer distance at all times so that the force applied on the biomolecules stays the same. The isolation legs optimizes for the heat transfer rate. They also determine the stiffness of the transducer to make sure the deflection of the stage is due to thermal fluctuations but not biomolecular interactions. In this method, the shift in zero-force level set for the cantilever can also be corrected by reading the displacement of the transducer. Displacement of the transducer is read out by optical means simultaneously with the AFM cantilever to measure and control the force on biomolecules accurately. Rapid compensation for ambient changes will allow long-time scale measurements at near physiological rates.
The objective of the proposed research is two-folds: (1) to develop micromachined transducers as ultra-stable AFM-based biomolecular assays and (2) to utilize these assays in long-time scale experiments to analyze the kinetics and mechanics of single pairs of interacting biomolecules. The specific aims are:
1. Develop micromachined transducers as sample holders in conjunction with AFM cantilevers:
a. Systematically analyze the drift problem in AFM
b. Design, fabricate and test micromachined transducers to eliminate drift in AFM
2. Run biological experiments using the new system:
a. Determine the force history dependence of bond off-rates using lifetime and unbinding force
b. Test the feasibility of the new system for protein folding/unfolding experiments
The research conducted in the first period of the project addressed important aims for the success of this project. Analytical and finite-element method based models were developed to predict thermal drift in AFM cantilevers. Special consideration was given for the case of liquid immersion, which is essential for biological experiments. The models were tested against experimental data obtained using commercially available microcantilevers commonly used for biomolecular research. These models served as design tools for the conforming micro-scale transducers. Thermo-mechanical response of the transducers in conjunction with AFM cantilevers was optimized for the cancellation of the effects of thermal drift at steady state and during transient period. First generation devices were fabricated using the cleanroom facility at Boğaziçi University.
In the second period, the specific aims of the project are:
1. to characterize the fabricated devices
2. to develop optimized second generation transducers
3. to perform single-molecule experiments using the transducers
First, a dedicated experimental setup has been constructed to characterize the devices. Thermal, mechanical and electrical characteristics of the devices have been measured. The thermally induced deflection values of the devices were measured as a few hundreds of nanometers per Kelvin, that are comparable with commonly used cantilevers. It is feasible to match the thermal displacement of a specific cantilever by using the transducers designed for the cantilever. Rise time values of the transducers were measured as 0.2-1 ms. The match in rise time values of commonly used cantilevers and the microstages is sufficient considering 1 kHz bandwidth for a typical force spectroscopy experiment. In a specific experiment, a reduction in the value of thermally induced deflection for a cantilever was demonstrated as 54% using the transducers.
Then, the fabrication process of the transducers was optimized and second generation devices were fabricated. Now, the process flow allow for the fabrication of new devices within a week time frame with high yield.
Finally, single-molecule biomolecular experiments were performed in the second period. Interactions of biotin/streptavidin pairs were probed using the introduced method. Functionalized cantilevers with biotin were used together with transducers functionalized with streptavidin during the experiments. The results of biomolecular force spectroscopy experiments are in agreement with the results published in the literature, demonstrated that the surface of microstages can be functionalized successfully for biomolecular experiments.
The outcomes of the research project have strong potential for commercialization. The researcher was awarded an US patent (Patent no: 8,239,968) in August 2012 regarding the novel method described here. In addition, the research on development of AFM methods resulted in another patent application. A Turkish patent was filed to protect the intellectual property developed at the host institute and this will be followed by a PCT application. Finally, applications to USPTO and EPO will be completed in the following periods. In addition to these activities, a commercialization plan has been developed. Bogazici University Technology Transfer Office supports the commercialization plan that will be implemented in in the following periods.
A conventional AFM system uses a micro-scale cantilever as a force sensor. The cantilever, coated with biomolecules is actuated against a sample surface using a piezo actuator. The interaction force between the biomolecules on the cantilever and the sample surface are recorded by measuring the deflection of the cantilever. In this method, mechanical properties of the biomolecular bonds are measured directly.
Long-time scale biomechanical experiments require stability and control of drift to minimize the detrimental effects of changes in ambient conditions. Thermal drift of the cantilever due to ambient temperature fluctuation is a significant source of drift in AFM along with mechanical vibrations, material creep, and surface stress changes. This is a significant bottleneck in current AFM technology that restrains the measurement window from minute-long experiments. Yet, there is a strong need for novel instruments with low instrumental drift to expand the capabilities of AFM. Long-time scale measurements with ultra-high stability will allow conducting experiments at physiological rates in low-force regime.
The goal of the proposed research is to systematically analyze drift problem in AFM and to develop micromachined transducers that will cancel the drift when used in conjunction with microcantilevers. Thermal drift in cantilever is inevitable due to thermal bimorphic structure of the cantilever and the large mechanical loop in AFM. The proposed approach is to design the transducer that thermo-mechanically matches the cantilever. The transducer comprises a micro-stage anchored to its substrate using bimaterial and isolation legs. Bimaterial legs are made of two different materials with different values of coefficient of thermal expansion (CTE). Due to CTE mismatch, these legs deflect under thermal fluctuations. It is guaranteed by design that the micro-stage deflects identically with the AFM cantilever. This provides constant tip-to-transducer distance at all times so that the force applied on the biomolecules stays the same. The isolation legs optimizes for the heat transfer rate. They also determine the stiffness of the transducer to make sure the deflection of the stage is due to thermal fluctuations but not biomolecular interactions. In this method, the shift in zero-force level set for the cantilever can also be corrected by reading the displacement of the transducer. Displacement of the transducer is read out by optical means simultaneously with the AFM cantilever to measure and control the force on biomolecules accurately. Rapid compensation for ambient changes will allow long-time scale measurements at near physiological rates.
The objective of the proposed research is two-folds: (1) to develop micromachined transducers as ultra-stable AFM-based biomolecular assays and (2) to utilize these assays in long-time scale experiments to analyze the kinetics and mechanics of single pairs of interacting biomolecules. The specific aims are:
1. Develop micromachined transducers as sample holders in conjunction with AFM cantilevers:
a. Systematically analyze the drift problem in AFM
b. Design, fabricate and test micromachined transducers to eliminate drift in AFM
2. Run biological experiments using the new system:
a. Determine the force history dependence of bond off-rates using lifetime and unbinding force
b. Test the feasibility of the new system for protein folding/unfolding experiments
The research conducted in the first period of the project addressed important aims for the success of this project. Analytical and finite-element method based models were developed to predict thermal drift in AFM cantilevers. Special consideration was given for the case of liquid immersion, which is essential for biological experiments. The models were tested against experimental data obtained using commercially available microcantilevers commonly used for biomolecular research. These models served as design tools for the conforming micro-scale transducers. Thermo-mechanical response of the transducers in conjunction with AFM cantilevers was optimized for the cancellation of the effects of thermal drift at steady state and during transient period. First generation devices were fabricated using the cleanroom facility at Boğaziçi University.
In the second period, the specific aims of the project are:
1. to characterize the fabricated devices
2. to develop optimized second generation transducers
3. to perform single-molecule experiments using the transducers
First, a dedicated experimental setup has been constructed to characterize the devices. Thermal, mechanical and electrical characteristics of the devices have been measured. The thermally induced deflection values of the devices were measured as a few hundreds of nanometers per Kelvin, that are comparable with commonly used cantilevers. It is feasible to match the thermal displacement of a specific cantilever by using the transducers designed for the cantilever. Rise time values of the transducers were measured as 0.2-1 ms. The match in rise time values of commonly used cantilevers and the microstages is sufficient considering 1 kHz bandwidth for a typical force spectroscopy experiment. In a specific experiment, a reduction in the value of thermally induced deflection for a cantilever was demonstrated as 54% using the transducers.
Then, the fabrication process of the transducers was optimized and second generation devices were fabricated. Now, the process flow allow for the fabrication of new devices within a week time frame with high yield.
Finally, single-molecule biomolecular experiments were performed in the second period. Interactions of biotin/streptavidin pairs were probed using the introduced method. Functionalized cantilevers with biotin were used together with transducers functionalized with streptavidin during the experiments. The results of biomolecular force spectroscopy experiments are in agreement with the results published in the literature, demonstrated that the surface of microstages can be functionalized successfully for biomolecular experiments.
The outcomes of the research project have strong potential for commercialization. The researcher was awarded an US patent (Patent no: 8,239,968) in August 2012 regarding the novel method described here. In addition, the research on development of AFM methods resulted in another patent application. A Turkish patent was filed to protect the intellectual property developed at the host institute and this will be followed by a PCT application. Finally, applications to USPTO and EPO will be completed in the following periods. In addition to these activities, a commercialization plan has been developed. Bogazici University Technology Transfer Office supports the commercialization plan that will be implemented in in the following periods.
