Direct Measurement of the Thermal Hysteresis of Antifreeze Proteins

Article pubs.acs.org/ac

Direct Measurement of the Thermal Hysteresis of Antifreeze Proteins (AFPs) Using Sonocrystallization Andrea Gaede-Koehler,† Alexej Kreider,† Peter Canfield,‡ Malte Kleemeier, and Ingo Grunwald* Fraunhofer Institute for Manufacturing Technology and Advanced Materials (FhG IFAM), Wiener Strasse 12, 28359 Bremen, Germany ABSTRACT: Antifreeze proteins (AFPs) are of great importance for applications in cryomedicine or the food industry. They are frequently used to lower the freezing point by preventing the growth of larger ice crystals; thus, it is paramount to determine their thermal hysteretic characteristics. However, the experimental analysis of the thermal hysteresisan effect that is characteristic for AFPsremains a challenging process. An easyto-use test method for measuring the thermal hysteresis of AFPs was developed and tested with the type III AFPs. Traditional methods that have been used until now have their disadvantages and limitations. The new measurement method described in this paper allows detection of the complete cooling, freezing, heating, and melting process in a single measurement. This makes it possible to directly determine the thermal hysteresis as a functional effect of the antifreeze proteins. Measurements of the thermal hysteresis were performed by applying ultrasound to initiate the crystallization process of the antifreeze protein solution. This ultrasound technique also allows a crystallization process to be performed at defined temperature. The demonstrated results were highly reproducible and could be clearly read off the measurement curves. As a future perspective, this enables the design of automatic test devices that can be also miniaturized.

A

area shows an atomic order with approximately hexagonal structure.34 Common to all AFPs is that they cause noncolligative freezing point depression1 by bonding to the ice crystal nuclei, thus hindering the growth of the ice crystals,3 while the melting point remains unchanged. A colligative effect in a solution depends solely on the number of molecules and not on their physical and chemical properties, whereas a noncolligative effect depends on both the concentration of the solute and its chemical properties. The extent of the thermal hysteresis2,3more specifically, the temperature difference between the freezing point and melting pointnot only depends on the AFP type34 concentration, but also on the size of the initial ice crystals. Thermal hysteresis can even differ within the single species, depending on the isoform.34 One model for thermal hysteresis has been described in more detail by Kristiansen and Zachariassen.3 In view of the huge interest in AFPs, it is necessary to have a simple and easy-to-use experimental method that allows determination of the freezing point depression and thermal hysteresis in a single measurement procedure. Previous measurement methods that have been used until now have all had their limitations. The method used by Scholander et al.35 and Fenney et al.36 was based on observing ice crystal formation and the melting process by microscope. This was a

ntifreeze proteins are of huge commercial interest, because of their ability to cause noncolligative freezing point depression,1 which is usually called thermal hysteresis.2,3 Studies have been undertaken on their use in the food industry,4 and patents have been granted in this area.5,6 Their potential applications for several other areas have also been investigated, including use in cryosurgery,7 cryopreservation for agriculture,8 aquaculture,9−11 the gas industry as kinetic gas hydrate inhibitors, and surface coatings (for example, paints or lacquers) for antifreeze protection.12,13 A variety of lifeforms have been shown to utilize antifreeze proteins (AFPs) as a survival strategy in environments that are permanently or periodically at temperatures below freezing. For example, AFPs14,15 have been found in numerous types of fish,16,17, arthropods,18−22 plants,23,24 and bacteria.25,26 The AFPs found in fish have differing primary and secondary structures and are subdivided into glycoproteins (AFPG) and polypeptides (AFP types I−IV).2,27,28 The structure of type III AFP from the ocean pout (Macrozoarces americanus), which was used for the present work, has previously been studied and reported in various publications.29−33 Similar to other AFPs, this protein type III functions at the interface between water and ice crystals, which forces a difference between the melting point and the freezing point (the so-called “thermal hysteresis”). For the considered AFP, the hydrophobic and hydrophilic side groups on the α-peptide residues develop intermolecular interactions (van der Waals forces or hydrogen bonds) between the water molecules and AFPs ice binding area. This functional © 2012 American Chemical Society

Received: July 11, 2012 Accepted: November 5, 2012 Published: November 5, 2012 10229

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bubble after the implosion acts as a crystallization nucleus and hence serves as a support for the subsequent ice crystals.46 The measurement method presented in this paper allows the complete cooling, freezing, heating, and melting process to be recorded, plotted in a single measurement curve, hence directly indicating the thermal hysteresis. In order to prevent the described disadvantages of mechanical excitation of the test solution or seeding with crystallization nuclei, the crystallization is initiated by an impulse from an ultrasonic generator.

very direct method, but it was also time-consuming and required great care. The work of Chakrabartty and Hew37 involved observing a single ice crystal under a microscope. The freezing point and melting point were defined as the temperature at which the crystal starts growing or starts melting, respectively, as determined by the operator of the apparatus. Most frequently, the antifreeze activity or thermal hysteresis is measured by using a Clifton nanoliter osmometer. In the Clifton nanoliter osmometer, a small drop (∼1 nL) of sample is frozen rapidly and melted again until a single ice crystal is obtained. The Clifton nanoliter osmometer is equipped with a light microscope, which allows direct observation of ice crystal morphology.38 Furthermore, a thermal analysis method, the socalled “differential scanning calorimetry (DSC)”, was also used to determine the thermal hysteresis of various AFPs.39−41 Another device for the AFP characterization was the freezing point osmometer used by Feeney et al.42 Here, the sample was supercooled and crystallization was initiated by mechanical excitation. The temperature increase due to the heat of crystallization was measured during the ice formation by a temperature sensor. It was not possible at that time to determine the melting temperature in the same experiment. Mulvihill et al.43 extended the method by, among other things, using a recorder, which allowed continuous recording of the freezing curves of samples. A further modification of the measurement method with a freezing point osmometer was carried out by Slaughter and Hew.44 As in the work of Feeney and Yeh,42 the crystallization was initiated by mechanical excitation. Slaughter and Hew44 used silver iodide (AgI) as crystallization nuclei. The test solution was placed in a test tube, which was inserted into a larger osmometer tube filled with air. A temperature sensor in the test solution was attached to a recorder unit. This modified test method allowed determination of the freezing and melting points and, hence, measurement of the thermal hysteresis. However, two separate measurements were necessary to determine the freezing and melting points.44 Slaughter and Hew44 described a technique to initiate crystallization at low supersaturation using externally added seed crystals. This method leads to an additional contamination of the solution, which can adversely affect the reproducibility of the crystallization process. This problem can be obviated using sonocrystallization.45 Parameters affecting sonocrystallization include not only the degree of supersaturation and supercooling of the solution, contaminants, interfaces, and mechanical actions (shaking the vessel),46 but also the ultrasound frequency, power, and duration.45 Hence, customization of the sonocrystallization parameters allows customized initiation of the crystallization.43 However, the precise physical processes that trigger the formation of crystallization nuclei are still not clearly understood.45,46 A direct relationship between acoustic cavitation and the formation of crystallization nuclei has been shown. In sonocrystallization, the sound waves pass directly into the supercooled liquid.46 If the sound wave of the ultrasonic impulse has sufficient amplitude, then an underpressure develops whose strength is sufficient to initiate the expansion of microscopically small gas bubbles, which are assumed to be present in the solution. These cavitation bubbles grow to macroscopic size before imploding because of the surrounding external pressure.47 It appears that the residual cavitation

2. MATERIALS AND METHODS 2.1. Chemicals. The antifreeze protein (AFP) type used in the tests was AFP type III from the ocean pout, Macrozoarces americanus. The source of this AFP type III was A/F Protein, Inc. (Waltham, MA, USA). The unit calibration was undertaken using the Osmolality Linearity Set supplied by ADVANCED Instruments, Inc. (Norwood, MA, USA). The potassium dihydrogen phosphate (KH2PO4) and sodium hydrogen phosphate (Na2HPO4) used to make the phosphate buffer were supplied by AppliChem (Germany). The salts were in a water-free form and had a purity of >98%. 2.2. Measurement Method. The HAAKE K15 external cooling bath (Haake, Germany) provides the coolant (ethanol/water mixture) that is cooled by the HAAKE DC 50 cooling thermostat (Haake, Germany) to −16 °C and circulated in the cooling block CB (see Figure 1). The heating unit HU (Figure 1, copper coil) sits in a plastic tube that is sunk into the cooling block. A heated wire with an electrical resistance of 9 Ω is wrapped around the copper coil. Energy is supplied to the heating unit via an adjustable power supply unit (Delta Elektronika, Germany). In addition, the copper coil contains an insert for a 2°mL polyethylene reaction vessel (Eppendorf Protein

Figure 1. Experimental setup within the cooling chamber. The cooling chamber itself consists of an aluminum cooling block (CB) surrounded by a layer of insulating wool, and then Styrofoam insulation (SI). The ultrasonic sonotrode (US) was used to initialize the ice crystallization, heating unit (HU), and thermometers (T1 and T2) are parts of the control unit (see the detailed explanation in the Materials and Methods section). 10230

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Table 1. Results of the Calibration Using the Osmolality Linearity Set for Verification of the Measured Freezing Point, in Comparison with the Target Values Freezing Point [°C] concentration of the calibration solution (mOsm/kg)

measurement 1

measurement 2

measurement 3

average value

target value

0 (water) 100 500 900 1500 2000

−0.03 −0.20 −0.98 −1.75 −2.86 −3.74

0.01 −0.20 −0.99 −1.76 −2.88 −3.74

0 −0.20 −1.00 −1.75 −2.85 −3.76

−0.01 −0.20 −0.99 −1.75 −2.86 −3.75

0.00 −0.19 −0.93 −1.67 −2.79 −3.72

LoBind Tube, Germany) and a hole at the edge for insertion of the Pt100 resistance thermometer T2 (JUMO, Germany) (see Figure 1). In order to start the crystallization using ultrasound technique in a small vessel (diameter of 1 cm and length of 4 cm) and detect the temperature of the protein solution during the measurement, the ultrasonic sonotrode (tip diameter = 3 mm) is inserted 3 cm deep and the thermometer Pt-100 (diameter = 1 mm) is inserted 3.5 cm deep into the protein solution without touching each other. In this process, a lateral area of 3.14 cm2 is required to fix the thermometer in the upper region of the vessel. Furthermore, in a larger vessel, a good distribution of the proteins in the solution during measuring process is ensured, avoiding the possibility that the magnetic stir bar comes into contact with the thermometer (T1) or with the ultrasonic sonotrode (US). Thermometer T2 serves to monitor the heating profile. The heating profile is shown in light gray in the graph shown later in Figure 3). The starting temperature for the measurements was set at +15 °C (see Figure 3) and the minimum temperature was set at −6 °C. After reaching the minimum temperature of −6 °C, this was held for 200 s. Heating to +0.4 °C then occurred. This temperature was held for 600 s, before heating once again to the starting temperature of +15 °C. The cooling and heating rates were 1.5 °C/min. The thermometer T1 (Figure 1) measured the temperature of the test solution. The temperatures of T1 and T2 were measured using an Almemo 2390-3 multimeter (Ahlborn, Germany) having a resolution of 0.01 °C and were transferred to a data recording program that we developed ourselves. The crystallization of the test solution (volume = 1 mL) was triggered with an ultrasonic sonotrode (US, Model CV 188) (Figure 1) at −5 °C using an ultrasound amplitude of 30%, as prescribed by the Sonics Vibracell control unit and using impulse duration of 0.1 s. In order to guarantee a uniform temperature distribution in the test solution, the solution was stirred with a polytetrafluoroethylene (PTFE)-coated magnet stirrer throughout the measurements. 2.3. Instrument Validation. Thermometer T1 (Figure 1) was calibrated to the freezing point of pure water. The calibration of the test unit was undertaken prior to determination of the thermal hysteresis of the type III AFP. Further test unit calibration using the Osmolality Linearity Set from Advanced Instruments subsequently allowed comparison of the expected and actually measured freezing points (see Figure 2) at solution concentrations of 100, 500, 900, 1500, and 2000 mOsm/kg. Several measurements (N ≥ 3) were carried out, with pure water and with each solution concentration (see Table 1). In Table 1, three measurements of the calibration solution are shown. This reveals that the deviation from the target value is minimal. 2.4. Sample Preparation and Test Procedure. The type III AFP was soluble in a phosphate buffer without other additives. The phosphate buffer was produced from 1.48 mmol KH2PO4 and 8.52 mmol Na2HPO4, which were dissolved in deionized water to give 1 L solution. The phosphate buffer solution had a pH of 7.4. Multiple measurements (N ≥ 3) on the pure phosphate buffer using this test unit should indicate that there is no thermal hysteresis for the pure phosphate buffer. For these measurements, 1 mL of phosphate buffer solution was pipetted into a 2-mL Eppendorf tube.

Figure 2. Comparison of the actual and theoretical calibration values. The large graph shows the measurements with freezing points as results (gray triangles) for validation carried out with pure water (first measurement point) and the Osmolality Linearity Set. The black circles indicate the target values. The small graph in the inset shows the difference between the calibration measurements and the actual values, along with the standard deviations of the measurement values. In order to demonstrate that thermal hysteresis can be detected using this test unit, different type III AFP concentrations were tested (3, 5, and 7 mg/mL). In addition, a type III AFP sample that had a concentration of 3 mg/mL and was denatured as a negative protein control at 60 °C for 30 min was tested.

3. RESULTS 3.1. Instrument Validation. The instrument calibration results (Figure 2) showed that, with increasing concentration of the calibration solution up to 900 mOsm/kg, there is an increasing difference between the measured and desired calibration values. A maximum temperature difference of, on average, −0.081 °C was observed at a concentration of 900 mOsm/kg. This temperature difference decreases again at high concentrations of the calibration solution, and, at a concentration of 2000 mOsm/kg, was −0.03 °C (on average). 3.2. Measurement Curves. Figure 3 shows the results for a complete type III AFP test run involving cooling, freezing, heating, and melting processes. The temperature of the test solution follows the prescribed heating and cooling program of the test unit. After the cooling process (a) ice crystal growth was initiated at −5 °C by the ultrasonic pulse (b) and the temperature of the sample was elevated by the release of latent heat (c). In the presence of AFP, the nonequilibrium freezing point temperature was reached at which the ice crystals did not grow further (d). When the temperature was elevated externally, these crystals started to melt at the equilibrium melting point temperature (f). The difference between melting 10231

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Figure 3. Direct measurement of the thermal hysteresis of AFP III using the sonocrystallization device. Measurement results for type III AFP with magnified section (light gray dotted line): (a) cooling process, (b) initiation of sonocrystallization, (c) liberated heat of crystallization, (d) freezing point plateau, (e) thermal hysteresis, (f) melting point plateau, and (g) heating process.

concentration. Each type III AFP concentration was tested at least three times and the thermal hysteresis was measured. The reference measurement (a type III AFP concentration of 0 mg/ mL) was carried out with pure water and gave, as expected, a thermal hysteresis of 0 °C. The type III AFP concentration of 3 mg/mL gave an average thermal hysteresis of 0.240 ± 0.021 °C, that of 5 mg/mL gave an average thermal hysteresis of 0.370 ± 0.016 °C, and that of 7 mg/mL gave an average thermal hysteresis of 0.473 ± 0.005 °C. As expected for the pure phosphate buffer and denatured type III AFP sample, no thermal hysteresis was observed.

point temperature and freezing point temperature is the thermal hysteresis (e). If the thermal equilibrium can no longer be maintained, the temperature (g) of the type III AFP solution once again follows the temperature of the heating unit. To reassess the reproducibility of the measurements, three measurements with the same protein solution were carried out. In Figure 4, the result of this measurements are shown. The temperature profiles of the protein solution after the initiation of the crystallization are identical. The thermal hysteresis at measurement 1 amounts to 0.36 °C, 0.37 °C at measurement 2, and 0.36 °C at measurement 3. The standard deviation of measurements 1, 2, and 3 accounts for 0.01 °C. Figure 5 shows the curve for pure water or pure phosphate buffer solution. As expected, there was no thermal hysteresis for pure water and the phosphate buffer. The freezing point and melting point have the same temperature; therefore, there is a continuous temperature plateau. In all other respects, the profiles for pure water and the pure phosphate buffer graphs have the same features as described for the type III AFP measurements. 3.3. Thermal Hysteresis. Figure 6 clearly shows the increase in thermal hysteresis with increases in the type III AFP

4. DISCUSSION The direct measurement of the thermal hysteresis of AFP using sonocrystallization is a new technique that will broaden the already existing methods for the analysis of antifreeze substances. This method is a fast and reliable technique that can be automated but is currently limited to sample volumes of at least 750−1000 μL. If recombinant or synthetic-produced AFP are used, a few milligrams are not the limiting factor; however, for fluids extracted from smaller species, such as insects, this is limiting. In those cases, and if the crystal growth 10232

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Figure 6. Correlation between thermal hysteresis and type III AFP concentration. Three different type III AFP concentrations have been measured using the sonocrystallization device. The average measurement values for the different type III AFP concentrations (3.0, 5.0, and 7.0 mg/mL) were plotted on the graph, and the relevant standard deviations were calculated. The standard deviation for the type III AFP concentration of 7 mg/mL was so small that it disappears behind the data points.

Figure 4. Reproducibility of the measurements for type III AFP. The experiments have been repeated three times, and the curves have been put on top of each other. Type III AFP with 5 mg/mL dissolved in phosphate buffer solution was used. The curve profile is the same for all type III AFP measurements. Solely, the freezing point temperature varies depending on the type III AFP concentration. Each measurement takes ∼45 min and is fully automated.

prolongs the test procedure and increases the risk of premature crystallization. A considerably higher heating rate makes it difficult to achieve a uniform temperature in the solution. The ultrasound crystallization is initiated at −5 °C to be able to record differing freezing points for different solution concentrations without having to change the test parameters. After crystallization there is a temperature plateau (Figure 3) at the freezing and melting points of the solution, which represents the thermal equilibrium. In order to obtain a clearly visible plateau, a minimum temperature of the heating unit of −6 °C should be selected. This prescribed minimum temperature of −6 °C was held for 200 s. During this 200 s, the plateau (Figure 3) for the freezing point develops. If the temperature difference between the minimum temperature of the heating unit and the crystallization temperature is too large, then a shortening of the length of the plateau results, because the thermal equilibrium would develop for a greater temperature difference. If the time for plateau development is chosen to be too long, the solution can no longer maintain the thermal equilibrium (and, hence, the plateau). The solution cools again and approaches the minimum temperature of the heating unit on the graph. In this case, it is not possible to easily measure the thermal hysteresis of the solution, because there is no longer a distinct transition between the plateaus of the freezing point and melting point. In order to measure the melting point, the heating unit is heated to +0.4 °C after holding for the prescribed period at the minimum temperature. This temperature was chosen in order to achieve a relatively quick transition between the thermal equilibrium at the freezing point and the thermal equilibrium at the melting point and thus for an accurate observation of thermal hysteresis. 4.2. Measurement Curves. As already mentioned, the measurement method presented here records the complete cooling, freezing, heating, and melting process in a single measurement curve (Figure 3) and directly indicates the thermal hysteresis. The time required for a test run depends on the settings and, in this work, ∼45 min were neededshorter measurements are possible. This result means that it is no longer necessary to undertake time-consuming observation of the test solutions under a microscope, as is the case with other methods.35,37,42

Figure 5. Control experiment with pure water. The same parameters, like those for the type III AFP measurements, have been used for the control experiment. Instead of a type III AFP solution, pure water was analyzed with the measuring device. The magnified section clearly shows that the plateaus for the freezing point and melting point join together with no thermal hysteresis.

is of interest, devices such as the Clifton nanoliter osmometer should be used. 4.1. Instrument Settings. The method described herein is quite fast and can be accelerated within a distinct range. In order to keep the time needed to reach the crystallization temperature as short as possible, yet guarantee uniform temperature change in the solution, the starting and end temperatures were set at ∼15 °C, with cooling and heating rates of 1.5 °C/min. A smaller cooling rate unnecessarily 10233

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After being started, the test runs automatically, meaning that during this time, other tasks can be undertaken. One disadvantage of the measurement technique proposed in this work is that it requires a relatively large amount of sample solution, whereas a small amount of protein solution (∼nL) is sufficient for microscopic observation of ice crystal morphology. A volume of 1 mL is required in this work in order to guarantee uniform mixing of the test solution and uniform quality of the measurements. This is mainly because of the selfbuild prototype devices used for the described experiments. A sample reduction to a few hundred microliters is possible with smaller device parts, e.g., stirrer, sonotrode, and thermometer. The use of the ultrasonic generator removes several disadvantages of freezing point osmometers.36,37 For instance, our device does not require mechanical excitation to start the crystallization process. In the suggested method, the use of the ultrasonic generator in turn allows for a uniform crystallization temperature to be attained. Sonocrystallization also avoids contaminating the samples with salts, such as those used by Slaughter and Hew44 as seed crystals. The experimental results obtained by means of the proposed method proved very reproducible, while many of the results of Slaughter and Hew could not be repeated with a high level of precision, as noted by the authors themselves.44 4.3. Thermal Hysteresis of Type III AFPs. To illustrate the accuracy of the results obtained by the proposed method, different type III AFP concentrations were characterized, and the characterization was compared to results obtained from the literature of Baardsnes and Davies34 and Hew et al.30 A data comparison is displayed in Figure 6. The work of Baardsnes and Davies34 cited molecular weights for the wild ocean pout, Macrozoarces americanus, and its variants between 6951.2 Da and 7035.7 Da. The type III AFP used in this work was analyzed by matrix-assisted laser ionization desorption-time of flight mass spectrometry (MALDI-ToF MS) (data not shown). The value of 7033.4 Da determined in the current work agrees with these literature values. The experimental results described by Baardsnes and Davies34 exhibit wide variation in the thermal hysteresis (Figure 7), depending on the particular mutant of the ocean pout, Macrozoarces americanus. For a type III AFP concentration of 3.0 mg/mL, the thermal hysteresis was between ∼0.1 °C and ∼0.6 °C. In turn, our own results on the same sample resulted in a thermal hysteresis of 0.24 °C (see section 3.3), which hence lies in the range of results from the Baardsnes and Davies work.34 For their work, Hew et al.30 used a type III AFP concentration from Macrozoarces americanus of 5.0 mg/mL. The measured thermal hysteresis was 0.44 °C. In our work, the thermal hysteresis for a type III AFP concentration of 5.0 mg/ mL was 0.37 °C (see section 3.3). Considering the range of variation of the thermal hysteresis, and if one compares all the curves, then both the value determined by Hew et al.30 and our value are in a realistic range.

Figure 7. Comparison of thermal hysteresis values from the literature and our own measurements. The graphs according to Baardsnes and Davies34 show the thermal hysteresis of AFP as a function of the protein concentration. The graphs (light gray and white symbols) show the type III AFP wild form and several type III AFP variants of the ocean pout having a type III AFP concentration up to 3.5 mg/ mL.34 The solid black triangle represents the measurement result of Hew et al.,30 and the solid black squares show our own results of the measurements using the sonocrystallization device.

technique was tested and validated with a commercially available antifreeze protein. The results gained are within the values described in the literature for this protein. If only small sample volumes are available or if the crystal shape and growth is of high importance, other methods such as the Clifton nanoliter technique are more favorable. Major fields of application of the technique explained herein will be the simple and scalable screening of the thermal hysteresis activity from natural or synthetic (like peptides) antifreeze substances, which are available in amounts of a few milligrams. The comparable fast method described here can be even more accelerated and allows the design of a (semi)automated high-throughput screening system if an auto sampler technique is implemented.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 49(0)421 2246 630. Fax: 49(0)421 2246 430. E-mail: [email protected].

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Present Address ‡

Center of Applied Space Technology and Microgravity, Research Department Fluid Dynamics and Multiphase Flow. Am Fallturm, 28359 Bremen, Germany.

CONCLUSIONS Direct measurement of the thermal hysteresis can be performed using a combination of a computer-controlled cooling/heating device with a sonotrode for initializing the ice crystal formation. This technique offers reproducible measurements within less than three-quarters of an hour, in which the process can be repeated automatically (for example, overnight). The described

Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest. 10234

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(33) Salvay, A. G.; Gabel, F.; Pucci, B.; Santos, J.; Howard, E. I.; Ebel, C. Biophys. J. 2010, 99, 609−618. (34) Baardsnes, J.; Davies, P. L. BBAProteins Proteomics 2002, 1601, 49−54. (35) Scholand, P. F.; Maggert, J. E. 1971 Cryobiology 8, 371. (36) Feeney, R. E.; Yeh, Y. Adv. Protein Chem. 1978, 32, 191−282. (37) Chakrabartty, A.; Hew, C. L. Eur. J. Biochem. 1991, 202, 1057− 1063. (38) Celik, Y.; Graham, L. A.; Mok, Y. F.; Bar, M.; Davies, P. L.; Braslavsky, I. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 5423−5428. (39) Hansen, T. N.; Baust, J. G. Biochim. Biophys. Acta 1988, 957, 217−221. (40) Hansen, T. N.; DeVries, A. L.; Baust, J. G. Biochim. Biophys. Acta 1991, 1079, 169−173. (41) Mao, X.; Liu, Z.; Li, H.; Ma, J.; Zhang, F. J. Therm. Anal. Calorim. 2011, 104, 343−349. (42) Feeney, R. E.; Hofmann, R. Nature 1973, 243, 357−359. (43) Mulvihill, D. M.; Geoghegan, K. F.; Yeh, Y.; Deremer, K.; Osuga, D. T.; Ward, F. C.; Feeney, R. E. J. Biol. Chem. 1980, 255, 659−662. (44) Slaughter, D.; Hew, C. L. Anal. Biochem. 1981, 115, 212−218. (45) Inada, T.; Zhang, X.; Yabe, A.; Kozawa, Y. Int. J. Heat Mass Transfer 2001, 44, 4523−4531. (46) Chow, R.; Blindt, R.; Chivers, R.; Povey, M. Ultrasonics 2005, 43, 227−230. (47) Yount, D. E. J. Acoust. Soc. Am. 1979, 65, 1429−1439.

ACKNOWLEDGMENTS This project was funded by the Federal Ministry of Education and Research (BMBF, Funding Reference No. 13N9788). The authors would like to thank the project partnersOHB System AG, Liebherr GmbH, Bergolin GmbH, and the VDI TZfor their helpful support.

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dx.doi.org/10.1021/ac301946w | Anal. Chem. 2012, 84, 10229−10235