International Journal of Biological Macromolecules 52 (2013) 353357

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Monitoring protein denaturation using thermal conductivity probe

Byoung Kyoo Park a , Namwoo Yi a , Jaesung Park b , Dongsik Kim a,
a
Department of Mechanical Enigneering, POSTECH, Pohang 790-784, Republic of Korea
b
School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang 790-784, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: We propose a method for probing denaturation of proteins by measuring the thermal conductivity of
Received 31 May 2012 the solution. We use the three-omega method with a microfabricated ac thermal sensor to measure the
Received in revised form 25 August 2012 thermal conductivity of lysozyme, -lactoglobulin, and bovine serum albumin protein solutions over a
Accepted 10 October 2012
range of temperature and pH. Results suggest that conformation transformation of the protein during
Available online 1 November 2012
denaturation changes the thermal network in protein solutions and thus changes the thermal conductiv-
ity for all the tested proteins. The proposed method of denaturation monitoring requires much simpler
Keywords:
experimental setup than conventional methods such as differential scanning calorimetry and circular
Protein denaturation
Thermal analysis
dichroism detection. We also demonstrate that the proposed analytical technique can detect the protein
Thermal conductivity denaturation in real time. Consequently, it is expected to be useful in lab-on-a-chip (LoC) applications as
Three-omega method the probe can be easily miniaturized for integration into LoC devices and allows real-time analysis.
2012 Elsevier B.V. All rights reserved.

1. Introduction dichroism (CD) is a powerful technique that is sensitive to the sec-

Proteins are essential components of biological cells. Proteins
perform physiological activities including catalysis of chemical
reactions, cell signaling, signaling transduction, immune response,
and cell adhesion; some have structural functions [1,2]. Generally,
proteins exist in a native state, which is the most stable three-
dimensional folded structure, and their tertiary folded structure
determines their specic functions [3]. Proteins that are exposed to
external stress or to compounds may lose their tertiary structure,
i.e., become denatured. Denaturation causes the protein to adopt
a random coil conformation [4]. This structural change may dis-
rupt cell activities, and can cause cell death. Understanding of the
effects of external inuences on denaturation may provide valuable
physiological insight. A rst step to such understanding is to detect
denaturation accurately and in real time. To investigate the physics
of conformational change in proteins, various methods have been
employed to detect the denaturation state [521]. Differential scan-
ning calorimetry (DSC) has been widely used to investigate the
thermodynamics of protein denaturation [710,20,21]. DSC can
measure the change in heat capacity and enthalpy of protein
solutions during denaturation. However, insufcient resolution
makes DSC inadequate for the dilute (0.110 mg ml1 ) protein solu-
tions typical of biological systems. Also, the DSC curve changes
with the scan rate of a device, so precisely determining dena-
turation temperature is difcult [20]. Far-ultraviolet (UV) circular
Corresponding author. Tel.: +82 54 279 2179; fax: +82 54 279 3199.
E-mail address: dskim87@postech.ac.kr (D. Kim).
ondary structure of a protein. CD is based on differential absorption
of left- and right-circularly polarized light; the signal reects the
proteins secondary structure. Far-UV CD is a powerful tool to inves-
tigate secondary structure of protein because specic structures
such as the -helix of protein and the double helix of DNA have their
own representative CD spectral signatures [1416]. By analyzing
CD spectra, the fraction of conformations such as -helix, -sheet,
-turn, or random coil in the protein can be estimated. However,
measurement of CD is complicated because oxygen and aqueous
buffers often absorb light in the spectral range that exhibits the
proteins structural features. Tris, dithiothreitol, histidine, and chlo-
ride buffers must be avoided, and phosphate, sulfate, carbonate,
and acetate buffers can be used only when they are diluted to
<50 mM. An alternative method of detecting protein denaturation
that does not suffer from these limitations of DSC and CD would be
a signicant advance. Recently, there were several investigations
concerning the feasibility of sensing biochemical reactions based
on thermal properties measurement [2224].
Among the studies, we reported that thermal conductivity k
[W m1 K1 ] of bovine serum albumin (BSA) solutions has a strong
correlation with conformational change of BSA [22]. k changes dur-
ing denaturation for two reasons; change in the proteins structure
causes changes in: (1) k of a protein itself because the vibrational
energy transport mechanism is altered and (2) the thermal network
in a solution including the thermal contact resistance between
protein and the buffer. In its tertiary structure, a protein usually
has a hydrophilic surface with a hydrophobic core. Intermolecular
hydrogen bonds make a protein solution stable. However, pro-
tein denaturation changes the degree of hydrogen bonding and

0141-8130/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijbiomac.2012.10.027
354 B.K. Park et al. / International Journal of Biological Macromolecules 52 (2013) 353357
Fig. 1. (a) Schematic diagram of 3 technique for measuring k of liquid sample and
(b) schematic diagram of thermal conductivity sensor.
non-polar hydrophobic interaction; these changes reduce the pro-
teins solubility and communal aggregation. We suggested that
these mechanisms make k of a protein solution (ksol ) change. Nev-
ertheless, that study [22] was restricted to only a single protein,
BSA, so further investigations are required to generalize the con-
clusion that the conformational dynamics of common proteins can
be monitored by measuring ksol .
In the present work, ksol was measured in solutions of -
lactoglobulin and lysozyme which are structurally distinct from
BSA. Whereas the structure of BSA is dominated by -helix, -
lactoglobulin has 1015% -helix and 43% -sheet, and lysozyme
has 40% -helix, 12% -sheet, and 48% random coil [25]. The present
work also introduces a measurement scheme that can be used to
monitor protein denaturation in real time.
2. Materials and methods
2.1. Sample preparation
-Lactoglobulin (SigmaAldrich L3908) was obtained as a chro-
matographically puried lyophilized powder from bovine milk.
It was dissolved (2.72 103 M, 50 mg ml1 ) in distilled water;
the pH of the solution was adjusted to 7.00, then stirred for
2 h. Lysozyme solution was prepared by the protein obtained
from chicken egg white (SigmaAldrich, 62971). It consists of a
single 14.7-kDa polypeptide chain. The solution was dissolved
(3.17 104 M, 2.18 mg ml1 ) in 50-mM sodium phosphate buffer
with pH 6.20. And the solution was prepared daily. The two con-
centrations were chosen to compare the results with the existing
DSC or far-UV CD data [16,19].
2.2. Measurement
To measure ksol and k of base uid kb we used the three-omega
method [2631], which is a well-known technique to measure heat
diffusion in bulk solid, solid thin lms or liquid, and is particularly
suited for measuring k of liquid samples which are electrically con-
ducting or as small as sub-nanoliter in volume [2931]. The sensing
device is composed of a thin-lm metal strip and a dielectric layer
on a glass substrate (Fig. 1). The metal strip serves as a line heater
as well as a temperature sensor. When ac current of frequency
[s1 ] is applied to the line heater, it generates Joule heating at
the frequency 2. Thus, temperature T and resistance of the heater
also oscillate at the same frequency 2. The resistance multiplied
by original current produces the 3 voltage signal. The average
thermal response of the sensor, more precisely, the amplitude of
temperature oscillation can be expressed analytically as a function
of the thermal properties of the substrate and the liquid sample in
contact with the sensor [29]. The response is reduced to a simple
form as a function of the qb, i.e., the half-width of the heater in units
of q1 under the condition that specic heat C and k are real-valued
quantities [26]:
lk 
T = ln(qb) + (qb) i, (1)
P 4
where P [W] is heating power, l [m] is the length of the heater, b
[m] is the half width of the heater, and q = 2/ is the inverse of
thermal penetration depth where [m2 s1 ] is thermal diffusivity.
Because (qb) approaches a real constant for qb  1, k of a liquid
sample kl can be determined in two ways, the slope method and
the single-frequency method.
2.2.1. Slope method
To obtain k of a liquid sample kl using the slope method that is
conventionally used, temperature responses of the sensor should
be obtained by varying the modulation frequency. The thermal con-
ductivity of the liquid sample kl can be determined from the slope
of the thermal-response vs. modulation frequency curve in a plot of
the real (in-phase) part of temperature Treal vs. ln() [29]; neglect-
ing boundary mismatch, the measured k can be approximated as
ks + kl :
P d ln
k = ks + kl = (2)
2l dTreal
where ks is the k of the sensor substrate (1.083 W m1 K1 for
Borooat glass substrate) [32]. Although substrates with lower
thermal conductivities give better sensitivity, glass was chosen to
be the substrate material for easy fabrication.
2.2.2. Single-frequency method
Alternatively, kl can be determined by using the imaginary (out-
of-phase) part of temperature oscillation Timag at a single frequency;
in this method, measured k can be also replaced by ks + kl [31]:
P 1
k = ks + kl = , (3)
4l dTimag
The slope method is generally more sensitive than the single-
frequency method because the real part of the temperature
response has much greater magnitude than does the imaginary
part. Nevertheless, it is advantageous in small volume measure-
ment like sub-nanoliter scale liquid [31]. Also, we can get k
in real-time because this scheme yields k from the tempera-
ture response of single frequency without scanning temperature
responses of a certain range of frequencies.
2.3. Experimental methods
The lift-off process was used to fabricate a thin-lm Au/Cr strip
heater/sensor on a Borooat glass substrate (Schott 33). A 200 nm-
thick Au lm was deposited with a 20-nm-thick chromium layer
as an adhesion layer using an electron beam evaporator. The width
and length of the metal strip are 20 m and 1 mm, respectively.
After patterning the heater/sensor, the PECVD (plasma-enhanced
chemical vapor deposition) process was employed to deposit a
silicon nitride (Si3 N4 ) dielectric layer of 200 nm in thickness for
electrical insulation of the heater/sensor. An electrical current of
frequency from an internal source of a lock-in amplier (Stan-
ford Research Systems, model SR810) was supplied to the Au strip.
The component of the signal was eliminated by adjusting the
gain of a differential amplier (Analog Devices, model AD620) with
an input impedance 10 G, leaving only the 3 signal that bears
the thermal information. The 3 signal was measured using the
lock-in amplier, and the amplitude and phase of the tempera-
ture signal were determined from the signal. The measurement
was repeated by varying the modulation frequency from 5000 to
1.5 Hz. In the case of DI water, the thermal penetration depth at
 B.K. Park et al. / International Journal of Biological Macromolecules 52 (2013) 353357 355

Fig. 2. Experimental setup: (a) sensor holder and (b) thermal conductivity sensor.

a frequency 1.5 Hz is about 87 m. The sensor was inserted into a

copper block inside a test chamber (Fig. 2). Although there was no
direct deposition of the protein on the sensor surface as the sensor
was vertically mounted, there was a possibility of long-term den-
sity variation due to protein settling. Therefore, the measurement
using the slope method was conducted shortly (within 5 min)
after sample preparation to avoid the settling effect. Neverthe-
less, some denatured proteins at high temperatures adhered to the
chamber and sensor surfaces and changed thermal responses of the
sensor. We thus inspected the surface by optical microscopy after
each measurement and excluded the data obtained when adhesion
occurred. A water bath was used to control T in the chamber to
within 0.1 C.
The device was rst calibrated by measuring k of DI water,
methanol, and ethanol. All measurements were repeated 10 times
for each sample to assess the random error; deviations from ref-
erence values [33] were all <3%. Maximum standard deviation
was 2.24% and average standard deviation was 1.29%. In the pro-
tein solutions, random error was higher than those of standard
materials: using slope method, maximum standard deviation was
3.68% and average sd was 1.48%; this increase in variability was
mainly due to inhomogeneity in the protein solution. Especially, the
error of measurements increased at T > 5060 C, probably because
agglomeration and precipitation of denatured protein increases the
solutions inhomogeneity.
ksol of BSA and lysozyme solutions were measured at
25 T 85 C in increments of 10 C. To observe k variation near
denaturation temperature Td minutely, ksol of -lactoglobulin solu-
tion was measured at 50 T 85 C in increments of 5 C. When
T > 85 C, some denatured proteins adhered to the sensor surface
and sensor chamber. As T increased, kb increased almost linearly.
ksol also increased with the same slope as kb until T reached a crit-
ical point Ti at which denaturation began. Here, Ti was dened as
the temperature at which

k = ksol kb k > 0.005 W m1 K1 , (4)

Fig. 3. (a) kDI and ksol , (b) k, (c) dk/dT of BSA and -lactoglobulin solution.

where k= ksol kb at the lowest temperature used for (25 C

BSA and lysozyme solution and 55 C for -lactoglobulin solu-
tion). When T was further increased above Ti , the slope of each
ksol curve deviated from that of its associated kb curve. The dif-
ference between ksol and kb signicantly increased around Td and
then stabilized at saturation temperature Ts . Because we assume
that k indicates the effect of protein denaturation, the value can
be used as an index to measure the progress of denaturation; this
means that k represents the accumulated quantity of denatured
proteins up to that T, and that dk/dT can be considered to represent
the instantaneous denaturation rate at T. Therefore, the meaning
of the dk/dT graph is similar to that of the endothermic heat ow-
temperature curve in DSC, in which Td is generally taken as T at
which the peak point of the DSC curve occurs. To be consistent
with DSC data, we dene Td as the temperature that corresponds
to the maximal dk/dT.
3. Results and discussion
3.1. Measurements using slope method
The slope method (non-real time thermal analysis) was used
to obtained k, k, and dk/dT graphs (Fig. 3) for BSA and -
lactoglobulin solutions, which use DI-water as the base uid. Curves
in the k, and dk/dT graphs are obtained using cubic spline inter-
polation. Before the denaturation started, k was 36% lower than
kb in each solution, because of the relatively low k of pure protein.
Numerically calculated k of proteins at 300 K is 0.27 W m1 K1 ,
which is less than half that of DI-water kDI [34]. We previously
estimated a similar value by linear extrapolation based on exper-
imental data with various BSA concentrations [22]. As T increased
from 25 to 85 C, kDI increased almost linearly from 0.608 to
356 B.K. Park et al. / International Journal of Biological Macromolecules 52 (2013) 353357

Fig. 4. Comparison between dk/dT and DSC curve (Q10, TA instrument, scan
rate 1 C min1 , temperature range 2080 C) of BSA solution (concentration
50 mg ml1 ).

0.674 W m1 K1 , and ksol of each sample increased with same slope

as that of kDI until Ti , and then deviated from that slope. Although
the tendencies of the graphs were similar, Ti , Td , and Ts values
changed with protein type and solution pH. This change shows
that the structural differences among proteins affect ksol and that
protein conformation is inuenced by pH as well as by T.
To verify the reliability of this analysis, we compared the dk/dT
graph with the DSC result (Fig. 4). DSC measurement of BSA solution
(pH 7.00 and 50 mg ml1 concentration with same preparation pro-
cedure) was conducted at 20 < T < 80 C at a scan rate of 1 C min1
(Q10, TA instrument). Although the endothermic heat ow curve of
DSC is less sensitive than dk/dT graph of our analysis, both curves
reect the same Ti , Td , and Ts trends as did protein in solution.
However, in the DSC measurement, Ti and Td could not be deter-
mined precisely without numerically compensating for the data
variation with scan rate [20], possibly due to the timeresponse of
the device. Whereas using the proposed method on -lactoglobulin
solution obtained Td = 71 C, DSC values of Td varied from 67 to
77 C as scan rate increased from 2 C h1 to 90 C h1 . Numeri-
cal methods to analyze the DSC curve [35,36] can be employed
to precisely determine Ti and Td but those analyses require an
appropriate model for each protein. Therefore, instead of DSC, we
employed a far-UV (ultraviolet) CD to more precisely probe the Fig. 5. (a) CD spectra of lysozyme in denaturation process, (b) k and (c) dk/dT of
accuracy of the proposed analysis method using k. We prepared lysozyme solution.
a highly dilute (3.17 104 M, 2.18 mg ml1 ) lysozyme solution in
50 mM sodium phosphate buffer of pH 6.2; this is close to the we compared them to verify the reliability of k analysis. The two
protein concentration in biological systems. We monitored dena- graphs were very similar (Fig. 5); this means that the ksol data can
turation of lysozyme solution using far-UV CD and compared the be used to detect the conformational change of a protein which
result to that obtained by our analysis using k. CD spectra (Fig. 5a) can be observed using a CD spectrometer.
were measured at a wavelength of 230 nm with a bandwidth of Our experimental results suggest that a proteins conformation
1 nm using a spectrometer (Jasco J-815) at 20 C < T < 90 C and a determines ksol by changing the thermal network in the protein
heating rate of 2 C min1 . The CD signal was recorded at 2 C inter- solution. Ti and Td of each protein solution (Table 1) differ from
vals. From room temperature to 60 C, CD was almost uniform at previous results by <5 C except for Td of BSA solution at pH 5.05
about 180 mdeg. As T increased above 60 C, CD started to signi- [8,16,19]. In this case, denaturation occurs over a relatively wide
cantly change to 100 mdeg; this means that the tertiary structure range of T with relatively small change in k. We assume that this
of lysozyme transformed to another structure at that temperature
range. The change of the value saturated at 85 C because the pro- Table 1
tein was fully denatured. Thus the CD value at a specic T represents Ti at which protein denaturation begins and Td at which it is maximal for each protein
solutions.
the cumulative proportion of protein denatured up to that T, as does
k of our method. Protein Measurement
We measured ksol of the same lysozyme sample at Ti (5 C) Td ( C)
20 C < T < 90 C in increments of 10 C; it was almost the same as
BSA (pH 5.05) [22] 44.0 50.0, 70.0
that of the sodium phosphate buffer base uid up to 55 C but BSA (pH 7.00) [22] 53.0 68.0
deviated signicantly from it at T > 55 C. Graphs of k and dk/dT BSA (pH 8.20) [22] 39.0 57.0
(Fig. 5b and c) were obtained employing the same methods used for BSA (pH 10.05) [22] 47.0 56.0
BSA and -lactoglobulin. The lysozyme solution had Ti 60 C and -Lactoglobulin 61.0 71.0
Lysozyme 60.0 71.0
Td 71 C. Because the k and CD curves have similar meaning,
 B.K. Park et al. / International Journal of Biological Macromolecules 52 (2013) 353357
Fig. 6. dk/dT curves of (a) BSA, (b) -lactoglobulin, and (c) lysozyme obtained by
single frequency method.
denaturation characteristic of the sample causes the relatively large
discrepancy between our Td measurements and previous results.
3.2. Measurements using single-frequency method (real-time
thermal analysis)
Temperature responses at = 3.53 and 5.40 Hz were used to
obtain ksol of BSA, lysozyme, and -lactoglobulin solutions. All
measurements were repeated ve times. The maximum sd was
3.96% and average sd was 3.13% (Fig. 6); these values are slightly
larger than those of the slope method. Although the measurements
are less precise than those obtained using the slope method, the
effect of denaturation on k could be distinguished for every protein
solution in real time. Most Td values obtained using the single-
frequency method differ by <5 C from those obtained using the
slope method (Fig. 6).
4. Conclusion
This work demonstrates that measuring k is an effective method
to detect change in protein conformation. It was previously shown
that k of BSA solution has strong correlation with structural
change of the protein [22]. In the present work, we generalize the
357
correlation between the conformational dynamics of common pro-
teins and the k by measuring ksol of -lactoglobulin and lysozyme
which are structurally distinct from BSA.
The proposed method of using ksol to analyze protein dena-
turation has several advantages over conventional methods such
as DSC and CD. (1) It has the potential to be integrated into a
lab-on-a-chip device because the measuring device has much sim-
pler structure than conventional devices, and thus it can be easily
miniaturized. (2) The method is not affected by the heating rate
(scan rate) as is DSC measurement. (3) This method can analyze
highly diluted protein solutions which are typical in biological
systems whereas DSC requires relative high concentration of the
protein solution to monitor the denaturation. (4) The method is not
affected by buffers and additives as is CD analysis. (5) The proposed
technique achieves real-time monitoring and analysis of protein
denaturation.
Acknowledgement
This work was supported by the NRF Basic Research Program
(No. 2012-0005649, No. 2011-0028845).
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