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BNL-113700-2017-JA
Micro-differential scanning calorimeter
for liquid biological samples
Shuyu Wang, Shifeng Yu, Michael S. Siedler,
Peter M. Ihnat, Dana I. Filoti, Ming Lu, Lei Zuo
Submitted to the Review of Scientific Instruments
October 2016
Center for Functional Nanomaterials
Brookhaven National Laboratory
U.S. Department of Energy USDOE Office of Science (SC), Basic Energy Sciences (SC-22)
Notice: This manuscript has been authored by employees of Brookhaven Science Associates, LLC under
Contract No. DE- SC0012704 with the U.S. Department of Energy. The publisher by accepting the
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Micro-differential scanning calorimeter for liquid biological
samples
Shuyu Wang1, Shifeng Yu2, Michael S. Siedler3, Peter M. Ihnat4, Dana I. Filoti4, Ming Lu5, Lei Zuo2*
1 Department of Mechanical Engineering, Stony Brook University, Stony Brook, NY 11794, USA
2 Department of Mechanical Engineering, Virginia Tech, Blacksburg, VA, 24061, USA
3AbbVie, Deutschland, 67061 Ludwigshafen, Germany
4 AbbVie Bioresearch Center, Worcester, MA 01605, USA
5Center for Functional Nanomaterials, Brookhaven National Laboratory, Upton, NY 11973, USA
*corresponding author: leizuo@vt.edu
Abstract: We developed an ultrasensitive micro-DSC (differential scanning calorimeter) for liquid
protein samples characterization. This design integrated vanadium oxide thermistors and flexible polymer
substrates with microfluidics chambers to achieve high sensitivity (6V/W), low thermal conductivity
(0.7mW/K), high power resolutions (40nW) and well-defined liquid volume (1µL) calorimeter sensor in a
compact and cost-effective way. We further demonstrated the performance of the sensor with lysozyme
unfolding. The measured transition temperature and enthalpy change were in accordance with the previous
literature data. This micro-DSC could potentially raise the prospect of high-throughput biochemical
measurement by parallel operation with miniaturized sample consumption.
Keywords: microcalorimeter, differential scanning calorimeter, protein unfolding, flexible substrate,
microfluidics, MEMS (micro electro-mechanical systems)
Introduction:
Kinetic processes are playing a critical role in
physical transformations, such as glass transition,
and in macromolecule transformations, such as
protein denaturation. Understanding the effects of
external influences on the transformation may
provide valuable insights into the process[1]. The
first step to such understanding is to detect the
reaction accurately in the real time. Calorimetry
is the technique that has been widely used to
study such kinetic process and thermodynamic
properties of the materials or biological system[2].
Among several different operation modes of
calorimetry, differential scanning calorimetry
(DSC) is a well-known dynamic calorimetric
method. It imposes a temperature ramp
continuously and measures heat capacity change
of the material (both in liquid and solid forms).
For the past decades, the micro/nanofabrication
technology has been enabling calorimeters to be
smaller, faster and more accurate. As a result, the
conventional bench-top scale instruments were
miniaturized into microelectromechanical sensor
systems and some may integrate microfluidics
systems [3-5]. These miniaturized calorimeters
were often called micro/nanocalorimeter or chip
calorimeters. Normally, they were based on the
thin films, either made of silicon/silicon nitride [6,
7] or polymers [5, 8]. These chip calorimeters
were capable of measuring samples with very
small thermal mass due to significantly reduced
addenda[9] and may measure the energy on the
order of nanojoule[4] accounted from the superb
thermal insulation. We have already seen chip
calorimeters’ application to study thin films [8],
polymer [10], cells [11],protein [12] and so on.
Such studies in biological systems can be very
crucial to the pharmaceutical industry as several
critical steps in the drug discovery process are
associated with calorimeters. Currently, the long
measurement time of the conventional scanning
calorimeters often requires years to finish
thousands of the chemical compounds[13] and
the liquid volume of protein sample required is
very large(500µL-2mL). Chip calorimeter might
be a potential solution since they use very small
amount of protein sample and can easily provide
parallel operations to achieve high-throughput
[12]. However, chip calorimeter as DSC to study
biological system still remains very scarce in
academics compare to micro isothermal titration
calorimeter (ITC) [4, 12, 14, 15] due to
technology difficulties to develop them[13].
Several different configurations of micro-DSC
for liquid sample appeared in recent years. Olson
[2] and Youssef[16] studied the scanning
calorimeter with liquid samples, yet their uses are
limited to measure latent heat of transformation
due to evaporation. Yao [17] built an AC
calorimeter with glass capillary tube that
consumed 10µL liquid. The device (heat capacity
resolution ±300nJ/K) successfully tested the
lysozyme solution’s heat capacity when
denaturing. Garden’s [18] scanning AC
calorimeter has also measured 5µL biological
liquid sample’s heat capacity with high
resolution(±150nJ/K). They used a gasket and
polyimide membranes (supported by micro-posts)
to construct the chamber. However, their design
suffered from large thermal conduction
(30mW/K) which limited the sensitivity. Lee[19]
constructed an enclosed parylene chamber (1µL)
to tested DNA and hydrocarbon yet the signal-to-
noise ratio is too low. Lin’s group successfully
integrated microfluidic systems in the DSC using
polydimethylsiloxane (PDMS) microfluidics
chamber (1.2µL) and SU8 diaphragm to and
reached high detection limit (30nW)[20]. They
further developed AC-DSC[5] and changed
substrates to flexible polyimide substrates. These
devices reached very high performance
(sensitivity 4-8V/W) when demonstrating with
protein samples[21]. Yet, the thermopiles
deposited on the flexible substrate could not
determine absolute temperature, and therefore the
transition temperature might not be accurately
measured.
In this paper, we reported the fabrication and
operation of a micro-DSC for liquid biological
samples which can address the above problems.
We showed how to make a flexible calorimeter
by microscale fabrication technology and
integrate it with PDMS microfluidics chamber
(1µL). Due to the highly temperature-sensitive
vanadium oxide thermistors and superb thermal
insulation of polyimide thin films, the device had
proven to be very sensitive to thermal events.
This enabled the sensor to be used for protein
interaction detection at low cost and disposable
way. This technology could readily scale up to
array format to apply in high-throughput drug
discovery.
Theory
Virtually all DSC measures the temperature
difference between the sample and reference
region since this temperature difference can be
converted proportionally to heat flow rate
difference based on a simple linear model. This
conversion is the zeroth approximation, and more
accurate conversions can use higher-order
approximations[22].
Within one chamber, the differential heat
equation can be described as Eq.1, where Cp is the
heat capacity, G is the heat conduction, and P is
power or heat flow.
0( ( ) ) ( )p
dTC G T t T P t
dt (1)
The time constant τ is determined by 𝐶𝑝
𝐺 . When
time constant is much smaller than the
biomolecular thermal event (time constant of
guanidine induced unfolding is on the order of
20-50s[17]), the process can be considered as in
the steady state. Therefore, ∆T=Ts-Tr can be
related to ∆P=Ps-Pr with a linear relationship
∆P=G∆T. Using temperature sensors to generate
voltage signal ∆U proportional to the ∆T, we can
have
∆𝑈 = 𝑆∆𝑃 (2)
where S is the sensitivity of the calorimeter.
Consequently, the thermal insulation of the
chamber (G) and temperature sensor’s sensitivity
can determine the device’s sensitivity. Based on
the mathematical model, the heat capacity
difference between the sample and reference
solution ∆C=Cs-Cr can be obtained from Eq3
where is the scanning rate of the calorimeter.
∆𝐶 =∆𝑃
[20] (3)
Since the differentiation method used in the DSC
measurement, the symmetry of the sample and
reference can be crucial to the final results.
Device description and fabrication
Figure 1and Figure 2 showed the 3D schematic
view and the cross section view of the
microcalorimeter respectively. The device
consisted of two parts: the PDMS microfluidic
chamber and the flexible calorimeter sensor.
They were fabricated separately and later bonded
together to formulate the whole device. There
were two identical microfluidic chambers: one
for protein liquid sample and the other for
reference buffer liquid sample. The chamber had
a volume of 1µL (height 200µm, radius 1.25mm)
and was surrounded by air gaps to reduce the
thermal mass of the close chamber and increase
the thermal insulation. We cast the liquid PDMS
onto the master pattern made from SU8 100
photoresist and then solidified it by soft baking.
Last, we drilled the tiny holes for inlet and outlet
of the chambers. The microfluidic chamber could
serve to prevent liquid evaporation and provide
well-defined liquid volume.
The flexible calorimeter sensor used polyimide
substrate (7µm) and integrates the thermistors
and the microheaters for sensing and calibration.
ANSYS simulation was used to assist the pattern
design of the microheater: the widths of the heater
in each circle were finely tuned to reach the
maximum temperature uniformity while heating.
The Au traces (width and gap were 10µm) were
the metallization features and served for electrical
interconnection of the thermistor layer. This
design was amount to parallel connection of
multiple resistors and could significantly lower
the resistance of the thermistor.
We spun coat PI 2611(HD microsystem)
polyimide on a silicon wafer and ramping
annealed it from room temperature to 350oC to
make it chemical inert. The solidified polymer
layer would have very low adhesion with the
silicon wafer but enough to bond them together
during fabrication so that easy separation of them
could be achieved (Figure 3(b)). We fabricated
the Au microheaters (Figure 3 (c)), the polyimide
dielectric layer, sputtered vanadium oxide
thermistors, Au traces for thermistor (Figure
3(d)), and the polyimide moisture protection
layer sequentially. The 100nm vanadium oxide
layer was deposited by DC sputter and Au trace
was deposited by e-beam evaporation. In order to
enhance the bonding strength of the flexible
sensor and microfluidics chamber, a thin layer of
PDMS diluted with toluene would be spun coated
before peeling off from the silicon substrate.
After oxygen plasma treatment for 30s under
20mTorr, the microfluidic chamber and flexible
sensor would be bonded together. Figure 3 (a)
showed the fabricated device after bonding.
Figure 1.Schematics of the micro-DSC, showing the
overall structure
Figure 2. Cross-section view of the micro-DSC design
Figure 3. (a) Fabricated micro-DSC device (b) flexible
substrate peeling off from the silicon wafer (c) Optical
microscopy image of the heater (d) thermistor metal trace
Experimental methods
The electrical circuit connection was illustrated in
Figure 4. The two thermistors for sample and
reference liquid and two outside variable resistors
were connected as a Wheatstone bridge in order
to provide common mode rejection. Such bridge
circuit could pick up the temperature difference
between the sample and reference side. The
voltage output of the bridge circuit represented
the temperature difference between the sample
and reference chamber. We used the lock-in
amplifier (Stanford SR830) to provide the voltage
input (V+) and differentiate the voltage output
from the bridge(Vout). The sourcemeter (Keithley
2636) was the power source for the microheaters
and measures the resistance change during the
thermal response test. To calibrate the resistance
of the thermistors and do temperature scanning,
we placed the microDSC inside a high
temperature probe station and further connect the
wire outside to the equipment. This high
temperature probe station could raise the
temperature with a program and provide the
thermal shielding at the same time. We used
lysozyme samples (molar weight is14307Da) at a
concentration of 100mg/mL to demonstrate the
device’s performance. The buffer used was 10
mM HEPES pH 7.4 and 150 mM NaCl. The
whole experimental measurement and control
were achieved with LabVIEW programs.
Figure 4. Schematic view of the micro-DSC circuit
connection. The thermistors and outside variable resistors
form a Wheatstone bridge.
Results and discussions
Before doing the measurement, we first
calibrated the vanadium oxide thermistors. The
resistance and temperature’s relation is plotted in
log scale (Figure 5). The thermistor’s resistance
is normally in the range of 15-40KΩ, meaning the
designed gold trace pattern can effectively reduce
the resistance to an appropriate range. The
temperature coefficient of resistance (TCR) can
be obtained from the slope of LnR versus
temperature as -2.8%/oC, which is one order
higher than a traditional platinum thermistor. We
also observed good uniformity of the thermistor
material; the sample and reference thermistors
could have 1-2% variation in resistance.
Although the thermistors could introduce self-
heating during measurement, the thermistor on
the reference side can counterbalance it, and such
self-heat can be further neglected during
temperature scanning. Unlike the thermopiles
that require large numbers of them to gain high
sensitivity, the high-performance thermistors can
achieve compactness with simplified
fabrication[23]. Moreover, thermistors can detect
the absolute temperature within the small
chamber, which is necessary for DSC
measurement.
Figure 5. Temperature calibration of a thermistor and
vanadium oxide resistivity measurement as a function of
temperature
We further calibrated the microcalorimeter using
electric power applied to the microheater. The
thermal response to a step function power can be
predicted by /( )( ) (1 )tP t
T t eG
. By fitting curve
of data in Figure 6 to exponential increase
function, we could extract the thermal time τ
constant to be 6 s. Due to the well-designed air
gap and the high thermal insulation of PDMS, the
affected addenda thermal mass was minimized to
be much smaller than the liquid samples
especially when small amount of heat was
applied. Therefore, the thermal conductance
(G=𝐶
τ )could be calculated to be around
0.7mW/K. Such low thermal conduction could be
comparable to the open-type calorimeters [12,
24].
Figure 6. Thermal response of the device after applying a
constant power from the microheater
We could also obtain the sensitivity of the device
by varying the power dissipated in the heater. The
data showed good linearity and we measured the
slope of the plotted data (Figure 7) which
indicated the sensitivity to be 6V/W. The result
proved the device was highly sensitive to
measure a small amount of heat and it was further
used to determine the differential thermal power
between the calorimeter chambers for protein
tests.
Figure 7. Micro-DSC’s voltage output responding to a
constant differential power input from the heater. sensitivity
of the microcalorimeter is obtained from the slope
The performance about noise level of the device
was also evaluated. We filled the two chambers
with deionized water and tested it at a constant
temperature in a well-defined environment. Due
to the inevitable asymmetry of the two sides, the
measured voltage signals might drift. Figure
Figure 8 was the result after subtracting the raw
data from three order fit of the data. The rms noise
at 1 Hz bandwidth is 250nV, corresponding to
40nW of power resolution and 60µK of
temperature resolution (the results are calculated
based on the sensitivity S and thermal conduction
value G). This noisy behavior could be related to
the environment temperature fluctuation control,
the extent of the common mode rejection and the
thermistors’ Johnson noise.
Figure 8 noise measurement of the micro-DSC at a constant
temperature (raw data subtracted from three order fit)
We demonstrated the micro-DSC’s performance
with the lysozyme protein (100mg/mL) for
consecutively two times. We raised the
temperature of the chamber from 25oC to 80oC at
the rate of 7.5 oC /min. After baseline subtraction,
the measured voltage outputs from the
Wheatstone bridge showed the typical DSC curve
of protein denaturation. We could further obtain
the partial specific heat capacity of lysozyme as a
function of temperature (Figure 9) based on Eq3.
The melting temperature were at around
61oC.These results were further integrated to
obtain the enthalpy change (∆H) of the lysozyme
samples (Figure 10). The values obtained in this
study were around 415KJ/mol, which
corresponded well with the published data 377-
439 KJ/mol [25]. Apart from these, we could see
the second test could almost repeat the first time
test. Such variation of the results might be related
to the difference in the protein samples.
Figure 9. micro-DSC’s two tests with lysozyme sample
(molar weight:14307 Da). Specific heat capacity of
lysozyme vs. Temperature. (baseline subtracted)
Figure 10 enthalpy change of two lysozyme samples vs.
Temperature during unfolding. The enthalpy is obtained by
integrating the data in figure 9.
Conclusion
This paper reported a micro-DSC applied for
liquid biological sample characterization. This
sensor implemented temperature sensitive
vanadium oxide as the thermistor material with
TCR value of -2.8%/oC. With the help of the high
thermal insulation provided by the thin polyimide
membrane and the well-designed microfluidics
chamber, a sensitivity of 6V/W and power
resolution of 40nW were achieved. This high-
performance micro-DSC successfully tested the
lysozyme unfolding with the minimum sample
consumption (1µL) and this technology could be
further applied to expand the devices into arrays
so that high throughput measurement in the drug
discovery industry might be attained.
Acknowledge
The authors thank the funding support from NSF
(IDBR #1530508) and AbbVie Inc. The
fabrication was carried out at the Center for
Functional Nanomaterials (CFN) at Brookhaven
National Laboratory, which is supported by the
U.S. Department of Energy, Office of Basic
Energy Sciences, under Contract No. DE-AC02-
98CH10886. We also thank Dr. Fernando
Camino and Kuan Hu for the help and useful
discussion.
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