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7.4

Nano-Calorimetry
S. VAN HERWAARDEN Xensor Intergration Delft, Netherlands

Around 1780 Lavoisier and Laplace developed the so called ice calorimeter which determined the heat evolved by a living organism within it fIom measurements the of quantity of ice the organ melted. In particular, it was used to determine the metabolism of entire living animaIs, about (1012to 1015)celIs, placed within the

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calorimeter [276] and a variant of this type of calorimeter is still in use [277]. Bataillard [278] reports that a nanocalorimeter was used to study the metabolism of lessthan 106bacteria which is a reduction in cell sample size over the animal within the ice calorimeter by a factor of between(106to 109).The advent of micro electromechanical systems(MEMS), also known as rnicro-system technology (MST), that bas evolved from the methods used to manufacture integrated circuits (IC) bas permitted the developmentof calorimeters that cao operate with significantly smaller sample sizes.MEMS cao be manufactured with dimensions from (10-3 to 10-9)m with high precision and reproducibility. MEMS calorimeters have been fabricated with dimensions on the order of 10-3m that incorporate heating elementswith weIl defined thermal properties, thermometers and mechanical structures that cao hold the samples.The term 'nanocalorimeters', consistent with the current and accepted definition of microcalorimeter, is used to describethesecalorimeters becauseof the power detected by the instrument rather than the dimensions of the object formed with integrated circuit technology. Nanocalorimeters have been used to measure thermodynamic properties of fluids. The main application of these devices is outside those traditional measurementsand differs from those of differential scanning calorimeters (DSC). The devicesreported at the time of this writing operate over a rather limited range of temperature but the small size provides both rapid response times and high sensitivity. 7.4.1 Micro Electro-Mechanical Systems(MEMS)

MEMS is a technology used to manufacture three-dimensional silicon based structures with specific geometrical, mechanical and electrical properties to execute certain tasks [279]. In a MEMS nanocalorimeter the microstructure includes on ODe substrate all the required components, including a heating resistor, a temperature sensor, a sensor to determine temperature differences, a well-defined thermal conductanceand a container [280-282]. In a calorimeter constructed with traditional methods, for example the DSC describedin Section 7.3, the assemblyis formed from severalindividual componentsthai are mechanicallyjoined to farm ODe calorimeter. In the case of a DSC it consists of the following individual components: an oven surrounding the instrument; a thermocouple to measure the temperature of the crucible; and a sample holder thai is usually a metal foil (that acts as athermal conductor) with two cup-like indentations to hold the sample. SiIicon-BasedMEMS With MEMS technology, the nanocalorimeter is formed as a single integrated structure including heater, sensor,and sampleholder on a Si base.Typically, MEMS usescircular silicon wafers about 100mm in diameter and about 0.5mm thick as the substrate.An individual devicebas dimensionson the order of 10-3mand up to 105 devicesmay be processedsimultaneouslywith the number dependingon both device

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sizeand wafer quantity. Resistorsand therrnopiles are forrned atop the silicon wafer by depositing specific substances that are then subsequently shaped with a combination of photolithography and either wet or dry etching. The fabrication stepsfor a membranetype calorimeter are shown, not to scale,in Figure 7.22. In this nanocalorimeter, the membranewith a heater at the centre is the sampleholder. The heat flows laterally through the membraneto the support rim, which is the heat sink, and creates a temperature difference across the membrane that is deterrnined with the thermopile. The membrane is forrned by removing the silicon beneath the therrnopile with a processcalled back etching. The membrane so forrned is about 5 Jlm thick, and comparable with the thickness of the therrnopile. The sensitivity of this calorimeter increases about a factor of 100compared with what it would have by had with a 500Jlm thick membrane. The silicon membrane can be between (5 to

He,ter

Oij:lectric isolation layer

Aluminium

heat sink

11

-~,-

Etch-resistent masking layer Etched cavity

Figure 7.22 Schematicofthe sequence usedto processa membrane-typenanocalorimeter. (a) Starting material silicon wafer 100mm diameter, 0.5 mm thick; (b) lithography: after spinning photo-sensitive resist on the wafer, the resist is exposed,with UV-radiation through a glass mask, and subsequentlydeveloped;(c) silicon thermopile and heating resistor formed after ion implantation again using photolithography with a photoresistant material and mask; (d) fabrication of Sia2 dielectric isolation layer and aluminium interconnection layer {with lithographic methods as shown in (b)}; and (e) a completed nanocalorimeter after etching away silicon underneath the thermal path, and application of an enzyme laver.

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371

50)~m thick with a cross-sectionalarea between (1 and 100)mm2, and becausethe thermal conductivity of silicon is about 150W.K-1.m-l, the thermal resistanceis of the order (10 to 100)K.W-1. The thermopile integrated into the membrane typically bas a sensitivity between(10 to 100)mV.K-I. The heating resistor, which is usually integrated into the middle of the membrane,bas a resistanceof about 1kg and, when supplied with voltages from (1 to 10)mV, caD dissipate between (1 to 100)nW. This particular design is appropriate foT liquids. Many nanocalorimeters caD be fabricated on ODewafer and the individu al devices are separatedfrom each other with a diamond cutting machine. Electrical connections to the MEMS are achieved by ultrasonically welding wires between contact pads and an extemal mount. MEMS usually require packaging that is specific to their end use and standard plastic IC packages are not usually used. Further details on MEMS and their processingmay be found in reference[283]. For gases,where the fluid thermal conductivity K is on the order of 10 times less than the corresponding liquid, for example, K(H2O, 1,373K)jK(H2O, g, 373K) ~ 30, the heat generatedby the resistor tendsto flow into the silicon support rather than the fluid. Increasingthe thermal resistance the membraneincreases sensitivity of the of the device.Typically, this is achievedby replacing the 5 ~m thick silicon membranewith a 0.5 ~m thick non-stoichiometric form of Si3N4,which bas athermal resistance500 times that of silicon. A thermopile of similar thickness caD also be fabricated from poly-silicon. Thesethin membranesare, necessarily, more fragile and sensitivethan the thicker counterpart. A device with the non-stoichiometric silicon nitride membrane,
with an area of 1 mm2, bas an overall sensitivity of 50 V . W-1
at a thermopile

sensitivity of 10mV. K-1 and a membrane thermal resistanceof 5000K. W-I.

Micro-Devices Based 00 Flexible Film

The membrane used in the nanocalorimeter caD also be formed from thin plastic membraneswith low thermal conductivity, such as Mylar. This approach eliminates the Deedfor micro-machining to createthe membrane.The nanocalorimetersformed from polymer films caD be mechanically more robust then the MEMS fabricated device. Muehlbauer et al. [284,285]developeda bio-nanocalorimeter from a thermopile formed from 50 bismuth-antimony junctions, each I ~m thick, evaporated onto a 40 ~m thick Mylar film. This sheetwasbent into a 3 mm diameter cylinder, asshown in Figure 7.23, with the thermopile on the inside. A glucosesensitiveenzymewas placed on the outside ofthe Mylar tube. This arrangementis a calorimetric-basedbiosensor. A thermoelectric generator bas been constructed from bismuth-telluride thermopiles sputtered onto a Kapton foil [286]. When the Kapton foil is folded into a block with linear dimensions of (9.5 by 7.5 by 2.8) mm and aluminium-oxide deposited on bath the top and bottom of the block, a generator was obtained. In this casethe internal resistancewas 1M.Q and an output of approximately 4 V and 16~W obtained when a 20 K temperature difference was applied between the top and bottom of the block. This approach could be used in nanocalorimeters.

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~~~~-E=~~~]
Polyethylene tube Epoxy Rolledmylarwith polyurethane interior faam Solution

Gluco..+O.

Produc'. + heat

~ Heat ~"""" """""" "" ""'" Thennopile~ '" '" '" "",,"-i '" Faam insu!.tion

Mylar

Figure 7.23 TOP: Schematic of the Mylar film based calorimeter for the determination of glucose concentration. BOTTOM: Cross section of the active element of the calorimeter. Mylar-film based thermopile-enzyme glucose sensor fabricated by Muehlbauer et al. [284, 285].

7.4.2

Nanocalorimetersas Sensors

Calorimeters can be used to determine physical properties, including the enthalpy of chemical reaction, biochemical transformation, and the heat capacity of a material, from measurements a temperature difference acrossathermal resistancethrough of which heat flows. The temperature difference is usually determined from measurements of the electrical characteristics of, as examples, thermocouples, resistors, or transistors. Nanocalorimeters are, necessarily, physically small with low intemal heat capacities so that they can be used with small sample volumes maintaining a necessary sensitivity without the calorimeter dominating the thermal process. Nanocalorimeters are usually operated isothermally and have been used for the study of biological systems.

Nanocalorimetry in Biology Bataillard [278] bas reviewed the use of nanocalorimeters in biomedical and biochemical research.ane application reported in reference[278] is the detection of the quantity ofboth glucoseand urea in blood from measurements ofthe enthalpy of an enzymatic reaction. Another important application is the useof nanocalorimeters with living celis, where either the sensitivity of cells to medication or toxins can be determined from measurement of the heat, which can be up to 300pW per cell, produced during metabolism. In addition, the concentration of nutrition in the fluid surrounding the cells can be determined from the metabolic heat output. The measurement of cell metabolism is the nanoscale altemative of the whole-body calorimeter of two centuries ago.

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Muehlbauer el al. [285,286]bas described a nanocalorimeter suitable foT such measurements thai is based on a flexible (Mylar) foil and was shown in Figure 7.23 above. Bataillard [278] used a MEMS, with a silicon membrane, integrated silicon thermopiles, and an enzyme coating thai was placed on the opposite side of the membrane to thai of the thermopiles, to detect constituents in blood and other liquids. Both of these devices could be used foT medical diagnostics techniques including, as examples,the determination in blood of the concentration of glucose, which is significant foT diabetes,urea, penicillin and creatinine, which is an indicator of kidney malfunction. To detect glucoseconcentration the reaction
C6H1206
o~

illr4 C6HI207 + H202

.71)

caD be used, foT which the enthalpy of reaction drH = +79 kJ. mol-I. In Equation (7.71), GOD is an acronym foT glucose-oxidase(P-D-Glucopyranose aerodehydrogenase),an enzymethat catalysesthe glucose-specific reaction and, thus, makes the sensorselective.The sensitivity of the sensorcaDbe enhancedby adding a secondenzyme, catalase,which decomposesthe hydrogen peroxide according to the reaction
H202 -+ H20
lA,

2'--'.

(7.72)

for which L\f~ = + 100kJ mol-I. Thus the total enthalpy of reaction is about 180kJ - mol-I. The nanocalorimeter describedin reference[278] bas a sensitivity of about 50mV - mol-1 - L -I, which gave a minimum glucose concentration detection threshold of20J.lmol- L -I. However, H2O2caDbe formed from reactions other than Equation (7.70) and a systematicerror is introduced when the sensoris coated with bath enzymes. When the calorimeter membrane is coated with the enzyme urease, the concentration of urea caD be determined from the enthalpy of reaction of L\rH = + 61kJ- mol-I, while the concentration of Penicillin G caD be obtained when the membrane is coated with the reagent enzyme /J-lactamasefor which L\rH = + 67kJ- mol-I. Other speciescaDbe detectedwith other membrane coatings [278]. Kidney diseasecaD lead to an increasein creatinine concentration in blood, from 30 J.lmol L -I to 1000J.lmol L -I, and in urine where the normal range is (4 to 18) mmol- L -I and the elevatedconcentration between(40 to 50) mmol- L -I.
0'

These variations of creatinine caD be determined with the enzyme creatinine deiminasecoated onto a nanocalorimeter membrane.The enthalpy of reaction gives a concentration resolution of between(5 to 10)J.lmol-L -1 and a sensitivity of about I mV - mmol-1 - L -I. The advantage of using a calorimetric technique is thai no energy is releasedin the sensorother than by the enzymatic oxidation. Verhaegenel al. [287]have developeda nanocalorimeter matrix of 96, essentially identical, calorimeters on a single l50mm diameter silicon wafer. In ibis calorimeter,
livin~ c~ll~ :JT~ k~nt in :J fT:Jction of th~ ~l~m~nt~ whil~ th~ T~m:Jinci~T :Jct :J~ :J

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Calorimetry

reference. The temperature difference between the living cell filled and reference cavities is determined with a thermopile. The calorimeter is basedon a large 0.61!m thick dielectric membrane formed from silicon oxide and silicon nitride which is coated in a 20 I!m thick layer of rubber to support the otherwise very fragile dielectric membrane. The thermopile consists of 666 p-type polysilicon-aluminium thermocouples with a combined sensitivity of 130mV. K -I. The calorimeter bas a sensitivity ofabout 20V.W-1. With ~107 cells in the cavity, and cell metabolism ofbetween (100 to 300)pW, an output on the order of lOm V bas beenobtained for a single-elementnanocalorimeter [287]. In this apparatus, the effect of medication or other alterations in the cell's environment on the cells metabolism cao be observed from voltage measurements with mV resolution. This sensormay be used for rapid drug screening. Nanocalorimetersas Gas Detector Calorimeters can be used for real-time in situ detection of specific gaseousspecies within a gas mixture, such as detecting potenti al illness based on a chemicals concentration within exhaled breath. Mitrovics el al. bas describeda modular basedsensorsystemfor the detection of gages gasmixtures [288]. In this so called electronic nose,there are elementsbased in on the following methods: quartz crystal microbalance and surface acoustic wave device, foT the determination of variations in mass; metal oxide sensors,for which specific gages alter the electrical resistance; and nanocalorimeters [289]. The nanocalorimeters included in this instrument are shown schematically in Figure 7.22. The membrane in thesecalorimeters is formed from a 6!lm thick, 3.5mm wide and 3.5mm deep silicon membrane. Four of these calorimeters are mounted in a single ceramic pin grid array (PGA) of a standard IC housing. Each nanocalorimeter in the matrix bas a different catalytic or enzymatic coating so that each gives a unique response to specific gases.This method gives a multi-dimensional signal allowing the determination of type and concentration of components in a gas mixture.

7.4.3

NanocalorimetersCorMaterial Properties Determination

The methods usedto fabricate MEMS are particularly useful fot the measurementof the thermal properties of compounds that may be vapour deposited atop, an otherwise essentially identical, nanocalorimeter membranes. The variation of the calorimeters thermal characteristicscaDbe usedto evaluate the thermal properties of additionallayers. The thermal properties of monocrystalline silicon, polycrystalline silicon as weIl as the dielectric layers Si3N4 and SiO2, and conductive layers of Al have been determined with nanocalorimeters [290]. Literature in this field bas been reviewed by von Arx who also reported measurementsof the Seebeckcoefficient [290].

376

Calorimetry

those shown in Figure 7.22 except that a cavity was etched below the polysilicon beam from atop the wafer rather than below. This fabrication technique is knowas surface micromachining, while that used to form the cavity shown in Figure 7.22 is called bulk micromachining. The former can pro vide smaller cavities within the substrate and, becausethe silicon substrate is not completely removed, the device is more robust. However, the fabrication process is somewhat more complicated becauseof the requirements placed on the photolithography alignment and the compatibility of the etching processes with the existing layers. Measurementswith devices have provided the thermal properties, listed in Table 7.3, of materials and layers commonly used in MEMS and nanocalorimeters.

Portable Nanocalorimeters Setarambas developeda portable calorimeter, similar in cross-sectionto that shown in Figure 7.22. It bas a square 10mm by 10mm silicon substrate in which was fabricated 8.5mm by 8.5 mm square membrane 50 J.lm thick [282,291]. This calorimeter bas a sensitivity of ~ 1V' W-1 [292]. A monocrystalline siliconaluminium thermopile with 164 junctions, with an estimated sensitivity of about

ot-junction of Ihennocouple

Ni tbin film
Healer electr

X'

v
Cavity (sample-holder) (300 ~m by 300~by 3~)

n-Si(IOO) subsb11te

Figure 7.25 TOP: Schema of a MEMS nanocalorimeter where the sample holder is tic thermally isolated from its surroundings. A 300~m by 300~m squaremembrane3 ~m thick, is suspendedby a 100~m wide and 3 ~m thick wire above an etched square cavity 2.5mm by 2.5 mmo A heater and gold-nickel thin-film thermocouple are integrated into the membrane [293]. BOlTOM: Cross-sectionalong X-X' showing the pyramidal shape of the cavity over which the membrane is suspended[293].

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100mV' K -I, and a heater are integrated into the membrane. The nanocalorimeter is mounted in standard ceramic PGA housing. Kimura et al. [293] have developed a MEMS nanocalorimeter in which the sample holder (membrane) is almost thermally isolated from its surroundings as shown in Figure 7.25. A 300J.lm by 300J.lm square membrane 3 J.lm thick, is suspendedby a 100J.lmwide and 3 J.lmthick wire above an etched square cavity 2.5mm by 2.5 mmoA heater and gold-nickel thin-film thermocouple, which is usedto measurethe temperature increase,are integrated into th~ membrane.This devicebas a low mass,and is thermally disconnectedfrom the surroundings. Therefore it is very sensitive with a very short time constant of response.Temperature-controlled gas sensorshave also been constructed [294].

Nanocalorimeter Probe tor Thermal Analysis An apparatus basedon atomic force microscopy methods, shown in Figure 7.26,bas been developed to determine the thermal conductivity, thermal diffusivity and heat capacity of a solid by TA Instruments (J.lTA2990Micro-Thermal Analyser) [295]. In this apparatus a probe, with a tip of radius 50nm, is lowered close to the sample surface and moved in the x, y plane while maintaining a fixed di stance z from the surface. These measurements vide the topography of the surface which must be pro flat to better than 10J.lm.In this particular device, a laser is used to determine the vertical displacement.The tip is a Wollaston wire, which is coated platinum that acts as bath the resistive heater and thermometer. This probe can be used to determine the thermal properties of the sample in 30 J.lmby 30 J.lmsquare blocks with a spatial resolution < 1 J.lm,and a total scanning area of (100 by 100)J.lm.Constant probe heating pro vides values of the surface thermal conductivity while ac heating yields thermal diffusivity and heat capacity.

Figure 7.26 Schematicofthe TA Instruments Micro-Thermal Analyser with platinum probe and laser used to determine its vertical disolacement.

Calorimetry 385 276. Hemminger, W. and Hhne, G., Ca/orimetry, Florida: Weinheim, Sect. 1.1.1, 1984. 277. McLean, J.A. and Tobil, G., Anima/ and Human Ca/orimetry, Cambridge University Press,UK, 1987. 278. Bataillard, P., Trends in Ana/. Chem. 12,387, 1993. 279. Sze, S.M., ed., SemiconductorSensors,New York, Wiley and Song, 1994. 280. Meijer, G.C.M. and van Herwaarden, A.W. eds., Therma/ Sensors,Bristol, lap, 1994. 281. van Herwaarden, A.W., Sensorsand Materia/s 8, 373, 1996. 282. van Herwaarden, A.W., Sarro, P.M., Gardner, J.W. and Bataillard, P., Sensorsand Actuators A43, 24, 1994. 283. Madou, M., Fundamenta/s Microfabrication, CRC Press,Baca Raton, 1997. of 284. Muehlbauer, M.J., Guilbeau, E.J. and Towe, B.C., Sensors and Actuators B2, 223, 1990. 285. Muehlbauer, M.J., Guilbeau, E.J. and Towe, B.C., Ana/. Chem. 61, 77, 1989. 286. Stordeur, M. and Stark, I., Proc. I6th Int. Conf on Thermoe/ectrics Dresden,August 2629, 1997, IEEE cat no 97TH8291, p. 575-577, 1997. 287. Verhaegen, K., Baert, K., Simaels, J. and van Driessche, W., Proc. IOth Int. Conf on So/id-State Sensorsand Actuators, June 7-10, 1999, Sendai, Japan, 2P5.4, 1999. 288. Mitrovics, J., Ulmer, H., Weirnar, U. and Gpel, W., Acc. Chem. Res. 31, 307, 1998. 289. Lerchner, J., Seidel, J., Wolf, G. and Weber, E., Sensors~nd Actuators B32, 71, 1996. 290. van Arx, M., Therma/ Properties of CMOS Think Fi/ms, Thesis ETH Zurich, Switzerland, No 12743, 1998. 291. Lerchner, J., Oehmgen, R., Wolf, G., Le Parlouer, P. and Daudon, J.-L., High TempHigh Press 30, 701, 1998. 292. Xensor Integration Data SheetLCM-2524, Xensor Integration, Delft, The Netherlands. 293. Kimura, M., Furuya, M., Aizawa, T. and Hayasak, J., Proc. IOth Int. Conf on So/idState Sensorsand Actuators, June 7-10, 1999, Sendai, Japan, 2P4.17, 1999. 294. Kunt, T.A., McAvoy, T.J., Cavicchi, R.E. and Semancik,S., Sensors and Actuators B53, 24,1998. 295. Lever, T.J. and Price, D.M., American Laboratory 30, 15, 1998.