JP5754693B2 - Calorie sensor, calorific value detection device using the same, and method of manufacturing caloric sensor - Google Patents

Description

  The present invention relates to a heat quantity sensor capable of detecting a temperature change due to heat generated from one cell of a living body, a heat quantity detection device using the same, and a method of manufacturing the heat quantity sensor, for example.

  In recent years, research on biological functions has progressed in the biotechnology field, and the target has shifted from tissues and organs to single cells and single biomolecules. Among these, for example, measurement of a thermal reaction at a single cell level is required. In particular, when performing thermal analysis of a single cell or a single biomolecule, it is necessary to measure the microcaloric value of a microvolume sample.

  Patent Document 1 discloses a biosensor that measures a temperature from a change in oscillation frequency using a crystal resonator temperature sensor.

Conventionally, tabletop calorimeters are commercially available, but the price is as high as 10 million yen or more, the measurement sample is 1 cm ³ , and only 10 ⁶ averaged calorie data is obtained for the number of cells. Absent.

  In recent years, research on elucidation of biological phenomena has been conducted in the biotechnology field, and the demand for nanodevices has also increased. Heat is an important parameter for living organisms, and thermal properties of cells, biomolecules such as proteins and DNA are measured (see Non-Patent Document 1).

  However, these measurements are performed in bulk, that is, in a large number of cell masses. If thermal measurement at the single cell or single biomolecule level is realized, it is expected to be applied to elucidation of biological thermal phenomena through biochemical reactions and monitoring of abnormal cells. For example, brown adipocytes are a representative sample having a heat generation function, and the temperature and heat generation in a bulk state are measured (see Non-Patent Documents 2 to 5). The value is estimated to be about 5 nW per cell, but there is no report that actually measured the calorific value of a single cell.

  On the other hand, various micro calorimetric sensors in which micro heaters and thermopiles are integrated on a thin film using a fine processing technique have been reported (see Non-Patent Documents 6 to 14).

Olson et al. Fabricated a Si chamber with a capacity of 35 nL (1 nL = 1 × 10 ⁻⁹ liter) on a Si ₃ N ₄ substrate and a metal heater that also operates as a temperature sensor, and the sensitivity of melting heat of 52 nL of indium to 3 μW. (Refer nonpatent literature 7).

Johannessen et al. Produced a thermopile and a heater on the upper surface of Si ₃ N ₄ and measured the heat of production when one mouse hepatocyte was hydrolyzed (see Non-Patent Document 8). At this time, a chamber made of polyimide having a capacity of 720 pL (1 pL = 1 × 10 ⁻¹² liters) was used, and the calorimetric resolution was 10 to 25 nW. A thermal analysis system using a microfluidic chip frequently used in the field of micro integrated analysis system (TAS) has also been reported.

Zhang et al. Bonded a substrate having a thermocouple array and a heater on a Si ₃ N ₄ film to a glass microchannel (see Non-Patent Document 11). At this time, the sample volume is increased, but a plurality of fluids can be controlled by providing a plurality of inlets. Taking advantage of this advantage, we measured the heat of catalytic reactions using enzymes.

There is also a report that differential scanning calorimetry was performed by forming a microchannel, a sample chamber, and a reference chamber using polydimethylsiloxane (PDMS) (see Non-Patent Document 14). Considering that the volume of brown adipocytes is about 1.3 fL (1 fL = 1 × 10 ⁻¹⁵ liters) and the calorific value is about 5 nW, further improvement in the capacity and sensitivity of the chamber is necessary.

  In addition, when observing the response due to reagent stimulation, it is necessary to provide a microchannel to facilitate solution replacement. However, a calorimetric sensor using a microfluidic chip has nW order sensitivity and pL order sample capacity. No one has been reported.

Further, in order to perform heat measurement while maintaining the activity of the cell, the cell needs to be in the liquid. However, it has been reported that heat diffusion occurs in the liquid (see Non-Patent Document 15).

The present inventors heated a thin-film bimetallic cantilever type thermal sensor made of Si ₃ N ₄ with a thickness of 175 nm and Au (gold) with a thickness of 50 nm with a focused laser, and measured the displacement in the air. Compared the heat escape in 82% of the heat escape occurred in air relative to the vacuum. It is expected that about 96% of heat escape occurs in the liquid (see Non-Patent Document 16). For this reason, the heat input to the sensor is reduced due to the diffusion of heat, and there is a problem that the measurement is performed with a value smaller than the actual calorific value.

JP-A-6-294863

The Japan Society for Thermal Measurements, “The calorimetric / thermal analysis handbook”, Maruzen, 2010 J. Nedergaard, B. Cannon, O. Lindberg, “Microcalorimetry of isolated mammalian cells”, Nature, Vol.267, p.518-p.520, 1977 N. J. Rothwell and M. J. Stock, “Whither Brown Fat”, Biosciqnce Reports, Vol. 6, p.3-p.18, 1986 D. G. Clark, M. Brinkman and S. D. Neville, “Microcalorimetric measurements of heat production in brown adipocytes from control and cafeteria-fed rats”, Biochemical Journal, Vol.235, p.337-p.342, 1986 K. Inokuma, Y. Ogura-Okamatsu, C. Toda, K. Kimura, H. Yamashita, and M. Saito, “Uncoupling Protein 1 Is Necessary for Norepinephrine-Induced Glucose Utilization in Brown Adipose Tissue”, Diabetes, Vol.54 , p.1386-1391, 2005 K, Verhaegen, K. Baert, J. Simaels, W. V. Driessche, “A high-throughput silicon microphysiometer”, Sensors and Actuators, Vol.82, p.186-p.190, 2000 EA Olson, M. Yu. Efremov, AT Kwan, S. Lai, V, Petrova, F. Schiettekatte, JT Warren, M. Zhang, and LH Allen, “Scanning calorimeter for nanoliter-scale liquid samples”, Applied Physics Letters, Vol. 77, No. 17, p.2671-p.2673, 2000 E. A. Johannessen and J. M. R. Weaver, P. H. Cobbold, J. M. Cooper, “Heat conduction nanocalorimeter for pl-scale single cell measurements”, Applied Physics Letters, Vol. 80, No. 11, p.2029-p.2031, 2002 J. L. Garden, E. Chateau, and J. Chaussy, “Highly sensitive ac nanocalorimeter for microliter-scale liquids or biological samples”, Applied Physics Letters, Vol.84, No.18, p.3597-p.3599, 2004 EB Chancellor, JP Wikswo, and F. Baudenbacher, M. Radparvar and D. Osterman, “Heat conduction calorimeter for massively parallel high throughput measurements with picoliter sample volumes”, Applied Physics Letters, Vol.85, No.12, p.2408 -p.2410, 2004 (11) Y. Zhang, S. Tadigadapa, “Calorimetric biosensors with integrated microfluidic channels”, Biosensors and Bioelectronics, Vol. 19, p.1733-p.1743, 2004 Osamu Nakabeppu, Junichi Sakayoro, “Metabolic fever monitoring of small number of cells with MEMS sensor”, Thermal Science & Engineering., Vol. 14, No.4, p.115-p.120, 2006 KS Suh, HJ Kim, YD Park and KH Kim, “Development and Characterization of a Microcalorimeter Based on a Si-N Membrane for Measuring a Small Specific Heat with Submicro-Joule Precision”, Journal of the Korean Physical Society, Vol. 49, No. 4, p.1370-1378, 2006 M. Toda, N. Inomata, T. Ono, “Bimorph Cantilevers Actuated by Focused Laser from the Side”, IEEJ Transactions on Sensors and Micromachines, in press L. Wang, B. Wang, Q. Lin, “Demonstration of MEMS-based differential scanning calorimetry for determining thermodynamic properties of biomolecules”, Sensors and Actuators B, Vol. 134, p.953-p.958, 2008 M. Toda, T. Ono, F. Liu and I. Voiculescu, “Evaluation of biomaterial cantilever beam for heat sensing at atmospheric pressure”, Review of Science Instruments, Vol. 81, 055104, 2010

  When handling cells or biomolecules, it is necessary to observe temperature measurement or the like in liquid or gas in order to maintain activity. Many conventional thermal analysis devices have a heater or temperature sensor with a thin metal wire on a silicon nitride thin film with a relatively low thermal conductivity, and a microfluidic chip is used to produce a microchamber and a microchannel. It can be seen. However, it requires a large sample volume and is not suitable for measurement of single cells or biomolecules having a volume of about several tens of pL. Even if the cell generates heat in the liquid, it diffuses around it, making accurate calorimetry difficult.

  There is also a conventional device in which only a microchamber is manufactured without using a microfluidic chip. In this device, the sample volume is reduced and the thermal sensitivity is improved, but it cannot be said that the sample volume and the thermal sensitivity are sufficient. Furthermore, since a microchannel is not provided, it is impossible to introduce a reagent necessary for cell stimulation. In addition, since the chamber is an open system, liquid evaporation, contamination, and disturbance are problematic.

  In view of the above problems, the present invention has as its first object to provide a calorific sensor capable of measuring the calorific value from a cell in a liquid with high accuracy while maintaining the activity of the cell. A second object is to provide a calorific value detection device, and a third object is to provide a method of manufacturing a caloric sensor.

  The present inventors have sealed one end of a cantilever-type calorimeter sensor in a vacuum micro-chamber to suppress the escape of heat to the surrounding environment, thereby generating a cell having a volume of less than 1 nL as heat generation from the measurement target in the liquid. The present inventors have realized a heat quantity sensor capable of measuring one heat quantity more accurately and with high sensitivity, and arrived at the present invention.

  In order to achieve the first object, a calorimetric sensor according to the present invention includes a measurement chamber, a side wall disposed adjacent to the measurement chamber, a microchannel disposed adjacent to the side wall, and a measurement chamber. A beam disposed on the side wall and the microchannel, the beam is floated in the measurement chamber, and the microchannel contact portion is disposed in the microchannel, A connecting portion that is disposed on the side wall and connects the measurement portion and the microchannel contact portion, and has a configuration that detects heat generated in the microchannel as a temperature change of the beam. Yes.

In the above configuration, the beams are preferably made of the same material, but may be made of two or more materials. The microchannel preferably has an inlet and an outlet.
A mirror layer is further formed on the surface of the measurement portion of the beam.

  In order to achieve the second object, a heat quantity detection device of the present invention includes the heat quantity sensor and a detection unit that detects a temperature change of the heat quantity sensor.

In the above-described configuration, it preferably has an exhaust part for exhausting the measurement chamber.
Furthermore, it preferably has a vibration part for forcibly vibrating the beam. The vibration unit preferably includes any one of a piezoelectric actuator, an electrostatic attraction actuator, and an optical drive. The vibration unit preferably includes a self-excited oscillation circuit having a beam.
Preferably, the detection unit detects the frequency of the oscillation circuit including a capacitance generated between the heat quantity sensor and an electrode disposed adjacent to the heat quantity sensor. The detection unit preferably detects the resonance frequency of the beam by any one of a laser Doppler vibrometer, a laser interferometer, and an optical lever method.

In order to achieve the third object, a method for manufacturing a calorific sensor according to the present invention includes a measurement chamber, a side wall disposed adjacent to the measurement chamber, and a side wall adjacent to the first and second glass substrates. A step of forming a microchannel to be disposed; and a SOI substrate comprising a Si substrate, a SiO ₂ layer, and an upper Si layer on a surface on which a measurement chamber, a side wall, and a microchannel are formed. A step of bonding the upper Si layer, a step of removing the Si substrate and the SiO ₂ layer of the SOI substrate, a step of patterning the upper Si layer to form a beam for heat conduction, and a second glass substrate. The method includes a step of forming an exhaust port of the measurement chamber and an inlet and an outlet of the microchannel, and a step of joining the first glass substrate on which the beam is disposed and the second glass substrate. Yes.

In the above configuration, the method preferably includes a step of patterning the mirror layer after removing the Si substrate and the SiO ₂ layer of the SOI substrate.

  According to the heat quantity sensor of the present invention, it is possible to provide a low-cost heat quantity sensor with high sensitivity capable of measuring the heat quantity of one cell having a volume of less than 1 nL.

  According to the calorie detector using the calorie sensor of the present invention, it is possible to provide a low-cost calorie detector with high sensitivity capable of measuring the calorie of one cell having a volume of less than 1 nL.

  According to the method for manufacturing a caloric sensor of the present invention, a highly sensitive caloric sensor capable of measuring the calorific value of a cell having a volume of less than 1 nL can be manufactured at a low cost by a so-called micromachine manufacturing method.

It is a perspective view which shows the structure of the calorie | heat amount sensor of this invention. It is an enlarged view of FIG. 1, (a) is a top view, (b) is sectional drawing along the II line | wire of (a). It is a figure which shows the temperature change of the resonant frequency of the beam of the calorie | heat amount sensor of this invention. It is a figure which shows the result of having analyzed the temperature distribution of the cantilever when a measurement chamber is evacuated. It is a figure which shows the result of having analyzed the temperature distribution of the cantilever when a measurement chamber is made into vacuum or water. It is a block diagram which shows the structure of the calorie | heat amount detection apparatus using the calorie | heat amount sensor of this invention. It is a block diagram of the calorific value detection apparatus using a laser Doppler vibrometer. It is a block diagram of the calorie | heat amount detection apparatus using the electrostatic capacitance of a cantilever. It is typical sectional drawing which shows the manufacturing method of a calorie sensor sequentially. It is a figure explaining the manufacturing method when forming the beam of a calorific value sensor with a different material. It is a figure of the scanning electron microscope (SEM) image which shows the 1st glass substrate and cantilever of the manufactured heat quantity sensor. It is a figure of the optical microscope image which shows the produced heat quantity sensor, (a) is an optical microscope image, (b) is sectional drawing along the A-A 'line of (a). It is a figure which shows Q value of a cantilever, (a) is Q figure before introducing a solution into a microchannel, (b) is a figure which shows Q value after introducing a solution into a microchannel. It is a figure which shows the relationship between the width | variety of a side wall, and the calorie | heat amount transmitted to the cantilever by the side of a measurement chamber. It is the figure which investigated the fluctuation | variation of the resonant frequency of a cantilever. It is a figure which shows the temperature change of the brown fat cell before adding norepinephrine. It is a figure which shows a temperature change when the norepinephrine is added and the heat_generation | fever by fat burning is confirmed. It is a figure which shows the temperature change of an inactive (dead) cell by sodium azide.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
(Heat quantity sensor)
FIG. 1 is a perspective view showing the structure of a calorie sensor 1 of the present invention, FIG. 2 is an enlarged view of FIG. 1, (a) is a plan view, and (b) is a line I-I in (a). FIG.
As shown in FIGS. 1 and 2, the calorie sensor 1 of the present invention includes a measurement chamber 2, a side wall 3 disposed adjacent to the measurement chamber 2, and a micro flow disposed adjacent to the side wall 3. The channel 4 and the beam 5 disposed in the measurement chamber 2, the side wall 3, and the microchannel 4 are configured. The beam 5 is also called a micro cantilever or simply a cantilever.
The calorie sensor 1 of the present invention may further include a solution supply unit that flows into the microchannel 4.

  The micro flow path 4 is made of a material such as glass and is a flow path for flowing cells 6 and a solution. The flow path of the micro flow path 4 is, for example, on the order of a width of μm. The microchannel 4 is provided with an inlet 4a and an outlet 4b for a medium made of a solution or gas with the beam 5 interposed therebetween.

  The beam 5 enters the measurement chamber 2 and does not come into contact with the inner wall, that is, the measurement unit 5a arranged to float, and the beam 5 enters the microchannel 4 and floats without contacting the inner wall. The microchannel contact portion 5b and a connecting portion 5c that is disposed on the side wall 3 and connects the measurement portion 5a and the microchannel contact portion 5b. The beam 5 can be made of Si or the like. In the following description, the beam 5 will be described as a Si cantilever unless otherwise specified. The micro-channel contact portion 5b of the beam 5 is provided so that one end of the Si cantilever 5 protrudes into the micro-channel 4 and the other end, that is, the measurement unit 5a of the beam 5 protrudes into the measurement chamber 2 serving as a vacuum microchamber. The microchannel contact portion 5 b is a contact portion s with a medium such as a solution that flows through the microchannel 4. An observation object such as a cell mixed in the medium and flowing in may be placed on the microchannel contact portion 5b and its vicinity. In the heat quantity sensor 1 of the present invention, the heat generated in the micro flow path 4 is detected as a temperature change of the beam.

  In the calorie sensor 1 of the present invention, a side wall 3 serving as a partition is formed on a wall made of glass, for example, between the measurement chamber 2 and the micro flow path 4, and this side wall 3 supports the cantilever sensor 5. The measurement part 5a of the beam 5 disposed on the measurement chamber 2 side serves as a thermal sensor. The beam 5 disposed on the microchannel 4 side is the microchannel contact portion 5 b of the beam 5. Si having a higher thermal conductivity than that of the solution is used as a material for the cantilever 5, and the heat from the cells 6 is transmitted to the measurement unit 5a before diffusing into the surrounding solution.

  The escape of heat can be reduced by reducing the width of the side wall 3. Furthermore, by using a polymer having low thermal conductivity as an adhesive layer between the side wall 3 and the connecting portion 5c of the beam 5, the amount of heat escaping from the cantilever 5 to the side wall 3 made of glass can be reduced.

  The degree of vacuum, that is, the pressure of the measurement chamber 2 can be set to about 1 Pa to 200 Pa, for example. By disposing the measurement part 5a of the beam 5 in a vacuum, the escape of heat to the surroundings can be minimized. The inside of the measurement chamber 2 may be exhausted by an exhaust device using a vacuum pump from an exhaust port 2a provided in the measurement chamber 2 (not shown).

  Each part of the beam 5 may be formed of two or more different materials instead of a single material such as Si as described above. For example, the measurement part 5a and the microchannel contact part 5b may be formed of Si, and the connection part 5c may be formed of metal.

FIG. 3 is a diagram showing a temperature change of the resonance frequency of the beam of the heat quantity sensor 1 of the present invention.
FIG. 3 shows the temperature dependence of the resonance frequency of the cantilever 5 and shows that the resonance frequency decreases as the temperature rises. That is, the resonance frequency of the cantilever 5 of the heat quantity sensor 1 varies with temperature, and the resonance frequency decreases as the temperature increases. In the calorie sensor 1 of the present invention, one end of the cantilever 5 made of Si is enclosed in the measurement chamber 2, so that no heat escapes to the surroundings, and the temperature rise of the cantilever 5 increases, resulting in a large change in resonance frequency. It is done. In addition, by making the measurement chamber 2 vacuum or low pressure, vibration attenuation of the beam 5 by liquid or gas can be remarkably reduced. Thereby, in the calorie | heat amount sensor 1 of this invention, a temperature rise can be measured correctly and with high sensitivity.

(Analysis of heat sensor)
In order to confirm the usefulness of vacuum sealing of the measurement chamber 2, analysis was performed using software (COMSOL Multiphysics, manufactured by COMSOL) using a finite element method.
The cantilever 5 is made of Si and has a width of 30 μm, a length of 150 μm, and a thickness of 1.5 μm. The material of the microchannel 4 was glass. The measurement chamber 2 was 300 μm on a side and 20 μm in depth. In the middle of the Si cantilever 5, there is a region in contact with the side wall 3 made of glass having a width of 30 μm. When the ambient temperature is 25 ° C., a heat amount of 4.5 μW is applied to the microchannel contact portion 5b on the microchannel 4 side filled with water, and the measurement chamber 2 is in a vacuum and when it is filled with water Analysis was performed.

FIG. 4 is a diagram showing the results of analyzing the temperature distribution of the cantilever 5 when the measurement chamber 2 is evacuated. FIG. 5 shows the temperature distribution of the cantilever 5 when the measurement chamber 2 is evacuated and water. It is a figure which shows the result of having analyzed.
When the temperature of the cantilever 5 was compared, it was uniform at 35.7 ° C. when the measurement chamber 2 was vacuum. When the measurement chamber 2 was made water, the temperature decreased toward the tip of the cantilever 5 and was 26.4 ° C. From this result, it can be inferred that heat can be transferred to the measurement unit 5a of the cantilever 5 without being attenuated by setting the measurement chamber 2 to vacuum sealing or low pressure.

  In a laser Doppler vibrometer, which will be described later, the measuring laser is irradiated onto the cantilever 5. At this time, the temperature rises due to irradiation of the measuring laser to the cantilever 5. When measuring the temperature change of the cantilever 5, steady heat inflow does not significantly affect the actual measurement, but care must be taken when the absolute temperature of the cantilever 5 affects the measurement result. When measuring the absolute temperature of the cantilever, a mirror layer 8 (see FIG. 9C) for reflecting light on the measurement chamber 2 side of the cantilever may be provided so that the temperature of the cantilever 5 does not increase. .

(Heat quantity detection device)
Next, the heat quantity detection apparatus 10 using the heat quantity sensor 1 of the present invention will be described.
FIG. 6 is a block diagram showing a configuration of a heat quantity detection device 10 using the heat quantity sensor 1 of the present invention.
As shown in FIG. 6, the heat quantity detection device 10 of the present invention includes a heat quantity sensor 1 and a detection unit 12 of the heat quantity sensor 1.
The heat quantity detection device 10 may include a medium supply unit 14 that supplies a medium to the micro flow path 4 of the heat quantity sensor 1, and may further include an exhaust unit 16 that exhausts the inside of the measurement chamber 2. Moreover, you may provide the pressure control which controls the pressure of the measurement chamber 2. FIG. When supplying the cells 6 and the like to the medium of the microchannel 4, an observation unit 18 for observing the cells 6 and the like is provided. The observation unit 18 can use various microscopes, cameras, monitors using image sensors, and the like.

  The detection unit 12 is provided to detect a temperature change of the measurement unit 5a of the calorie sensor 1. The temperature change can be detected by utilizing the fact that the resonance frequency of the beam 5 of the calorie sensor 1 changes with temperature as described with reference to FIG. Any one of a laser Doppler vibrometer, a laser interferometer and an optical lever method can be used to measure the change in the resonance frequency of the beam 5.

(Laser Doppler Vibrometer)
The laser Doppler vibrometer causes a reference signal and a signal whose frequency is changed from the beam 5 serving as a vibration part to interfere with each other to generate a beat signal. Since the beat signal is modulated by the frequency change of the vibration part, that is, the frequency shift, the frequency shift of the vibration part can be measured.

(Laser interferometer)
The laser interferometer superimposes the signal from the laser beam that serves as the reference signal and the signal from the laser beam that is applied to the beam 5 that serves as the vibrating portion, and compares the phases. By measuring this phase difference, the distance to the object can be measured. The displacement of the beam 5 is obtained from this distance, and the temperature change can be calculated from the change in the frequency based on the displacement of the beam 5. When the laser interferometer is used, measurement is possible even with non-vibration other than the object to be vibrated like the beam 5, that is, temperature change of the object to be vibrated.

(Optical lever method)
The optical lever method is often used as a method for detecting the resonance frequency of the beam 5 due to temperature fluctuation. The displacement caused by the temperature variation of the beam 5 can be measured as a change in the position of the reflected light by the beam 5 of the laser beam irradiated on the measurement unit 5a of the beam 5.

  The detection part 12 is good also as a detection part using the electrostatic capacitance which arises between the beam 5 of the calorie | heat amount sensor 1, and the electrode arrange | positioned adjacent to the beam 5. FIG. The capacitance change may be measured by measuring the capacitance change or detecting the frequency of the oscillator using the capacitance. As such an oscillator, a CR oscillator including a capacitance (C) and resistance generated in the beam 5 and an LC oscillator including a capacitance and inductance (L) generated in the beam 5 can be used. The capacitance changes due to the temperature change of the beam 5, and the capacitance changes, for example, the frequency of the LC oscillator changes. If the temperature change of the inductance is negligible compared with the temperature change of the capacitance, if the relationship between the temperature change of the beam 5 and the change of the oscillation frequency is examined in advance, the temperature of the beam 5 can be determined from the frequency change of the LC oscillator. Can be measured.

  Furthermore, the calorific value detection device may have a vibrating part 22 for forcibly vibrating the beam 5. As the vibration unit 22, any one of a piezoelectric actuator such as a piezoelectric element for forcibly vibrating the beam 5, an electrostatic attraction actuator, and an optical drive can be used. A self-excited oscillation circuit including the beam 5 may be connected to the vibration unit 22.

  Furthermore, a reactant injection unit 24 that injects a reactant such as a reagent that gives a stimulus to a measurement object such as a cell 6 supplied to the microchannel 4 may be provided. In the illustrated case, the reactant injection unit 24 is connected to the medium supply unit 14.

(A calorific value detector using a laser Doppler vibrometer)
FIG. 7 is a block diagram of the calorific value detection device 10 using the laser Doppler vibrometer 28. As shown in this figure, the temperature measuring device 10 using the calorimeter sensor 1 includes a container 26 that houses the calorie sensor 1, a laser Doppler vibrometer 28 disposed on the cantilever 5, and a laser Doppler vibrometer 28. The phase shifter 32 and the phase synchronization circuit 34 (also referred to as a PLL circuit) to be connected are included. A plate made of a piezoelectric material is disposed as the vibrating portion 22 below the heat sensor 1. As the piezoelectric material, a PZT plate 22a can be used. The vibration unit 22 will be described as a PZT plate 22a. The PZT plate 22a is connected to an amplifier 35 with adjustable gain connected to the phase shifter 33. The laser Doppler vibrometer 28 outputs the vibration frequency of the cantilever 5 as a voltage signal. The output of the laser Doppler vibrometer 28 is input to the phase shifter 32 and the PLL circuit 33.

  The output of the laser Doppler vibrometer 28 output to the phase shifter 32 is phase-shifted by 90 degrees by the phase shifter 32 and further gain adjusted by the amplifier 35 so as to have an appropriate voltage amplitude, and input to the PZT plate 22a. Is done. When the PZT plate 22a forcibly vibrates the cantilever 5, the cantilever 5 self-oscillates.

  The PLL circuit 34 includes a phase comparator 36, a low pass filter 37 connected to the phase comparator 36, and a voltage controlled oscillator 38 connected to the low pass filter 37. A part of the output of the low-pass filter 37 is output as a frequency shift signal (dF) 39. The output signal of the low-pass filter 37 is converted into an AC signal by the voltage controlled oscillator 38 and output to the phase comparator 36 as a reference signal 38a.

  According to the calorific value detection apparatus 10 using the laser Doppler vibrometer 28, the temperature change of the resonance frequency of the beam 5 can be detected by the laser Doppler vibrometer 28.

(Amount of heat detection device 10A using the capacitance of the amount of heat sensor)
FIG. 8 is a block diagram showing a calorific value detection device 10 </ b> A using the capacitance 41 of the cantilever 5.
As shown in FIG. 8, the temperature measurement device 10 </ b> A using the heat quantity sensor 1 includes a heat quantity sensor 1, an inductance 42 connected to the cantilever 5, a frequency mixer 43, a local oscillator 44, and an FM demodulator 45. A frequency counter 46, a phase shifter 32 connected to the FM demodulator 45, an amplifier 35 with adjustable gain connected to the phase shifter 32, a PZT plate 22 a disposed below the heat sensor 1, It is comprised including. The amplifier 35 with adjustable gain connected to the phase shifter 32 is connected to the PZT plate 22a. An LC oscillator is constituted by the capacitance 41 and the inductance L42 generated in the cantilever 5 of the calorific value sensor 1. The output signal of the LC oscillator and the output signal of the local oscillator 44 are input to the frequency mixer 43, and the frequency difference between the output signal of the LC oscillator and the output signal of the local oscillator 44 is FM as an intermediate frequency signal (IF signal). It is output to the demodulator 45. The frequency of the IF signal is, for example, about 100 Hz to 10 MHz.

  According to the heat quantity sensor 1 of the present invention, the escape of heat to the surrounding environment can be minimized. Moreover, by making the measurement chamber 2 into a vacuum, the heat can be transmitted to the measurement unit 5a of the beam 5 before the heat escapes to the surrounding environment.

According to the calorific value detection devices 10 and 10A using the caloric sensor 1 of the present invention, the temperature of the solvent is conventionally increased by the heat generation of the cells 6 by using the caloric sensor 1 using the cantilever 5 in order to increase the thermal sensitivity. However, the heat generation can be directly measured by placing the cell 6 in contact with or near the calorific sensor 1.
Moreover, since the calorie sensor 1 includes the micro flow path 4, it is possible to easily replace the solution with the micro flow path 4 and prevent disturbance, contamination, and evaporation of the solution.

(Method of manufacturing calorific sensor)
FIG. 9 is a schematic cross-sectional view sequentially showing a method for manufacturing the heat quantity sensor 1.
As shown in FIG. 9A, first, regions to be the measurement chamber 2, the side wall 3, and the micro flow path 4 are formed on the glass substrate by etching. One set of the glass substrates, that is, the first and second glass substrates 51 and 52 (see FIG. 9E) are prepared.
As shown in FIG. 9B, an SOI substrate 60 is prepared and bonded to the first glass substrate 51 by anodic bonding. In the SOI substrate 60, a sacrificial layer 62 composed of a SiO ₂ layer and an upper Si layer 63 are sequentially laminated on a Si substrate 61. Specifically, for example, an upper Si layer 63 having a thickness of 1.5 μm is formed on the surface of a silicon substrate 41 having a thickness of 200 μm via a sacrificial layer 62 made of SiO ₂ having a thickness of 0.5 μm. Yes. Anodic bonding is performed so that the surface of the upper Si layer 63 of the SOI substrate 60 and the surface of the first glass substrate 51 where the measurement chamber 2 and the microchannel 4 are formed face each other.
Next, as shown in FIG. 9C, the silicon substrate 41 and the sacrificial layer 62 are removed, leaving only the upper Si layer 63. When the mirror layer 8 is formed, the mirror layer 8 is formed on the upper Si layer 63 at this stage. The mirror layer 8 can be formed by depositing a metal thin film such as Cr or Au to be the mirror layer 8 by vapor deposition or sputtering, and then patterning by photolithography.
Next, as shown in FIG. 9D, the upper Si layer 63 is patterned to form the cantilever 5. Further, on the first glass substrate 51, a pattern to be an anodic bonding adhesive layer 66 described later is also formed.
Next, as illustrated in FIG. 9E, the inlet 4 a and the outlet 4 b of the microchannel 4 are formed on one side of the second glass substrate 52. Sand blasting can be used for etching the glass. At this stage, an exhaust port 2a of the measurement chamber may be provided.
Finally, as shown in FIG. 9F, the first glass substrate 51 processed in FIGS. 9B to 9D, and the inlet 4a and the outlet 4b processed in FIG. 9E are formed. The second glass substrate 52 is bonded by anodic bonding. The atmosphere of anodic bonding may be performed by controlling the pressure in the measurement chamber 2. As an example of the conditions for anodic bonding, a temperature of 450 ° C. and a voltage of 500 V can be applied for 10 minutes.
Thus, the heat quantity sensor 1 is manufactured.

(Modified example of manufacturing method of heat quantity sensor)
As a modified example of the manufacturing method of the heat quantity sensor 1, a manufacturing method when the beam 5 is formed of different materials will be described.
FIG. 10 is a diagram for explaining a manufacturing method when the beam 5 of the heat quantity sensor 1 is formed of different materials.
As shown in FIG. 10A, the silicon substrate 41 and the sacrificial layer 62 of the SOI substrate 60 shown in FIG. 9C are removed, and a connection portion 5c is formed in the upper Si layer 63 of the SOI substrate 60. The upper Si layer 63 is etched.
Next, as shown in FIG. 10B, a metal layer 65 to be the connection portion 5 c is deposited on the upper Si layer 63.
Next, as shown in FIG. 10C, the metal layer 65 excluding the connecting portion 5c is removed by etching. Thereby, the connection part 5 c is formed of the metal layer 65. Further, the upper Si layer 63 is patterned to form the measurement part 5a and the micro-channel contact part 5b that become the beams 5 other than the connection part 5c. This step is the same step as FIG.
9E and 9F, the beam 5 including the measurement portion 5a and the microchannel contact portion 52b made of Si and the connecting portion 5c made of the metal layer 65 is performed. Can be manufactured.

(Production of heat quantity sensor)
The calorie sensor 1 was manufactured by the manufacturing method described in FIG. As a material, a glass plate and an SOI substrate 60 were used. The cantilever 5 is made of Si and has a width of 30 μm, a length of 150 μm, and a thickness of 1.5 μm. The measurement chamber 2 was a square having a side of 300 μm and a depth of 20 μm. The microchannel 4 has a width of 100 μm and a depth of 20 μm. The thickness of the side wall 3 between the measurement chamber and the microchannel 4 was changed to 150 μm, 100 μm, 50 μm, 35 μm, 15, 10 μm. Patterning was performed by light exposure with ultraviolet rays, and etching of Si or the like was performed by plasma etching.
FIG. 11 is a scanning electron microscope (SEM) image showing the first glass substrate 51 and the cantilever 5 of the manufactured heat quantity sensor 1. As shown in this figure, it can be seen that the cantilever 5 is formed on the side wall 3 of the first glass substrate 51 by the steps of FIGS.

  12A and 12B are views of an optical microscope image showing the manufactured heat quantity sensor 1. FIG. 12A is an optical microscope image, and FIG. 12B is a cross-sectional view taken along line A-A ′ of FIG. As shown in FIG. 12, the second glass substrate 52 is joined to the first glass substrate 51 by the steps of FIGS. 8E to 8F, and as shown in the side view for explanation, the beam It can be seen that the measurement unit 5 a of 5 is in a floating state in the measurement chamber 2 and the micro-channel contact unit 5 b of the beam 5 is in a floating state in the micro-channel 4. Further, it can be seen that a mirror layer 8 made of Au is formed on the measurement portion 5a of the beam 5.

(Change in Q value when liquid is introduced)
Next, liquid (water) was introduced into the microchannel 4 and the change in the Q value of the cantilever 5 was compared. FIG. 13 is a diagram illustrating the Q value of the cantilever 5, (a) is the Q value before introducing the solution into the microchannel 4, and (b) is the Q value after introducing the solution into the microchannel 4. FIG. The vertical axis represents the amplitude (mm) of the cantilever vibration, and the horizontal axis represents the resonance frequency (kHz).
First, before putting the liquid, the Q value was measured in a vacuum state in both the measurement chamber 2 and the microchannel 4 to be 4200. When water was introduced into the microchannel 4 and the Q value was measured, in the case of the measurement chamber 2, there was no change from 4188.

  There was no change in the Q value of the cantilever 5 on the measurement chamber 2 side before and after the introduction of the liquid. This indicates that the vacuum state is maintained even after the liquid is introduced. The measurement sensitivity of the cantilever 5 depends on the Q value. Even if one side of the cantilever 5 is exposed to a liquid, the sensitivity of the other cantilever 5 can be maintained, suggesting the possibility that the amount of heat of the sample in the liquid can be measured with higher sensitivity.

(Heat conduction through the glass wall of the cantilever)
At this time, the dependency of the width of the side wall 2 made of a glass wall supporting the cantilever 5 was also verified. The escape of heat not only to the Si cantilever 5 but also to the side wall 2 is conceivable.
On the other hand, if heat is transferred preferentially to the Si cantilever 5, the heat transfer to the cantilever 5 does not depend on the width of the side wall 2. The microchannel contact portion 5b was heated with a laser, and the change in the resonant frequency of the cantilever 5 on the measurement chamber 2 side was measured and converted into a temperature change. Since the microchannel contact portion 5b irradiated with the laser also has the cantilever 5 structure, the temperature change was obtained in the same manner. Here, the heat transfer efficiency was defined as (temperature change of cantilever sensor) / (temperature change of microchannel contact portion 5b), and the heat transfer efficiency was evaluated.

FIG. 14 is a diagram showing the relationship between the width of the side wall 3 and the heat transfer efficiency to the cantilever 5 on the measurement chamber 2 side. The horizontal axis of FIG. 14 is the width (μm) of the side wall 3, and the vertical axis is the heat transfer efficiency.
As shown in FIG. 14, the heat transfer efficiency toward the sensor side decreased as the width of the side wall 3 increased. In the heat quantity sensor 1 of the present invention, the maximum value of the heat transfer efficiency was 55%, and the width of the side wall 3 at that time was 10 μm. It is not preferentially transmitted to the Si cantilever 5, and it can be assumed that heat is also escaping from the side wall 3. Although the heat transfer efficiency of 55% is not necessarily a high value, it can be said that the heat transfer efficiency is sufficiently improved considering that 82% of heat diffuses even in the atmosphere.
Further, the efficiency could be maintained when the liquid was allowed to flow through the microchannel 4. As a cause of the heat transfer depending on the side wall 3, the escape of heat to Si of the side wall 3 supporting the cantilever 5 and the adhesive layer therebetween can be considered. By using a polymer having low thermal conductivity as the adhesive layer, the amount of heat escaping from the cantilever 5 to the glass side wall 3 can be reduced.

Further, a liquid consisting of deionized water (DI water) was introduced into the microchannel 4 and the same experiment was performed.
The heat transfer efficiency of the microchannel contact portion 5b was 48%. Compared to heat transfer in vacuum, it is slightly lower. As the cause, the input temperature change was measured in vacuum because the input temperature change in the liquid could not be measured. Further, since the laser could not be focused well in the liquid as compared to the vacuum, it is considered that the amount of heat actually input in the liquid is smaller than the amount of heat input in the vacuum. As described above, the reason why the heat transfer in the liquid is smaller than the heat transfer in the vacuum is highly likely that the actual input heat amount is small, and it cannot be said that the scattering into water is large. Moreover, the value of 48% of the heat transfer efficiency cannot be said to be large as an absolute value, but it can be said that it has been greatly improved considering the value of 94% of the conventional research.

(Verification of effect of mirror layer)
When the resonance frequency of the cantilever 5 was measured using the laser Doppler vibrometer 28, it was confirmed that the temperature was increased by the measuring laser. This is a disturbance when measuring the temperature. It has been found that the mirror layer 28 made of Au is effective in suppressing the temperature rise of the cantilever 5 by the measuring laser. From the above, it can be said that the mirror layer 8 is necessary to accurately measure the temperature by the resonance frequency measurement using the measurement laser.

(Change in resonance frequency of cantilever 5)
FIG. 15 is a diagram in which the variation of the resonance frequency of the measurement unit 5a of the cantilever 5 is examined. In FIG. 15, the horizontal axis represents signal integration time (seconds), the left vertical axis represents frequency variation (× 10 ⁻² ppm), and the right vertical axis represents Allan variance (Hz).
As shown in FIG. 15, in order to evaluate the stability of this device, when the Allan dispersion was determined, the value was 0.05 to 0.20 Hz.
Here, taking the calorific value of brown adipocytes (about 5 nW) as an example, the heat generation for 1 second becomes 5 nJ. From the Si density and specific heat, the amount of heat required to raise the temperature of the microchannel contact portion 5b is about 8 nJ. Assuming that the amount of heat from the cells 6 is all transmitted to the cantilever 5 and that the heat transfer efficiency of 50% can be maintained even when the liquid flows through the microchannel 4, the amount of heat transferred to the cantilever 5 is 4 nJ.
As shown in FIG. 15, on the basis of 0.015 Hz which is the minimum value of Allan dispersion, the thermal resolution on the microchannel contact portion 5b side where the S / N ratio is 1 or more (S / N> 1) is obtained. It was 1.6 mK, and the calorific value was 5.2 pJ. These values are of sufficient resolution to measure the calories of brown adipocytes.

  As a result of measuring the influence of heat transfer by the glass wall serving as the support part of the cantilever 5 from the above measurement results, the heat transfer efficiency was about 50%. When the liquid was allowed to flow to the microchannel 4 side and the change in the Q value of the cantilever 5 was measured, the Q value of the cantilever 5 on the measurement chamber 2 side remained at 4000. It was confirmed that the temperature rise can be improved by providing the mirror layer 8. As described above, the heat generation from the sample in the liquid could be measured by sealing the cantilever-type heat quantity sensor 1 in the measurement chamber 2 with low pressure or vacuum.

(Temperature measurement of brown adipocytes)
Actually, the calorific value from a single brown adipocyte in the liquid was successfully measured. Brown adipocytes are cells that burn their own fat and cause a fever phenomenon by the addition of a reagent (norepinephrine).
FIG. 16 is a diagram showing the temperature change of brown adipocytes before adding norepinephrine. The horizontal axis represents time (minutes), and the vertical axis represents temperature change (° C.). As shown in the figure, in the case of brown adipocytes before adding norepinephrine, a rapid temperature change was observed every few minutes.

FIG. 17 is a diagram showing a temperature change when norepinephrine is added and heat generation due to fat burning is confirmed. The horizontal and vertical axes are the same as in FIG.
As shown in FIG. 17, the exotherm was observed for several minutes and then decreased. That is, an exotherm of about 20 minutes was observed. A sudden temperature change was observed every few minutes before the addition of norepinephrine, and a gradual temperature change was confirmed after the addition of norepinephrine. Since such events were not observed in inactive brown adipocytes, it can be assumed that rapid temperature changes are also part of the biological response.

  The resolution of temperature by the calorific value detection device 10 using the calorific value sensor 1 was 1.6 mK, and a calorific value of 5.2 pJ could be observed. This calorific value corresponds to the calorific value of one cell. A rapid temperature change was confirmed every few minutes before the addition of norepinephrine, and a gradual temperature change was confirmed after the addition. Since such events were not observed in inactive brown adipocytes, it can be assumed that rapid temperature changes are also part of the biological response.

  FIG. 18 is a graph showing the temperature change of cells that are inactive (dead) by sodium azide. The horizontal and vertical axes are the same as in FIG. As shown in FIG. 18, it was found that there was no temperature change in the case of brown adipocytes inactivated by sodium azide.

  As shown in FIG. 17, not only a slow exothermic phenomenon due to fat burning was observed in brown adipocytes added with norepinephrine, but also a rapid exothermic phenomenon was observed in brown adipocytes as shown in FIG. In brown adipocytes inactivated with sodium azide (see FIG. 18), no temperature change as shown in FIG. 16 was observed, so it can be said that this rapid exothermic phenomenon is also part of life activity.

  From the above results, since the temperature of the solvent heated by the exotherm of the cells was conventionally measured, the time constant was not high. It is possible. In addition, the use of the microchannel 4 has solved the problems of liquid evaporation, contamination, and disturbance, which are problems in the open system.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these are also included in the scope of the present invention. .

1: Calorie sensor 2: Measurement chamber 2a: Exhaust port 3: Side wall 4: Microchannel 4a: Inlet 4b: Outlet 5: Beam 5a: Measuring unit 5b: Microchannel contact unit 5c: Connection unit 6: Cell 8: Mirror Layer 10, 10A: Calorific value detection device 12: Detection unit 14: Medium supply unit 16: Exhaust unit 18: Observation unit 22: Vibration unit 22a: PZT plate 24: Reactant injection unit 26: Container 28: Laser Doppler vibrometer 32: Phase shifter 33: Phase synchronization circuit 35: Amplifier 36: Phase comparator 37: Low pass filter 38: Voltage controlled oscillator 38a: Reference signal 41: Capacitance 42: Inductance 43: Frequency mixer 44: Local oscillator 45: FM demodulator 46: frequency counter 51: first glass substrate 52: second glass substrate 60: SOI substrate 61: Si substrate 62: sacrificial layer 63: upper Si layer 65: metal layer 6 : Adhesive layer

Claims (11)

A measurement chamber, a sidewall disposed adjacent to the measurement chamber, a microchannel disposed adjacent to the sidewall, a beam disposed in the measurement chamber, the sidewall, and the microchannel And including
The beam includes a measurement unit that is suspended in the measurement chamber, a microchannel contact unit that is suspended in the microchannel, and the measurement unit and the microstream that are disposed on the side wall. A connecting portion that connects the road contact portion,
A heat quantity sensor that detects heat generated in the microchannel as a temperature change of a beam disposed in the measurement chamber.   The heat sensor according to claim 1, wherein the beams are made of the same material or two or more different materials.   The heat sensor according to claim 1, wherein the microchannel has an inlet and an outlet.   The heat sensor according to claim 1, wherein a mirror layer is further formed on a surface of the measurement portion of the beam. A calorie sensor according to any one of claims 1 to 4,
A detection unit for detecting a temperature change of the heat quantity sensor;
A calorific value detection device using a caloric sensor.   The calorific value detection apparatus using the calorific value sensor according to claim 5, further comprising an exhaust part that exhausts the measurement chamber.   The heat quantity sensor according to claim 5, further comprising: a vibration part for forcibly vibrating the beam, wherein the vibration part includes any one of a piezoelectric actuator, an electrostatic attraction actuator, and an optical drive. Calorific value detection device using   The calorific value detection device using the calorific value sensor according to claim 7, wherein the vibration unit includes a self-excited oscillation circuit having the beam.   The detection unit detects a resonance frequency of the beam by any of a laser Doppler vibrometer, a laser interferometer, and an optical lever method, or between the heat sensor and an electrode disposed adjacent to the heat sensor. The calorific value detection device using the calorific value sensor according to claim 5, wherein the frequency of an oscillation circuit including an electrostatic capacitance generated therebetween is detected. Forming a measurement chamber, a side wall disposed adjacent to the measurement chamber, and a microchannel disposed adjacent to the side wall on the first and second glass substrates;
Bonding the upper Si layer of the SOI substrate comprising the Si substrate, the SiO ₂ layer, and the upper Si layer on the surface of the first glass substrate on which the measurement chamber, the side wall, and the microchannel are formed;
Removing the Si substrate and the SiO ₂ layer of the SOI substrate;
Patterning the upper Si layer to form a beam composed of a measurement part, a microchannel contact part and a connection part;
Forming an inlet and an outlet of the microchannel on the second glass substrate;
Bonding the first glass substrate on which the beam is disposed and the second glass substrate;
A method for manufacturing a calorific sensor. The manufacturing method of the heat quantity sensor according to claim 10, further comprising a step of patterning a mirror layer after removing the Si substrate and the SiO ₂ layer of the SOI substrate.