JP2009002677A - Surface acoustic wave device biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a surface acoustic wave device biosensor capable of detecting a process of propagation-growth-injury repairing of a cell, which is observed conventionally only by an optical microscope, by measuring it as a surface acoustic wave (SAW) propagation characteristic due to physical and chemical changes of the cell formed on an SAW propagation route, using an SAW generated on a piezoelectric substrate. <P>SOLUTION: A high-frequency current or voltage is applied/impressed to an IDT 23 joined to the piezoelectric substrate 22, the SAW 25 is generated in the vicinity of a surface of the piezoelectric substrate 22, and the physical and chemical changes of the cell of the SAW are detected based on the propagation characteristic of the SAW 25, in this SAW device biosensor 21 of the present invention. In this SAW device biosensor 21, SiO<SB>2</SB>is film-formed onto the IDT 23, a pseudo-matrix 27 is coating-treated thereafter on the piezoelectric substrate 22, the SAW propagation route 24 comprising a culture cell 28 is formed to be constituted into dual channels, a change caused by a temperature and a solution mass is corrected in one of the channels, and the physical and chemical changes of the cell are detected in the other one channel. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention provides a living body generated by performing pseudo-matrix processing on a propagation path of a surface acoustic wave, seeding and culturing cells, and loading various elements on an artificial tissue constructed on the propagation path. Focusing on surface acoustic wave propagation characteristics, various physical and chemical changes related to health effects and health effects are related to surface acoustic wave device biosensors with a non-powered and wireless characteristic that can be measured with high sensitivity .

  The ultrasonic waves currently used in the engineering and medical fields are mainly elastic waves (bulk waves) that propagate in water or solids. On the other hand, surface acoustic waves (SAW) that propagate by concentrating energy near the surface of an elastic body have recently been utilized as elements for electronic / communication equipment and have played an important role in the progress of this direction.

  One of the related technologies that are the basis for the development of functions of devices using such SAW is the piezoelectric effect. This piezoelectric effect is generally referred to as a piezoelectric effect, where a charge or a stress is induced when a strain or stress is applied (forward effect), and a strain or stress is generated when a voltage is applied (reverse effect). In particular, the forward effect was discovered by Curie brothers (J. Curie, P. Curie, 1880) for tourmaline, and the reverse effect was found the following year by G. Lippmann. Whether or not the crystal exhibits a piezoelectric effect is determined by the point group symmetry of the crystal, and among the 32 crystal groups, the crystal effect is the 20 crystal group.

Electromechanical transducers using the piezoelectric effect are the mainstream of current ultrasonic application parts. Piezoelectric elements include single crystals such as quartz, Rochelle salt (calcium sodium tartrate), LiTaO ₃ , LiNbO _3, etc., and polycrystals such as barium titanate (BaTiO ₃ ), lead zirconate titanate (PbZrO ₃ , PbTiO ₃ ), niobic acid Typical examples are salt. The piezoelectric vibrator is one elastic vibration body configured by using a piezoelectric body having electrodes as a whole or a part thereof, and has a function of electrically exciting and detecting its own elastic vibration by piezoelectric conversion.

  Table 1 shows the piezoelectric basic formula described above. Piezoelectricity is a linear interaction between electrical and mechanical systems, and is a type of energy conversion. In that type of classification, of the transformations that occur through changes in physical variables, it enters a quasi-static transformation in which the changes occur slowly (in a form close to thermal equilibrium). The physical variables are an electric field E and an electric flux density D (or polarization P) in an electrical system, and a stress T and a strain S in a mechanical system (dynamic system). In dealing with this interaction, it is necessary to distinguish between strong (inclusive) variables and explanatory (external) variables. Here, E and T are the former, and D (or P) and S are the latter.

  A surface acoustic wave is a sound wave in which energy concentrates and propagates only near the surface of a medium, and its wave phenomenon was theoretically derived in 1885 by Lord Rayleigh in the United Kingdom as a wave traveling on a semi-infinite solid surface. . There are various types of surface acoustic waves, but here we describe typical Rayleigh waves and SH waves (BGS waves).

  Rayleigh waves are surface acoustic waves discovered by Lord Rayleigh in 1885. This surface acoustic wave propagates along the free surface of the semi-infinite elastic body (the surface of a sufficiently thick plate). Wave energy is concentrated near the surface, and more than 90% is contained within one wavelength from the surface. In the case of a non-piezoelectric medium, the displacement part has only the wave traveling direction and the depth direction, and the phase difference between the two components is 90 °, so each point draws an elliptical orbit. The Rayleigh wave is always constant regardless of the frequency. Such constant velocity is said to be nondispersive, which is a major feature of Rayleigh waves. As in the case of an isotropic body, a Rayleigh wave propagates to the surface of a piezoelectric medium that is an anisotropic medium, but generally has all displacement components. However, it is the same that speed does not have frequency characteristics.

  SH waves (BGS waves) are surface acoustic waves discovered by Bleustein-Gulyaev-Shimizu in 1969. In non-piezoelectric media, surface acoustic waves (SH waves) do not exist in the direction perpendicular to the traveling direction, but since piezoelectric media are anisotropic, pure transverse waves that propagate energy concentrated on the surface are not present. Exists. As a feature of this wave, there is no speed dispersion like the Rayleigh wave, and the higher the electromechanical coupling coefficient, the higher the energy concentration on the surface. The SH wave exists on a substrate having a plane parallel to the c-axis or the polarization axis of hexagonal 6 mm piezoelectric crystal or piezoelectric ceramic. Also, since the particle displacement direction is parallel to the substrate surface (complete shear surface wave), the surface acoustic wave attenuation is relatively small even in moisture / viscous liquids, liquid identification, water measurement, evaluation of two mixed solutions, Application in liquid such as an automatic liquid measuring device is conceivable. FIG. 13 schematically shows the propagation pattern of the Rayleigh wave having the rotational component distribution in the depth direction on the surface, and FIG. 14 schematically shows the state of the SH wave (Shear-Horizontal) displacement distribution. This SH wave becomes a deformation mode with only horizontal and shear components, and can be propagated even in a moisture-humidity substance, so that the state of biological fluid and cell body can be detected.

The propagation characteristics of surface acoustic waves propagating through a piezoelectric substrate include values such as propagation velocity v, electromechanical coupling coefficient K ² , delay time temperature coefficient TCD, and power flow angle PFA. The characteristics of a SAW device using surface acoustic waves largely depend on the piezoelectric substrate used. Therefore, the characteristics required for a better piezoelectric substrate are listed as follows.
(1) The electromechanical coupling coefficient (K ² ) is large.
(2) Good temperature characteristics (TCD).
(3) The spurious response is small.
(4) The power flow angle (PFA) is zero.
(5) The propagation loss is small.

  The electromechanical coupling coefficient is a value indicating the conversion efficiency from electric energy to surface wave energy. TCD indicates the coefficient of variation of surface wave velocity or delay time with temperature. The spurious response is a phenomenon in which the attenuation amount deteriorates due to the unnecessary vibration mode. The power flow angle is an angle representing the difference between the direction of the phase velocity that propagates and the direction of the group velocity when surface acoustic waves are excited in the comb-shaped electrode. If the propagation velocity v is high, it is advantageous for high frequencies, and if it is slow, it is advantageous for delay lines.

  Devices that apply the piezoelectric effect and surface acoustic wave (SAW devices) contribute to technological innovation of devices used in mobile communication terminals, along with the rapid expansion of the mobile communication market represented by recent mobile phones. is doing. Such a SAW device is regarded as one of key parts for realizing miniaturization and high functionality of mobile communication terminals, and is mainly used as a band pass filter for RF and IF stages, and is manufactured by a photolithography process. Therefore, microfabrication is possible, and now it is put into practical use even in the several GHz band.

  FIG. 15A shows a schematic diagram of the SAW device 1. In this SAW device 1, a piezoelectric substrate 3 is used to excite and receive the surface acoustic wave 4. The piezoelectric substrate 3 is required to have a smoothness of the order of μm, which is the wavelength of the elastic wave, and a reproducibility of a frequency of several hundred ppm or less. A piezoelectric thin film formed in the above is used. As a means for exciting the surface acoustic wave 4, a comb-shaped electrode (IDT) formed with a predetermined pitch 5 intersecting plus (+) and minus (−) as shown in FIG. 2 is used. By using the frequency characteristic peculiar to this IDT2, a filter or a resonator can be configured.

  The SAW filter constituted by the SAW device 1 is generally constituted by the piezoelectric substrate 3 and an IDT 2 for transmission / reception, and the center frequency f is determined by the acoustic velocity of the surface acoustic wave v and the pitch 5 of the IDT 2. If the wavelength is λ, it can be expressed as f = v / λ. Therefore, an arbitrary frequency band can be taken out by designing so that this center frequency band is excited most. At this time, in order to obtain an arbitrary frequency with higher accuracy, several stages of filters are used. FIG. 16 shows a configuration example of a reception block of a mobile phone using the SAW filter. The radio wave taken in from the antenna 6 passes through the first-stage SAW filter 7a, and then drops the frequency through the amplifier 8 and the first-stage mixer 9a. To the baseband unit 11. Reference numeral 14 is a VCO, 12 is a PLL synthesizer, 15 is a TCXO, and 13 is a local oscillator. Since the SAW filters 7a and 7b cover a frequency range of several tens of MHz to several GHz, for example, 1.5 GHz digital mobile phones are used as first-stage IF filters of RF 1.5 GHz and 130 MHz.

FIG. 17 is a conceptual diagram showing stress on the SAW propagation path when cells extend and move on the SAW device. Here, (a) is an extension / contraction force immediately before cell movement, and (b) is a cell 17 extension / contraction force that generates a stress 17 in the SAW propagation path via cell-matrix bond after cell movement. It is the conceptual diagram which showed a mode to make it do. A stress 17 is applied to the surface of the SAW propagation path substrate of the adhesion spot 19 (pointing to a minute adhesion region) via the adhesion receptor 18 on the basal plane of the cell 16 (pointing to the side of the cell membrane facing the culture substrate). Arise. The cells 16 are extended and moved using the extension / contraction force 20 at that time.
Multi-functional Surface Acoustic Wave Sensor for Monitoring Environmental and Structural Condition, Y.Furuya, T. Kon T. Okazaki, Y. Saigusa and T.Nomura, On-line Proc.SPIE.vol.6170, 61700Q (2006) Q1-Q11 Furuya, Okazaki, Nomura, Ima, Saegusa, Metal 76-12 (2006) pp.1286-1291 T.Nomura, A.Saitoh, Wireless acoustic wave sensor system, 2002 Marc D. Schlensog, Thomas MA Gronewold, Michael Tewes, Michael Famulok, Eckhard Quandt: BIOSENSOR (2003) Katsuri Mochi, National Institute for Environmental Studies, WO2004 / 085606 Ken Imadai, Hirosaki University Graduate School Master's thesis (2006) Shibayama Inui, Acoustic Wave Element Technology Handbook, 1991

  As conventional biocell sensing / evaluation techniques, in many cases, using optical observation such as phase difference or fluorescence microscope, observation with transmission or scanning electron microscope, biochemical and molecular biological techniques, We have observed changes in cell growth, necrosis, morphology, biochemical characteristics, etc., against drugs, toxic substances, and environmental factors, and have detected and evaluated the effects from these changes. However, these techniques not only require time to obtain a conclusion from the preparation of the observation sample, but also tend to increase human labor and cost. In addition, it is not efficient to evaluate a large number of samples quickly and inexpensively. In addition, physical stresses are measured from the physical point of view, such as stress that occurs in the substrate as cells expand and contract during cell growth and migration, cell-cell or cell-substrate binding force, and the internal structure of organelles. At present, there are almost no research examples that have attempted.

  Cell sensing / evaluation techniques are often performed while culturing cells in a medium (a nutrient solution necessary for cell growth). However, when Rayleigh waves are selected as surface acoustic waves, the displacement is in the depth direction ( It is considered that the surface acoustic wave is greatly attenuated in the solution because it has a (longitudinal wave) component. Therefore, it is necessary to select a surface wave that does not attenuate the surface acoustic wave.

  The material of the piezoelectric substrate tends to react sensitively to changes in temperature due to the temperature coefficient of the piezoelectric material itself. However, when seeding and culturing cells on the surface acoustic wave propagation path and measuring the surface acoustic wave, the temperature that affects the surface acoustic wave, even if it is kept constant, is kept constant. It is assumed that changes will occur. It is necessary to consider the signal change due to this temperature change.

  In culturing cells on the propagation path of such surface acoustic waves, when cells are seeded and cultured directly on the surface of the device, between the cells and the piezoelectric substrate due to the piezoelectric substrate not being the original adhesion substrate for the cells. Due to instability of adhesion and contamination of the piezoelectric substrate, which may be toxic to the cells, the cells cannot be stably cultured, and the surface acoustic wave measurement accuracy may not be ensured. Therefore, in order to measure with high sensitivity with reliability, it is necessary to establish a technique for stably improving the adhesion between the cell and the piezoelectric substrate.

  According to the present invention, in a surface acoustic wave device that propagates energy concentrated near the surface of a piezoelectric material, an artificial tissue constructed by seeding and culturing cells on the propagation path of the surface acoustic wave is a drug, a toxic substance, or an environment. Surface acoustic wave propagation is caused by the addition of chemical substances and mechanical damage, and changes caused by stress due to cell expansion and contraction, cell-cell or cell-substrate binding force, and mechanical structures that support organelles. The present invention provides a surface acoustic wave device biosensor that can be measured with high sensitivity by paying attention to a change in characteristics.

  In order to solve the above-described problems, the surface acoustic wave device biosensor of the present invention causes a high-frequency current to flow through the IDT 2 bonded to the piezoelectric substrate 3 to generate SAW 4 on the piezoelectric substrate 3, and the propagation characteristics of the SAW 4 It is characterized by detecting physical and chemical changes in cells such as stress due to cell expansion and contraction, cell-cell or cell-substrate binding force, mechanical structure supporting intracellular organelles, and activity state. To do.

In the surface acoustic wave device biosensor of the present invention, the propagating surface acoustic wave component is a completely horizontal shear component (shear-horizontal) with low propagation loss in an aqueous solution.
In the process of repairing or recovering damage or biological effects caused to the artificial tissue fabricated on the piezoelectric substrate, stress caused by extension or contraction in the growth or movement of cells, cell-cell or cell -It is characterized in that the detection sensitivity of the surface acoustic wave propagation characteristics is improved so as to detect from the phenotypic changes such as the binding force between substrates, the mechanical structure supporting the organelles, and the activity state.

  By making the element (device) made of IDT bonded to the piezoelectric substrate into two channels, the change in the received signal due to temperature and solution mass is corrected for one channel, and the physical and chemical changes caused by cells in the other channel are corrected. The detection sensitivity is improved by extracting the signal reception simultaneously.

  In addition, after performing pseudo-matrix treatment (Katsumi Mochiri, National Institute for Environmental Studies, WO2004 / 085606) to fix and grow cells on the propagation path of the surface acoustic wave device biosensor of the present invention. By seeding and culturing the cells, the adhesion between the cells and the piezoelectric substrate is enhanced, the measurement sensitivity to the cells is improved, and the detection is performed.

  The surface acoustic wave propagation path is placed in a single device with single or multi-channel IDTs facing the planar X coordinate axis, and cells are seeded and cultured on the surface acoustic wave propagation path to create drugs and toxic substances. Alternatively, it is possible to evaluate in real time the degree of cellular activity comprising a shear horizontal surface acoustic wave device that detects changes in cell activity, damage repair and traits due to environmental chemicals, etc., and detects signal changes in each channel. .

  Furthermore, the device is arranged in multiple channels for a planar XY coordinate axis, and from the change in surface acoustic wave signal between the IDT channels facing each other in the X and Y coordinates, cell activity, injury repair, and trait Changes are visualized on an XY screen by image processing, and the influence of environmental factors on cell activity can be visually and quickly evaluated.

  In order to establish and grow cells in the propagation path of the surface acoustic wave, a pseudo-matrix process (Katsu Tatechi, National Institute for Environmental Studies, WO2004 / 085606) is applied, and SH-SAW made of a complete horizontal shear component is applied. It can be used for measurement in culture medium, and can be stressed by stretching and contraction in cell migration and proliferation in the living state, cell-cell or cell-substrate binding force, and mechanical support for organelles. Changes in structure, activity state, etc. can be measured and evaluated using surface acoustic waves.

  Designed to enable parameter sensing of multiple IDTs installed on the device. In addition, after constructing an artificial tissue by seeding and culturing cells on the propagation path of surface acoustic waves in a measurement channel that has been processed with a pseudo matrix, mechanical damage, biological effects or health effects due to drugs on the artificial tissue give. By capturing the change in SH-SAW at that time, it becomes possible to simultaneously measure the influence on the cells cultured on the piezoelectric substrate or the formed artificial tissue due to a plurality of factors.

  By adopting a wireless and non-powered system, measurement can be automated and simplified, such as measuring changes to cells / tissues caused by drugs, chemicals, and environmental factors while the device is in the culture device. . It also contributes to downsizing of the entire device. As a result, biological effects and health effects, and in general, effects on cells and tissues in the fields of toxicology and pharmacology can be remotely sensed with high sensitivity. Technical contributions can be realized by researching and developing fundamental technologies and developing them into systems that will be incorporated into future advanced analysis and measurement equipment.

  SAW devices composed of piezoelectric substrates and comb electrodes can be miniaturized by photolithography, and products have been developed as communication devices such as television receivers and mobile phones as amplifiers using resonance phenomena. It was. Recently, research on applicability to the field of sensor technology has been conducted, and bio-related research has also been conducted. However, there are almost no research examples in the field of cell biology.

  In the present invention, paying attention to changes in the surface acoustic wave (SAW) propagation characteristics, cells are cultured on the propagation path by the SAW, and the influence on cells and tissues due to various factors such as pharmacological, toxicological and health effects. Can be developed as a basic technology for a small artificial tissue SAW sensing system that can be measured mainly from a mechanical point of view. FIG. 1 is a schematic configuration diagram of a SAW device biosensor (hereinafter referred to as a SAW device sensor) using an artificial tissue of the present invention.

The SAW device sensor 21 includes a comb-shaped electrode (IDT) 23 formed on a piezoelectric substrate 22, and a high-frequency current or a high-frequency voltage is applied to the IDT 23 from the outside, whereby surface acoustic waves ( SAW) 25. Further, on the propagation path (hereinafter referred to as SAW propagation path) 24 by the SAW 25, the cells are cultured after the pseudo matrix 27 is processed. The pseudo-matrix 27 plays a role of enhancing the adhesion between the cultured cells 28 and the piezoelectric substrate 22 and is biochemical by drugs, environmental chemical substances (toxic substances such as heavy metals, oxidizing substances, chlorinated organic compounds, and environmental hormones). It is possible to detect (sensing) the influence of a plurality of external factors, such as an injury caused by a mechanical change or mechanical damage and a change in stress applied to the piezoelectric substrate 22 in the repair process. Reference numeral 26 denotes a SiO ₂ thin film covering the IDT 23.

  Also, by using one channel for reference, measurement errors due to minute temperature changes during culture can be reduced. The SAW device sensor 21 according to the present embodiment includes two channels of the SAW propagation path 24. However, the SAW device sensor 21 is not limited to this. By increasing the number of channels, it is possible to realize high-precision measurement with one chip. It is.

  Since the SAW device sensor 21 is manufactured by a photolithography process, the SAW device sensor 21 is easily reduced in size and is inexpensive, and the frequency characteristic thereof is determined by the interval between the linear electrodes of the IDT 23. For this reason, the frequency becomes higher with the development of microfabrication technology, and more advanced sensing with a biosensor or the like becomes possible.

In the design and trial manufacture of the SAW device sensor 21, a LiTaO ₃ (42 ° YX cut) piezoelectric substrate 22 having dimensions of 8 mm × 10 mm was used. Since it is necessary to measure in solution, the piezoelectric substrate 22 having the SH mode with little propagation loss in water was selected. As shown in Table 2, Au was used for the IDT 23 formed on the piezoelectric substrate 22 and was manufactured by photolithography so that P (pitch) was 10 μm, the crossing width was 3 mm, and the propagation distance was 4 mm. When the center frequency was measured with an oscilloscope, it was 108 MHz. FIG. 2A shows the shape of the SAW device sensor 21 actually designed and the dimensions of each part. FIG. 2B shows a 2-channel SAW device sensor in which the SAW device sensor 21 is mounted on two channels. This is an overview of

Since the entire SAW device sensor 21 was immersed in the solution at the time of measurement, the IDT 23 and the wiring part were insulated. The IDT 23 was covered with a SiO ₂ thin film, and the other portions were insulated using PDMS, which is a silicone resin. Further, as a method of forming a SiO ₂ thin film on the IDT 23, a magnetron sputtering method was used, and the substrate temperature was 40 ° C., the degree of vacuum was 1.5 × 10 ⁻⁴ Pa, and the argon pressure was 4.0 Pa. The thickness of the thin film was set to 400 nm. FIG. 3 shows an apparatus configuration by the magnetron sputtering method. In this magnetron sputtering method, the chamber 38 is first evacuated by the vacuum pump 37. Next, a leakage magnetic field connecting the center and the outer periphery of the target 35 is generated, a high voltage is applied to an inert gas such as the argon gas 31 using a high frequency power source 36, and glow discharge is generated, whereby ionized argon is generated. The gas 31 is made to collide with the target 35 and a part thereof is blown off. At that time, by operating the shutter 34 in the chamber 38, a thin film having the composition of the target 35 can be obtained on the substrate 33. At this time, the thin film can be formed at an arbitrary temperature by using the heater 32. The PDMS was coated by coating the entire area other than the propagation part, and was cured by keeping the temperature for about 1 hour in a high-temperature bath set at 80 ° C. PDMS was used after confirming that it was not toxic to cells.

  In the connection method, etching was performed on a polymer substrate on which a copper foil was attached, and the copper foil was formed into an arbitrary shape. Next, the prototype SAW device sensor 21 is fixed to the center of the polymer substrate, and the copper foil on the polymer substrate and the SAW device sensor 21 are wired with a copper tape so as to improve the flow of electricity at each joint. A conductive adhesive (doutite) was applied.

  In the present invention, instead of directly culturing cells on the piezoelectric substrate 22, a pseudo-matrix coating treatment was performed to improve cell adhesion. As a technique for culturing cells on the surface of this SAW device sensor, a technique developed by Dr. Kurichi of the National Institute for Environmental Studies was used (Katsumi Tatechi, National Institute for Environmental Studies, WO2004 / 085606). By using this technique, the adhesiveness of cells in the SAW propagation path is enhanced, and changes due to cells can be captured with high sensitivity as changes in SAW.

  The circuit used for the measurement is shown in FIG. A tone burst wave 44 as an input signal is obtained by multiplying the continuous wave from the reference signal generator 42 by the signal from the pulse generator 41 using the mixer 43. At this time, the frequency of the continuous wave from the reference signal generator 42 is 108 MHz which is the center frequency. A part of the continuous wave from the reference signal generator 42 was input to the digital oscilloscope 48 as a reference wave 47. The reception unit measures the phase difference between the output signal from the sensor device 45 that has passed through the amplifier 46 and the reference wave, thereby measuring the change in the propagation speed of the SAW.

  FIG. 5 is a conceptual diagram when a defective portion 29 is generated in the cultured cell 28 on the SAW device sensor 21 shown in FIG. 1, and FIG. 6 shows a procedure for measuring the progress of the cultured cell 28. It is explanatory drawing. As shown in FIG. 6, the progress state of the cultured cell 28 is measured by a measurement waveform 51 based on a time axis and an intensity axis. Next, the measurement waveform 51 is imaged 53 by the data analysis / image display unit 52. According to this imaging 53, it is possible to confirm as an image how the contraction force 54 as shown by the arrow acts as well as the position and size of the wound 30 in the cultured cell 28.

Hereinafter, conditions for measuring the cell culture state by the SAW device sensor 21 will be described. Type 2 alveolar epithelial cells (hereinafter abbreviated as T2 cells) (Reference: Clement et al.
SV40T-immortalized lung alveolar epithelial cells. Exp. Cell Res.
196: 198-205, 1991), in order to determine what kind of change occurs depending on the presence or absence, it was measured before culturing, after 1 day of culturing (after 24 hours), and after 2 days of culturing (after 48 hours). Further, in order to see the change in SAW due to the difference in cell culture state, T2 cells cultured on the SAW propagation surface were subjected to mechanical damage or chemical damage and measured.

  As a mechanical damage, using a plastic rod with a narrowed tip, a scratch was linearly formed with a width of about 500 μm perpendicular to the SAW propagation direction, and the change in SAW was measured. As chemical damage, treatment with the calcium chelating agent EDTA was performed, and changes in SAW due to reduction of binding between T2 cells were measured.

(Measurement of cell presence by cell culture)
FIG. 7 shows changes in SAW delay time before and after cell culture. In this graph, the change from the reference is shown with reference to the delay time before cell culture. The result shows that the delay time is decreased after the culture and the propagation speed is increased. From the relational expression (Equation 1) between the elastic modulus and density of sound waves, when E and ρ of water and cells are compared, E is higher in cells than water, whereas ρ is water: 1, cells: 1. There is almost no difference at 01. Thereby, it is considered that only the change in elastic modulus due to the culture of the cells acted.

  In addition, it was considered that E increased and the change increased because the planar density of cells and the degree of binding increased on the first and second days of culture. Images were taken with a reflection differential interference microscope to confirm that the cells were cultured. FIG. 8 shows photographs before (a) and after (b) culturing of cells on SAW. While there was nothing on the piezoelectric substrate before culture, it was confirmed that cells were growing on the entire piezoelectric substrate after culture.

  T2 cells were cultured on a piezoelectric substrate that had been subjected to a pseudo-matrix treatment. After the cells developed and grew over the entire surface, the cells were mechanically detached in a straight line with a width of about 500 μm. The change in SAW before and after this processing is shown in FIG. On the second day of cell culture, the SAW change immediately before cell detachment was taken as 100%, and compared with the SAW change immediately after cell detachment. From this result, the amount of change in SAW is reduced by peeling cells with a constant width perpendicular to the SAW propagation path, approaching the speed of SAW in the absence of cells. As the area occupied by the cells with respect to the SAW propagation path is reduced, the rate of change of the SAW is reduced, and it is considered that the cell approached.

  That is, the entire elastic modulus on the SAW propagation path was reduced by removing a part of cells having higher rigidity (elastic modulus) than the solvent. As a result, it is considered that the rate of change is reduced by approaching the case where the sound speed of the SAW becomes slow and there are no cells. FIG. 10 (a) shows a reflection differential interference micrograph in a state where T2 cells are linearly detached.

  FIG. 11 is a graph showing changes in the processing time with the calcium chelating agent EDTA and the SAW signal. The amount of change in SAW delay by T2 cells before EDTA treatment on the second day of culture was taken as 100%, and the amount of change in SAW after addition of 10 mM EDTA was measured over time. From this graph, it is shown that the amount of change in SAW by T2 cells cultured on the SAW propagation path is reduced by performing EDTA treatment. The change converged in about 10 minutes from the start of the treatment because the effect of the calcium chelating agent has subsided.

  FIG. 10 (b) shows a reflection differential interference micrograph of T2 cells treated with EDTA. Also from this photograph, it was confirmed that the cell-cell bond was broken and the total amount of T2 cell culture was reduced. In the case of chemical treatment, it is considered that the SAW signal was changed by cooperating with the influence of the decrease in the cell-cell binding force in addition to the decrease in the cell culture area as compared with the mechanical damage described above.

From the above, in this experiment,
(1) Since the propagation speed of SAW changes depending on the presence or absence of cells on the surface of the SAW propagation path (difference in culture time), measurement can be performed while observing the culture state of the cells.
(2) By culturing the cells cultured over the entire surface of the SAW propagation path as a mechanical damage in a straight line perpendicular to the propagation path, or by weakening (separating) the cell-cell bond by chemical treatment. The degree of influence could be measured by comparing the change in cell state with the signal before treatment. From this, the effectiveness of the SAW device sensor by the artificial tissue was confirmed.

  In order to further develop the system using the SAW device sensor of the present invention and to improve the reliability and versatility of the sensor device, the selection of a piezoelectric material having a high sensitivity and a low temperature coefficient as a piezoelectric substrate, and the cells by increasing the density of the IDT It is necessary to measure individual changes with high sensitivity, to improve the accuracy by increasing the number of measurement channels, and to simplify the system by applying wireless technology. In addition, in order to increase the value as a sensor, cell-cell binding force and change in the elastic modulus of the whole cell, density factor, measurement of the influence of cell morphology and internal structure, which are factors other than elastic modulus and density, For example, visualization of activity status.

  The surface acoustic wave propagation path is placed in a single device with a single channel or multiple channels facing the flat X-coordinate axis, and cells are seeded and cultured on the surface acoustic wave propagation path for drugs and toxic substances. It is possible to evaluate in real time the degree of cell activity comprising a shear horizontal surface acoustic wave device that detects changes in cell activity, damage repair, and traits due to environmental factors, etc., and detects signal changes in each channel.

  Furthermore, as shown in FIG. 12, the device is used for a planar XY coordinate axis constituted by an X-side SAW reflection array 64 made up of X transmitters 62 and a Y-side SAW reflection array made up of Y transmitters 63. Changes in activity, injury repair, and traits of cells 61 from SAW signal changes that propagate through the X-side propagation path 66 and Y-side propagation path 67 between the IDT channels facing each other in the X and Y coordinates. Can be visualized by image processing on an XY screen, and the effects of drugs, toxic substances, environmental factors, etc. on cell activity can be visually and quickly evaluated.

  By improving these technical issues, the effects on cells or artificial tissues in model experiments and field surveys in the fields of pharmacology / toxicology research and biological effects research related to environmental problems can be remotely controlled with high sensitivity. Research and development of the basic technology of a compact and lightweight SAW device sensor system that can be sensed and wirelessly developed and developed into a system that will be incorporated into future advanced analysis / measurement equipment can contribute technically.

The technical and social significance and application fields of the present invention are as follows.
The present invention relates to development of a new technology for detecting (sensing) and evaluating in real time pharmacological and toxicological effects on cells and tissues and biological effects due to environmental factors. It is possible to make the measurement short-range wireless (wireless), it is possible to measure a large number of samples quickly, and the social significance and application field of the SAW device sensor using the artificial tissue of the present invention has been used in pharmacological and toxicological research. Impact assessment from new indicators that were not present, systems that detect and warn of infections caused by viral diseases at an early stage, allergic diseases (eg, hay fever), and bioterrorism warning systems that use ultra-sensitive cells Application to such as can be expected. The significance and fields are listed below.
(1) Development of biological sensing technology (2) Proposal of surface acoustic wave device biosensor (3) Development of cell mechanical structure analysis technology (4) Cell culture and fixation technology (5) Bioterrorism countermeasure technology in safety measures Development (6) Simplification and cost reduction of evaluation processes related to biological sensing (7) Development of testing and evaluation technology in medical field development (8) Health management system technology (9) Contribution to a safe and secure society and international It can be developed and established as a unique and superior advanced measurement analysis technique and means.

The following can be considered as specific application fields of the SAW device sensor using the artificial tissue of the present invention.
(1) Measurement of cell mechanical elements (cell-cell or cell-substrate binding force, elastic modulus, density factor, cell internal stress).
(2) An image drawing system for cell shape change by multi-channel application (XY).
(3) Measurement of the distribution of mechanical elements (cell-cell or cell-substrate or cell-substrate binding force, elastic modulus, density factor, cell internal stress) in the process of repairing cell or tissue injury using an image drawing system .
(4) Real-time detection and evaluation of environmental factors by detecting the activity level of cells and tissues.
(5) Comprehensive detection of biological enzyme reactions, bacteria, and various viruses by combining various chemical reaction membranes with multiple channels and combining ion polarization, viscosity changes, and attenuation changes.
(6) Biohazard countermeasure detection in the bacteria management system.
(7) Bioterrorism detection detection using trace component sensing using cells.
(8) Real-time measurement of biological effects and health effects due to miniaturization.
(9) Detection and evaluation of pharmacological effects and side effects in real time in new drug development.

It is a block diagram of the SAW device sensor of this invention. It is a photograph of a prototype SAW device sensor and a two-channel SAW device sensor. It is a basic lineblock diagram of magnetron sputtering. It is a block diagram of the experimental system used by this invention. It is a conceptual diagram which shows that the defect | deletion part produced on the SAW propagation path | route by giving mechanical damage to an artificial tissue. It is the conceptual diagram which showed the method of measuring a mode that the surrounding cell stretches, moves, and proliferates for the injury repair in the artificial tissue which produced the defect | deletion by mechanical damage. It is the figure which showed the time change of the SAW propagation characteristic by seed | inoculating and culture | cultivating the cell on a SAW propagation path. It is a reflection type differential interference microscopic photograph comparing before and after seeding and culturing cells on a SAW propagation path. It is the figure which showed the change of the SAW propagation characteristic by giving the mechanical damage with respect to the cell seed | inoculated and cultured on the SAW propagation path. It is a reflection type differential interference micrograph at the time of giving mechanical damage or chemical damage to a cell. It is the figure which showed the change of the SAW propagation characteristic by giving the chemical damage with respect to the cell seed | inoculated and cultured on the SAW propagation path. It is a conceptual diagram of multi-channel imaging. It is a displacement distribution schematic diagram of a Rayleigh wave. It is a displacement distribution schematic diagram of SH wave. It is the schematic of IDT. This is the principle of a SAW filter using a SAW device sensor. It is the conceptual diagram which showed the mode of the cell-substrate binding force at the time of a movement and proliferation of a cell.

Explanation of symbols

1 SAW device 2,23 IDT (comb electrode)
3 Piezoelectric substrate 4 SAW (surface acoustic wave)
5 Pitch 6 Antenna 7a, 7b SAW filter 8 Amplifier 9a, 9b Mixer 10 Ceramic filter 11 Baseband part 12 PLL synthesizer 13 Local transmitter 14 VCO
15 TCXO
16 cells 17 stress 18 adhesion receptor 19 adhesion spots 20 stretching / contraction force 21 SAW device sensor (surface acoustic wave device biosensor)
22 Piezoelectric substrate 24 SAW propagation path 25 SAW (surface acoustic wave)
26 SiO ₂ (silicon dioxide)
27 Pseudo Matrix 28 Cultured Cells 29 Defects 30 Scratches 31 Argon Gas 32 Heater 33 Base 34 Shutter 35 Target 36 High Frequency Power Supply 37 Vacuum Pump 38 Chamber 41 Pulse Generator 42 Reference Signal Generator 43 Mixer 44 Tone Burst Wave 45 Sensor Device 46 Amplifier 47 Reference wave 48 Digital oscilloscope 51 Measurement waveform 52 Data analysis / image display section 53 Imaging 54 Contraction force 61 Cell 62 X transmitter 63 Y transmitter 64 X side SAW reflection array 65 Y side SAW reflection array 66 X side propagation path 67 Y Side propagation path

Claims (9)

Applying high-frequency current or voltage to an element composed of comb-shaped electrodes joined to a piezoelectric substrate, generating surface acoustic waves near the surface of the piezoelectric material, and seeding and culturing cells in the propagation path of the surface acoustic waves A surface acoustic wave device biosensor that detects physical and chemical changes to an artificial tissue or cell formed by the method described above. It consists of an element composed of comb-shaped electrodes bonded to the piezoelectric substrate, and the surface acoustic wave component that propagates consists of a completely horizontal shear component, and the signal change of the surface acoustic wave from a living cell or artificial tissue in a wet state The surface acoustic wave device biosensor according to claim 1, wherein the detection sensitivity is improved. An element composed of comb-shaped electrodes bonded to the piezoelectric substrate is multi-channeled, and one or more of the elements are subjected to surface acoustic waves caused by changes in the measurement environment such as temperature, solution mass, solute concentration fluctuations, etc. 3. The detection sensitivity of the surface acoustic wave of the physical and chemical change only by the factor derived from the artificial tissue or the cell is improved by using as a reference channel for removing or correcting the disturbance factor in the signal of claim 1 or 2. Surface acoustic wave device biosensor. On the surface acoustic wave propagation path consisting of comb-shaped electrodes joined to the piezoelectric substrate, the cells are seeded and cultured after pseudo-matrix coating treatment for fixing and growing the cells, and the adhesion of the cells is increased. The surface acoustic wave device biosensor according to any one of claims 1 to 3, which detects a growth process. The artificial elastic tissue is formed by seeding and culturing the cells on the propagation path of the surface acoustic wave composed of the comb-shaped electrodes bonded to the piezoelectric substrate, and then artificially forming cells by artificial seeding or culturing. The surface acoustic wave device biosensor according to any one of claims 1 to 4, which detects a process of tissue injury repair. The cells are seeded and cultured on the surface acoustic wave propagation path after being subjected to a pseudo-matrix coating, and a change in the activity of a cell or an artificial tissue due to an environmental factor load or administration of a drug or chemical substance is detected. 4. The surface acoustic wave device biosensor according to any one of 3 above. Cells are seeded and cultured on the surface acoustic wave propagation path after being subjected to a pseudo-matrix coating treatment, and using the acoustoelastic effect, traits of cells or artificial tissues, particularly cell-cell or cell-substrate binding force, 5. The surface acoustic wave device biosensor according to claim 1, wherein a change in internal stress is detected. The surface acoustic wave propagation path is arranged in a multi-channel for a flat X-axis or XY axis coordinate axis in one element, and cells are seeded and cultured on the surface acoustic wave propagation path to load environmental factors. Alternatively, the surface acoustic wave device bio according to any one of claims 1 to 7, wherein a change in signal of each channel is detected by detecting a change in a cell or artificial tissue activity, injury repair, or a trait due to administration of a drug or a chemical substance. Sensor. The surface acoustic wave propagation path is arranged in a single element in multiple channels for planar X-axis or XY-axis coordinate axes, and the activity of cells or artificial tissue, injury repair, and changes in traits are imaged from signal changes. The surface acoustic wave device biosensor according to claim 1, which can be visualized by processing.