A device and method for evaluating erythrocyte activity based on surface acoustic wave

Abstract

The application discloses a kind of based on surface acoustic wave's red blood cell activity evaluation device and method.Device includes injection pump, syringe, silica gel hose, red blood cell activity sensing platform, power signal generator, amplitude phase measurement circuit.Red blood cell activity sensing platform is three-layer structure, from top to bottom respectively microchannel layer, piezoelectric layer and temperature control layer.Temperature control layer uses indium tin oxide conductive glass, and realizes the stable control of temperature by designing corresponding circuit pattern.Lithium niobate is used as piezoelectric layer, and surface acoustic wave is generated.Surface acoustic wave acts on red blood cell in microchannel layer, and judges by measuring the amplitude of output surface acoustic wave to evaluate the activity of red blood cell.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of signal detection, sensing and physical parameter measurement, in particular to a device and method for evaluating the activity of red blood cells based on surface acoustic wave. BACKGROUND

[0002] In the treatment of diseases such as trauma, red blood cells play a very important role in the process of blood transfusion. Under normal circumstances, red blood cells in the blood bank are usually stored by low-temperature preservation method, and are used after rewarming and washing when needed. The physical properties of red blood cells stored by low-temperature preservation will change after rewarming. The activity of red blood cells after rewarming is directly related to blood coagulation and clinical complications. Therefore, it is of great significance to detect the activity of red blood cells after rewarming. The activity of red blood cells is largely reflected in mechanical properties such as viscoelasticity, aggregation and deformation. The commonly used methods for testing the survival rate of red blood cells, such as blood cell analyzer and ultraviolet spectroscopy, cannot sense the mechanical and physical parameters that can show the activity of cells, such as cell quality, viscoelasticity, deformation and aggregation, because there is no mechanical force acting on the red blood cells. The detection methods such as atomic force microscope and optical tweezers are only suitable for testing the properties of single red blood cells, and cannot meet the high-throughput activity evaluation. In addition, the different methods, processes and conditions of red blood cell death will result in different colors of the solution after hemolysis of red blood cells, so there is a large error in the measurement by ultraviolet spectroscopy. SUMMARY

[0003] The present application aims to overcome the shortcomings of the existing measurement methods, and provides a device and method for evaluating the activity of red blood cells from practical application, which realizes high-throughput activity evaluation of red blood cells by the mechanical radiation force generated by surface acoustic wave acting on red blood cells. The technical scheme of the present application is as follows:

[0004] The application discloses a red blood cell activity evaluation device based on a surface acoustic wave, which comprises an injection pump, an injector, a silica gel hose, a red blood cell activity sensing platform, a power signal generator, an amplitude phase measurement circuit and a temperature measurement and control circuit. The red blood cell activity sensing platform is a three-layer structure, and the microchannel layer, the piezoelectric layer and the temperature control layer are sequentially arranged from top to bottom. The microchannel layer comprises a microfluidic channel inlet, a microfluidic channel, a microfluidic channel outlet and a linear microfluidic channel block pipe. The microfluidic channel is composed of a round cornered serpentine microchannel and a linear microchannel in series and is bonded to the upper surface of the piezoelectric layer. The first interdigital transducer and the third interdigital transducer are arranged on the two sides of the linear microchannel, and the second interdigital transducer and the fourth interdigital transducer are arranged on the two sides of the linear microfluidic channel block pipe. The injector is fixed to the injection pump, the injector is connected to the microfluidic channel inlet through the silica gel hose, one end of the third interdigital transducer and the fourth interdigital transducer is connected to the positive electrode of the power signal generator, the other end of the third interdigital transducer and the fourth interdigital transducer is connected to the negative electrode of the power signal generator and grounded, one end of the first interdigital transducer and the second interdigital transducer is connected to the amplitude phase measurement circuit, and the other end of the first interdigital transducer and the second interdigital transducer is grounded. The temperature control layer is connected to the temperature measurement and control circuit.

[0005] Further, the surface acoustic wave propagation direction generated by the third interdigital transducer and the first interdigital transducer, the fourth interdigital transducer and the second interdigital transducer is 30° to the horizontal direction and 70° to the linear microchannel liquid flow direction.

[0006] Further, the microchannel layer material is polydimethylsiloxane, the piezoelectric substrate material is 128° YX cut lithium niobate, the first interdigital transducer, the second interdigital transducer, the third interdigital transducer and the fourth interdigital transducer material are gold, and the temperature control layer material is indium tin oxide conductive glass.

[0007] Further, the temperature control layer comprises a first round cornered serpentine heating wire, a third round cornered serpentine heating wire, a fourth round cornered serpentine heating wire, a U-shaped heating wire, a ∏-shaped heating wire, a first round cornered serpentine heating wire, a second round cornered serpentine heating wire, a round cornered serpentine temperature measurement wire, a third round cornered serpentine temperature measurement wire, a fourth round cornered serpentine temperature measurement wire, a U-shaped temperature measurement wire, a ∏-shaped temperature measurement wire, a first round cornered serpentine temperature measurement wire and a second round cornered serpentine temperature measurement wire deposited on a glass substrate.

[0008] The first round cornered serpentine heating wire, the second round cornered serpentine heating wire and the round cornered serpentine temperature measurement wire are located directly below the round cornered serpentine microchannel, and the round cornered serpentine temperature measurement wire is embedded between the first round cornered serpentine heating wire and the second round cornered serpentine heating wire.

[0009] The third angular serpentine temperature measuring line and the third angular serpentine heating line are located directly below the third interdigital transducer, and the non-metallic gaps of the third interdigital transducer and the third angular serpentine heating line are spatially staggered with each other, and the third angular serpentine temperature measuring line is embedded in the upper opening non-metallic gap of the third angular serpentine heating line;

[0010] The fourth angular serpentine temperature measuring line and the fourth angular serpentine heating line are located directly below the fourth interdigital transducer, and the non-metallic gaps of the fourth interdigital transducer and the fourth angular serpentine heating line are spatially staggered with each other, and the fourth angular serpentine temperature measuring line is embedded in the upper opening non-metallic gap of the fourth angular serpentine heating line;

[0011] The first angular serpentine temperature measuring line and the first angular serpentine heating line are located directly below the first interdigital transducer, and the non-metallic gaps of the first interdigital transducer and the first angular serpentine heating line are spatially staggered with each other, and the first angular serpentine temperature measuring line is embedded in the upper opening non-metallic gap of the first angular serpentine heating line;

[0012] The second angular serpentine temperature measuring line and the second angular serpentine heating line are located directly below the second interdigital transducer, and the non-metallic gaps of the second interdigital transducer and the second angular serpentine heating line are spatially staggered with each other, and the second angular serpentine temperature measuring line is embedded in the upper opening non-metallic gap of the second angular serpentine heating line;

[0013] The ∏-shaped heating line and the ∏-shaped temperature measuring line are located directly below the linear microchannel, the peripheral width of the ∏-shaped heating line and the width of the linear microchannel are spatially coincided with each other, and the ∏-shaped temperature measuring line is embedded in the lower opening non-metallic gap of the ∏-shaped heating line;

[0014] The U-shaped heating line and the U-shaped temperature measuring line are located directly below the linear microfluidic channel block pipe, the peripheral width of the U-shaped heating line and the width of the linear microfluidic channel block pipe are spatially coincided with each other, and the U-shaped temperature measuring line is embedded in the upper opening non-metallic gap of the U-shaped heating line.

[0015] Further, the temperature measurement and control circuit comprises the following technical solutions:

[0016] The upper end of the first round angle serpentine heating line is connected with the upper end of the second round angle serpentine heating line, the lower end of the first round angle serpentine heating line is connected with the drain electrode of the field effect transistor M1, the source electrode of the field effect transistor M1 is grounded, the gate electrode is connected with one end of the resistor R4, the other end of the resistor R4 is connected with the microcontroller MCU, and the lower end of the second round angle serpentine heating line is connected with the power supply V1;

[0017] One end of the third angle serpentine heating line is connected to the power supply V1, the other end of the third angle serpentine heating line is connected to one end of the fourth angle serpentine heating line, the other end of the fourth angle serpentine heating line is connected to the drain of the field effect tube M5, the source of the field effect tube M5 is grounded, the gate is connected to one end of the resistor R8, the other end of the resistor R8 is connected to the microcontroller MCU;

[0018] One end of the second angle serpentine heating line is connected to the power supply V1, the other end of the second angle serpentine heating line is connected to one end of the first angle serpentine heating line, the other end of the first angle serpentine heating line is connected to the drain of the field effect tube M2, the source of the field effect tube M2 is grounded, the gate is connected to one end of the resistor R5, the other end of the resistor R5 is connected to the microcontroller MCU;

[0019] One end of the ∏-shaped heating line is connected to the power supply V1, the other end of the ∏-shaped heating line is connected to the drain of the field effect tube M3, the source of the field effect tube M3 is grounded, the gate is connected to one end of the resistor R6, the other end of the resistor R6 is connected to the microcontroller MCU;

[0020] One end of the U-shaped heating line is connected to the power supply V1, the other end of the U-shaped heating line is connected to the drain of the field effect tube M4, the source of the field effect tube M4 is grounded, the gate is connected to one end of the resistor R7, the other end of the resistor R7 is connected to the microcontroller MCU;

[0021] The upper end of the rounded serpentine temperature measuring line is connected to the power supply V2 and one end of the resistor R1, the lower end of the rounded serpentine temperature measuring line is connected to one end of the switch K1, the other end of the switch K1 is connected to one end of the resistor R2 and the positive input end of the differential amplifier IC1, the other end of the resistor R1 is connected to one end of the resistor R3 and the negative input end of the differential amplifier IC1, the other end of the resistor R3 is connected to the other end of the resistor R2, the ground, the output end of the differential amplifier IC1 is connected to the A / D interface of the microcontroller MCU;

[0022] One end of the fourth angle serpentine temperature measuring line is connected to the power supply V2, the other end of the fourth angle serpentine temperature measuring line is connected to one end of the third angle serpentine temperature measuring line, the other end of the third angle serpentine temperature measuring line is connected to one end of the switch K5, the other end of the switch K5 is connected to the positive input end of the differential amplifier IC1;

[0023] One end of the second angle serpentine temperature measuring line is connected to the power supply V2, the other end of the second angle serpentine temperature measuring line is connected to one end of the first angle serpentine temperature measuring line, the other end of the first angle serpentine temperature measuring line is connected to one end of the switch K4, the other end of the switch K4 is connected to the positive input end of the differential amplifier IC1;

[0024] One end of the U-shaped temperature measuring line is connected with the power supply V2, the other end of the U-shaped temperature measuring line is connected with one end of the switch K3, and the other end of the switch K3 is connected with the positive input end of the differential amplifier IC1;

[0025] One end of the U-shaped temperature measuring line is connected with the power supply V2, the other end of the U-shaped temperature measuring line is connected with one end of the switch K3, and the other end of the switch K3 is connected with the positive input end of the differential amplifier IC1;

[0026] Further, the microcontroller MCU controls the on and off time of the field effect tubes M1, M2, M3, M4 and M5 through pulse width modulation (PWM) to realize the heating function of the first round cornered serpentine heating line, the second round cornered serpentine heating line, the first square cornered serpentine heating line, the second square cornered serpentine heating line, the third square cornered serpentine heating line and the fourth square cornered serpentine heating line; at the same time, the round cornered serpentine temperature measuring line, the third square cornered serpentine temperature measuring line, the fourth square cornered serpentine temperature measuring line, the U-shaped temperature measuring line, the ∏-shaped temperature measuring line, the first square cornered serpentine temperature measuring line and the second square cornered serpentine temperature measuring line are switched by the switches K1, K2, K3, K4 and K5, and are connected with the Wheatstone bridge composed of the resistors R1, R2 and R3, respectively, the output voltage of the Wheatstone bridge changes due to temperature change, the voltage amplified by the differential amplifier IC1 in the microcontroller MCU is collected to obtain the temperature value. According to the collected temperature value, the PWM is adjusted through the PID algorithm to realize the closed loop control of the temperature.

[0027] Further, the needle of the syringe is provided with a thread on the outer wall and is provided with a detachable screw cap.

[0028] A red blood cell activity evaluation method based on surface acoustic wave, comprising the following steps:

[0029] Step one, take 100 μL of red blood cell solution to be tested, add 300 μL of 0.9% NaCl solution, mix uniformly, and set the working temperature of the temperature control layer to 37℃;

[0030] Step two, load the mixed solution into a syringe, tighten the needle of the syringe with a screw cap, and centrifuge in a centrifuge at a speed of 3000 rpm for 5 minutes;

[0031] Step three, unscrew the screw cap on the needle of the syringe, fix the syringe on an injection pump, connect the needle of the syringe with a silica gel hose, and push the mixed solution in the syringe into a microfluidic channel with the injection pump at a pushing speed of 35 μL / min, and set the power of the power signal generator to 28 dBm;

[0032] Step four, the amplitude phase measurement circuit records the amplitude curve and calculates the red blood cell activity, and the red blood cell activity L RBCRepresented as:

[0033]

[0034] In the formula, T is the time required for all the red blood cell solution to pass through the linear microchannel, and Tx is the time required for the viable cells to pass through the linear microchannel.

[0035] The beneficial effects of this invention are: it can sense mechanical and physical parameters that can be expressed as red blood cell activity, such as red blood cell mass, viscoelasticity, and deformation, while being insensitive to the residues of hemolyzed red blood cells, thus overcoming the large errors that exist when measuring with ultraviolet spectroscopy; it can achieve high-throughput red blood cell activity assessment, avoiding the shortcomings of detection methods such as atomic force microscopy and optical tweezers, which are only suitable for testing the characteristics of a single red blood cell. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Appendix Figure 1 This is a block diagram illustrating the principle of a red blood cell viability assessment device.

[0038] Appendix Figure 2 This is a side view of the erythrocyte activity sensing platform.

[0039] Appendix Figure 3 This is a block diagram of the temperature measurement and control system of the red blood cell activity sensing platform.

[0040] Appendix Figure 4 This is a block diagram of the syringe structure.

[0041] Appendix Figure 5 This is a graph defining the parameters for the erythrocyte viability assessment test curve.

[0042] Appendix Figure 6 Load the output amplitude curve of frozen hemolyzed red blood cells onto the red blood cell viability sensing platform.

[0043] Appendix Figure 7 Load the output amplitude curve of pure water hemolyzed red blood cells onto the red blood cell activity sensing platform.

[0044] Appendix Figure 8 Load the output amplitude curve of hot water hemolyzed red blood cells onto the red blood cell activity sensing platform.

[0045] Appendix Figure 9 Output amplitude curves were obtained by loading a 6:4 ratio of pure water-lysed hemolyzed red blood cells and active red blood cells solution onto the red blood cell viability sensing platform.

[0046] Appendix Figure 10 A comparative graph showing the activity of mixtures of live and dead red blood cells measured using a erythrocyte viability assessment device (SAW) and ultraviolet spectroscopy (UVS).

[0047] Appendix Figure 11 The image shows a comparison of hemolyzed red blood cells obtained using the pure water method, freezing method, and hot water method, serving as a control group of live red blood cells.

[0048] Appendix Figure 12 This image shows a comparison of the absorbance of the supernatant of hemolyzed red blood cells in pure water, frozen water, and hot water, as measured by ultraviolet spectroscopy. Detailed Implementation

[0049] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more definite definition of the scope of protection of the present invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by this application.

[0050] like Figure 1 As shown, a surface acoustic wave-based erythrocyte activity assessment device includes an injection pump 9, a syringe 4, a silicone tubing 6, an erythrocyte activity sensing platform 5, a power signal generator 8, an amplitude and phase measurement circuit 7, and a temperature measurement and control circuit 10; Figure 2 As shown, the erythrocyte activity sensing platform 5 has a three-layer structure, consisting of a microchannel layer 1, a piezoelectric layer 2, and a temperature control layer 3 from top to bottom; Figure 1 , Figure 2As shown, the microchannel layer 1 includes a microfluidic channel inlet 11, a microfluidic channel 12, a microfluidic channel outlet 13, and a linear microfluidic channel plug 14. The microfluidic channel 12 is composed of a rounded serpentine microchannel 121 and a linear microchannel 122 in series and is bonded to the upper surface of the piezoelectric layer 2. The piezoelectric layer 2 includes a piezoelectric substrate 25, a first interdigital transducer 21, a second interdigital transducer 22, a third interdigital transducer 23, and a fourth interdigital transducer 24 deposited on the upper surface of the piezoelectric substrate 25. The first interdigital transducer 21 and the third interdigital transducer 23 are arranged on both sides of the linear microchannel 122, and the second interdigital transducer 22 and the fourth interdigital transducer 24 are arranged on both sides of the linear microfluidic channel plug 14. The injector 4 is fixed on the syringe pump 9, and the injector 4 is connected to the microfluidic channel inlet 11 through the silicone hose 6. One end of the third interdigital transducer 23 and the fourth interdigital transducer 24 is connected to the positive electrode of the power signal generator 8, and the other end of the third interdigital transducer 23 and the fourth interdigital transducer 24 is connected to the negative electrode of the power signal generator 8 and grounded. One end of the first interdigital transducer 21 and the second interdigital transducer 22 is connected to the amplitude-phase measurement circuit 7, and the other end of the first interdigital transducer 21 and the second interdigital transducer 22 is grounded. The temperature control layer 3 is connected to the temperature measurement and control circuit 10.

[0051] Further, as shown in FIG. 3, the third interdigital transducer 23 and the first interdigital transducer 21, the fourth interdigital transducer 24 and the second interdigital transducer 22 generate surface acoustic waves in a direction that forms a 30° angle with the horizontal direction and a 70° angle with the liquid flow direction of the linear microchannel. Figure 1

[0052] Further, the microchannel layer 1 is made of polydimethylsiloxane (PDMS), the piezoelectric substrate 25 is made of 128°YX-cut lithium niobate (128°YX-LiNbO3), the first interdigital transducer 21, the second interdigital transducer 22, the third interdigital transducer 23, and the fourth interdigital transducer 24 are made of Au, and the temperature control layer 3 is made of indium tin oxide (ITO) conductive glass.

[0053] Further, as shown in FIG. 3, the third interdigital transducer 23 and the first interdigital transducer 21, the fourth interdigital transducer 24 and the second interdigital transducer 22 generate surface acoustic waves in a direction that forms a 30° angle with the horizontal direction and a 70° angle with the liquid flow direction of the linear microchannel. Figure 3

[0054] ​​The first rounded serpentine heating line 31, the second rounded serpentine heating line 314 and the rounded serpentine temperature measuring line 315 are located directly below the rounded serpentine microchannel 121, and the rounded serpentine temperature measuring line 315 is embedded in the first rounded serpentine heating line 31 and the second rounded serpentine heating line 314;

[0055] The third angular serpentine temperature measuring line 32 and the third angular serpentine heating line 33 are located directly below the third interdigital transducer 23, and the non-metallic voids of the third interdigital transducer 23 and the third angular serpentine heating line 33 are spatially staggered with each other, and the third angular serpentine temperature measuring line 32 is embedded in the upper opening non-metallic void of the third angular serpentine heating line 33;

[0056] The fourth angular serpentine temperature measuring line 34 and the fourth angular serpentine heating line 35 are located directly below the fourth interdigital transducer 24, and the non-metallic voids of the fourth interdigital transducer 24 and the fourth angular serpentine heating line 35 are spatially staggered with each other, and the fourth angular serpentine temperature measuring line 34 is embedded in the upper opening non-metallic void of the fourth angular serpentine heating line 35;

[0057] The first angular serpentine temperature measuring line 310 and the first angular serpentine heating line 311 are located directly below the first interdigital transducer 21, and the non-metallic voids of the first interdigital transducer 21 and the first angular serpentine heating line 311 are spatially staggered with each other, and the first angular serpentine temperature measuring line 310 is embedded in the upper opening non-metallic void of the first angular serpentine heating line 311;

[0058] The second angular serpentine temperature measuring line 312 and the second angular serpentine heating line 313 are located directly below the second interdigital transducer 22, and the non-metallic voids of the second interdigital transducer 22 and the second angular serpentine heating line 313 are spatially staggered with each other, and the second angular serpentine temperature measuring line 312 is embedded in the upper opening non-metallic void of the second angular serpentine heating line 313;

[0059] The ∏-shaped heating line 39 and the ∏-shaped temperature measuring line 38 are located directly below the straight microchannel 122, the peripheral width of the ∏-shaped heating line 39 and the width of the straight microchannel 122 spatially coincide with each other, and the ∏-shaped temperature measuring line 38 is embedded in the lower opening non-metallic void of the ∏-shaped heating line 39;

[0060] The U-shaped heating line 37 and the U-shaped temperature measuring line 36 are located directly below the straight microfluidic channel block pipe 14, the peripheral width of the U-shaped heating line 37 and the width of the straight microfluidic channel block pipe 14 spatially coincide with each other, and the U-shaped temperature measuring line 36 is embedded in the upper opening non-metallic void of the U-shaped heating line 37.

[0061] Further, as Figure 3As shown, the temperature control circuit 10 comprises the following technical solutions:

[0062] The upper end of the first rounded serpentine heating wire 31 is connected with the upper end of the second rounded serpentine heating wire 314, the lower end of the first rounded serpentine heating wire 31 is connected with the drain of the field effect transistor M1, the source of the field effect transistor M1 is grounded, the gate is connected with one end of the resistor R4, the other end of the resistor R4 is connected with the microcontroller MCU, and the lower end of the second rounded serpentine heating wire 314 is connected with the power supply V1.

[0063] One end of the third rounded serpentine heating wire 33 is connected with the power supply V1, the other end of the third rounded serpentine heating wire 33 is connected with one end of the fourth rounded serpentine heating wire 35, the other end of the fourth rounded serpentine heating wire 35 is connected with the drain of the field effect transistor M5, the source of the field effect transistor M5 is grounded, the gate is connected with one end of the resistor R8, and the other end of the resistor R8 is connected with the microcontroller MCU.

[0064] One end of the second rounded serpentine heating wire 313 is connected with the power supply V1, the other end of the second rounded serpentine heating wire 313 is connected with one end of the first rounded serpentine heating wire 311, the other end of the first rounded serpentine heating wire 311 is connected with the drain of the field effect transistor M2, the source of the field effect transistor M2 is grounded, the gate is connected with one end of the resistor R5, and the other end of the resistor R5 is connected with the microcontroller MCU.

[0065] One end of the Π-shaped heating wire 39 is connected with the power supply V1, the other end of the Π-shaped heating wire 39 is connected with the drain of the field effect transistor M3, the source of the field effect transistor M3 is grounded, the gate is connected with one end of the resistor R6, and the other end of the resistor R6 is connected with the microcontroller MCU.

[0066] One end of the U-shaped heating wire 37 is connected with the power supply V1, the other end of the U-shaped heating wire 37 is connected with the drain of the field effect transistor M4, the source of the field effect transistor M4 is grounded, the gate is connected with one end of the resistor R7, and the other end of the resistor R7 is connected with the microcontroller MCU.

[0067] The upper end of the rounded serpentine temperature measurement wire 315 is connected with the power supply V2 and one end of the resistor R1, the lower end of the rounded serpentine temperature measurement wire 315 is connected with one end of the switch K1, the other end of the switch K1 is connected with one end of the resistor R2 and the positive input end of the differential amplifier IC1, the other end of the resistor R1 is connected with one end of the resistor R3 and the negative input end of the differential amplifier IC1, the other end of the resistor R3 is connected with the other end of the resistor R2, and the ground, the output end of the differential amplifier IC1 is connected with the A / D interface of the microcontroller MCU.

[0068] One end of the fourth square corner serpentine temperature measuring line 34 is connected with the power supply V2, the other end of the fourth square corner serpentine temperature measuring line 34 is connected with one end of the third square corner serpentine temperature measuring line 32, the other end of the third square corner serpentine temperature measuring line 32 is connected with one end of the switch K5, the other end of the switch K5 is connected with the positive input end of the differential amplifier IC1;

[0069] One end of the second square corner serpentine temperature measuring line 312 is connected with the power supply V2, the other end of the second square corner serpentine temperature measuring line 312 is connected with one end of the first square corner serpentine temperature measuring line 310, the other end of the first square corner serpentine temperature measuring line 310 is connected with one end of the switch K4, the other end of the switch K4 is connected with the positive input end of the differential amplifier IC1;

[0070] One end of the ∏-shaped temperature measuring line 38 is connected with the power supply V2, the other end of the ∏-shaped temperature measuring line 38 is connected with one end of the switch K2, the other end of the switch K2 is connected with the positive input end of the differential amplifier IC1;

[0071] One end of the U-shaped temperature measuring line 36 is connected with the power supply V2, the other end of the U-shaped temperature measuring line 36 is connected with one end of the switch K3, the other end of the switch K3 is connected with the positive input end of the differential amplifier IC1;

[0072] Further, the microcontroller MCU controls the on and off time of the field effect tubes M1, M2, M3, M4 and M5 through pulse width modulation (PWM), so as to realize the heating function of the first round corner serpentine heating line 31, the second round corner serpentine heating line 314, the first square corner serpentine heating line 311, the second square corner serpentine heating line 313, the third square corner serpentine heating line 33 and the fourth square corner serpentine heating line 35; at the same time, the round corner serpentine temperature measuring line 315, the third square corner serpentine temperature measuring line 32, the fourth square corner serpentine temperature measuring line 34, the U-shaped temperature measuring line 36, the ∏-shaped temperature measuring line 38, the first square corner serpentine temperature measuring line 310 and the second square corner serpentine temperature measuring line 312 are switched through the switches K1, K2, K3, K4 and K5, and are respectively connected with the Wheatstone bridge composed of the resistors R1, R2 and R3; the temperature change causes the output voltage of the Wheatstone bridge to change; the voltage amplified by the differential amplifier IC1 is collected by the A / D in the microcontroller MCU, and the temperature value is obtained. According to the collected temperature value, the PWM is adjusted through the PID algorithm to realize the closed-loop control of the temperature.

[0073] Further, as shown in Figure 4 The outer wall of the needle 41 of the syringe 4 is provided with threads, and is provided with a detachable screw cap 42.

[0074] A red blood cell activity evaluation method based on surface acoustic wave, comprising the following steps:

[0075] Step one, take 100 μL of the red blood cell solution to be tested, and add 300 μL of 0.9% NaCl solution and mix well, and set the working temperature of the temperature control layer 3 to 37°C;

[0076] Step two, load the mixed solution into the syringe 4, tighten the needle 41 of the syringe 4 with the screw cap 42, and centrifuge in the centrifuge at a speed of 3000 rpm for 5 min;

[0077] Step three, unscrew the screw cap 42 on the needle 41 of the syringe 4, fix the syringe 4 on the injection pump 9, connect the needle 41 of the syringe 4 with the silicone hose 6, and use the injection pump 9 to push the mixed solution in the syringe 4 into the microfluidic channel 12, the pushing speed of the injection pump 4 is 35 μL / min, and the power signal generator 8 is set to a power of 28 dBm;

[0078] Step four, the amplitude phase measurement circuit 7 records the amplitude curve and calculates the red blood cell activity. The output voltage of the sensing channel composed of the third interdigital transducer 23 and the first interdigital transducer 21 is denoted as Vs, the output voltage of the reference channel composed of the fourth interdigital transducer 24 and the second interdigital transducer 22 is denoted as Vr, and the amplitude curve is denoted as Vr / Vs. As shown in Figure 5 , the red blood cell activity L RBC is represented as:

[0079]

[0080] In the formula, T is the time required for the red blood cell solution to pass through the straight microchannel 122, and Tx is the time required for the active cells to pass through the straight microchannel.

[0081] As shown in Figure 6 , Figure 7 , Figure 8 , Figure 9 , it can be seen by comparison that the whole dead red blood cells after hemolysis by different methods such as freezing, pure water, and hot water have no obvious jump in the amplitude curve within T time after being measured by the red blood cell activity sensing platform, while the 6:4 ratio of hemolytic red blood cells and active red blood cell mixture has a significant jump in the amplitude curve within T time from 100 seconds to 284 seconds, which shows that although the whole dead red blood cells after hemolysis have cell shells and other substances, the mechanical force generated by the surface acoustic wave is not sensitive to the mechanics of the red blood cells after hemolysis, that is, the red blood cell activity sensing platform is not sensitive to the whole dead red blood cells after hemolysis, but only to the active red blood cells. As shown in Figure 10 , the red blood cell activity evaluation device (SAW) of the present application and the ultraviolet spectrum method (UVS) are used to measure different proportions of active red blood cells and dead red blood cell mixture, and there is no any impurity such as red blood cell shell in the mixture, and the actual measurement results show that the correlation coefficient R2 0.97283, indicating good correlation.

[0082] By Figure 11 , Figure 12 It can be seen that after red blood cells are hemolyzed by pure water, freezing, hot water method, etc., impurities such as red blood cell shells will be left at the bottom. When measuring the absorbance of the upper liquid of hemolyzed red blood cells by ultraviolet spectrometry (UVS), the test results obtained by different hemolysis methods are different. According to the red blood cell activity L RBC Calculation formula:

[0083]

[0084] In the formula, A0 is the negative control group, i.e. 0% survival and 100% hemolysis sample, A1 is the positive control group, i.e. 100% survival and no hemolysis sample, and A is the sample to be tested. It can be seen that different hemolysis methods of red blood cells will result in different A0 values, and the final red blood cell activity measurement value is not accurate. During low-temperature preservation, rewarming and washing of red blood cells, operations such as freezing, hot water rewarming, etc. are inevitable. These factors ultimately result in a large difference in the evaluation of red blood cell activity by ultraviolet spectrometry.

[0085] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any change or replacement without creative labor should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be limited by the protection scope defined in the claims.

Claims

1. A red blood cell activity evaluation device based on surface acoustic wave, characterized by: The application relates to a red blood cell activity sensing platform, which comprises an injection pump, an injector, a silica rubber hose, a red blood cell activity sensing platform, a power signal generator, an amplitude phase measurement circuit and a temperature measurement and control circuit; the red blood cell activity sensing platform is a three-layer structure, and the three layers are a microchannel layer, a piezoelectric layer and a temperature control layer from top to bottom; the microchannel layer comprises a microfluidic channel inlet, a microfluidic channel, a microfluidic channel outlet and a linear microfluidic channel block pipe; the microfluidic channel is composed of a round cornered serpentine microchannel and a linear microchannel in series and is bonded to the upper surface of the piezoelectric layer; the piezoelectric layer comprises a piezoelectric substrate, a first interdigital transducer, a second interdigital transducer, a third interdigital transducer and a fourth interdigital transducer deposited on the upper surface of the piezoelectric substrate; the first interdigital transducer and the third interdigital transducer are arranged on the two sides of the linear microchannel, and the second interdigital transducer and the fourth interdigital transducer are arranged on the two sides of the linear microfluidic channel block pipe; the propagation direction of the surface acoustic wave generated by the first interdigital transducer and the third interdigital transducer and the second interdigital transducer and the fourth interdigital transducer is 30 DEG to the horizontal direction and 70 DEG to the liquid flow direction of the linear microchannel; the injector is fixed to the injection pump, the injector is connected to the microfluidic channel inlet through the silica rubber hose, one end of the third interdigital transducer and the fourth interdigital transducer is connected to the positive electrode of the power signal generator, the other end of the third interdigital transducer and the fourth interdigital transducer is connected to the negative electrode of the power signal generator and grounded, one end of the first interdigital transducer and the second interdigital transducer is connected to the amplitude phase measurement circuit, and the other end of the first interdigital transducer and the second interdigital transducer is grounded; the temperature control layer is connected to the temperature measurement and control circuit. 2.The device for evaluating erythrocyte activity based on surface acoustic wave according to claim 1, characterized in that: The microchannel layer material is polydimethylsiloxane; the piezoelectric substrate material is 128 YX cut lithium niobate; the materials of the first interdigital transducer, the second interdigital transducer, the third interdigital transducer and the fourth interdigital transducer are gold; and the temperature control layer material is indium tin oxide conductive glass. 3.The device for evaluating erythrocyte activity based on surface acoustic wave according to claim 1, characterized in that: The temperature control layer comprises a first round cornered serpentine heating wire, a third square cornered serpentine heating wire, a fourth square cornered serpentine heating wire, a U-shaped heating wire, a Pi-shaped heating wire, a first square cornered serpentine heating wire, a second square cornered serpentine heating wire, a second round cornered serpentine heating wire, a round cornered serpentine temperature measurement wire, a third square cornered serpentine temperature measurement wire, a fourth square cornered serpentine temperature measurement wire, a U-shaped temperature measurement wire, a Pi-shaped temperature measurement wire, a first square cornered serpentine temperature measurement wire and a second square cornered serpentine temperature measurement wire deposited on a glass substrate. The first round cornered serpentine heating wire, the second round cornered serpentine heating wire and the round cornered serpentine temperature measurement wire are located directly below the round cornered serpentine microchannel, and the round cornered serpentine temperature measurement wire is embedded in the first round cornered serpentine heating wire and the second round cornered serpentine heating wire. The third square cornered serpentine temperature measurement wire and the third square cornered serpentine heating wire are located directly below the third interdigital transducer, the non-metallic gap of the third interdigital transducer and the third square cornered serpentine heating wire is spatially staggered, and the third square cornered serpentine temperature measurement wire is embedded in the upper opening non-metallic gap of the third square cornered serpentine heating wire. The fourth square corner serpentine temperature measuring line and the fourth square corner serpentine heating line are located directly below the fourth interdigital transducer, and the non-metallic gaps of the fourth interdigital transducer and the fourth square corner serpentine heating line are spatially staggered with each other, and the fourth square corner serpentine temperature measuring line is embedded in the upper opening non-metallic gap of the fourth square corner serpentine heating line; The first square corner serpentine temperature measuring line and the first square corner serpentine heating line are located directly below the first interdigital transducer, and the non-metallic gaps of the first interdigital transducer and the first square corner serpentine heating line are spatially staggered with each other, and the first square corner serpentine temperature measuring line is embedded in the upper opening non-metallic gap of the first square corner serpentine heating line; The second square corner serpentine temperature measuring line and the second square corner serpentine heating line are located directly below the second interdigital transducer, and the non-metallic gaps of the second interdigital transducer and the second square corner serpentine heating line are spatially staggered with each other, and the second square corner serpentine temperature measuring line is embedded in the upper opening non-metallic gap of the second square corner serpentine heating line; The ∏-shaped heating line and the ∏-shaped temperature measuring line are located directly below the linear microchannel, the peripheral width of the ∏-shaped heating line and the width of the linear microchannel are spatially coincided with each other, and the ∏-shaped temperature measuring line is embedded in the lower opening non-metallic gap of the ∏-shaped heating line; The U-shaped heating line and the U-shaped temperature measuring line are located directly below the linear microchannel blocking pipe, the peripheral width of the U-shaped heating line and the width of the linear microchannel blocking pipe are spatially coincided with each other, and the U-shaped temperature measuring line is embedded in the upper opening non-metallic gap of the U-shaped heating line. 4.The device for evaluating erythrocyte activity based on surface acoustic wave according to claim 3, characterized in that: In the temperature measurement and control circuit, the upper end of the first round corner serpentine heating line is connected with the upper end of the second round corner serpentine heating line, the lower end of the first round corner serpentine heating line is connected with the drain electrode of the field effect transistor M1, the source electrode of the field effect transistor M1 is grounded, the gate electrode is connected with one end of the resistor R4, the other end of the resistor R4 is connected with the microcontroller MCU, and the lower end of the second round corner serpentine heating line is connected with the power supply V1; One end of the third square corner serpentine heating line is connected with the power supply V1, the other end of the third square corner serpentine heating line is connected with one end of the fourth square corner serpentine heating line, the other end of the fourth square corner serpentine heating line is connected with the drain electrode of the field effect transistor M5, the source electrode of the field effect transistor M5 is grounded, the gate electrode is connected with one end of the resistor R8, and the other end of the resistor R8 is connected with the microcontroller MCU; One end of the second square corner serpentine heating line is connected with the power supply V1, the other end of the second square corner serpentine heating line is connected with one end of the first square corner serpentine heating line, the other end of the first square corner serpentine heating line is connected with the drain electrode of the field effect transistor M2, the source electrode of the field effect transistor M2 is grounded, the gate electrode is connected with one end of the resistor R5, and the other end of the resistor R5 is connected with the microcontroller MCU; One end of the ∏-shaped heating line is connected with the power supply V1, the other end of the ∏-shaped heating line is connected with the drain electrode of the field effect transistor M3, the source electrode of the field effect transistor M3 is grounded, the gate electrode is connected with one end of the resistor R6, and the other end of the resistor R6 is connected with the microcontroller MCU. One end of the U-shaped heating wire is connected to a power supply V1, the other end of the U-shaped heating wire is connected to the drain of a field effect transistor M4, the source of the field effect transistor M4 is grounded, the gate of the field effect transistor M4 is connected to one end of a resistor R7, and the other end of the resistor R7 is connected to a microcontroller MCU; One end of the upper end of the round serpentine temperature measuring wire is connected to a power supply V2 and one end of a resistor R1, the other end of the round serpentine temperature measuring wire is connected to one end of a switch K1, the other end of the switch K1 is connected to one end of a resistor R2 and a positive input end of a differential amplifier IC1, the other end of the resistor R1 is connected to one end of a resistor R3 and a negative input end of the differential amplifier IC1, the other end of the resistor R3 is connected to the other end of the resistor R2, and the output end of the differential amplifier IC1 is connected to an A / D interface of the microcontroller MCU; One end of the fourth square serpentine temperature measuring wire is connected to the power supply V2, the other end of the fourth square serpentine temperature measuring wire is connected to one end of the third square serpentine temperature measuring wire, and the other end of the third square serpentine temperature measuring wire is connected to one end of the switch K5, and the other end of the switch K5 is connected to the positive input end of the differential amplifier IC1; One end of the second square serpentine temperature measuring wire is connected to the power supply V2, the other end of the second square serpentine temperature measuring wire is connected to one end of the first square serpentine temperature measuring wire, and the other end of the first square serpentine temperature measuring wire is connected to one end of the switch K4, and the other end of the switch K4 is connected to the positive input end of the differential amplifier IC1; One end of the U-shaped temperature measuring wire is connected to the power supply V2, the other end of the U-shaped temperature measuring wire is connected to one end of the switch K3, and the other end of the switch K3 is connected to the positive input end of the differential amplifier IC1. The microcontroller MCU controls the on and off time of the field effect transistors M1, M2, M3, M4 and M5 through pulse width modulation (PWM), so as to realize the heating function of the first round serpentine heating wire, the second round serpentine heating wire, the first square serpentine heating wire, the second square serpentine heating wire, the third square serpentine heating wire and the fourth square serpentine heating wire; at the same time, the round serpentine temperature measuring wire, the third square serpentine temperature measuring wire, the fourth square serpentine temperature measuring wire, the U-shaped temperature measuring wire, the ∏-shaped temperature measuring wire, the first square serpentine temperature measuring wire and the second square serpentine temperature measuring wire are switched through the switches K1, K2, K3, K4 and K5, and are connected to the Wheatstone bridge composed of the resistors R1, R2 and R3, respectively, the output voltage of the Wheatstone bridge changes due to the temperature change, the voltage amplified by the differential amplifier IC1 is collected by the A / D in the microcontroller MCU, and the temperature value is obtained, and the temperature closed loop control is realized through the PID algorithm to adjust the PWM according to the collected temperature value. The outer wall of the needle of the syringe comprises a screw thread and is provided with a detachable screw cap. 5.The device for evaluating erythrocyte activity based on surface acoustic wave according to claim 1, characterized in that: The method comprises the following steps of:

6. An evaluation method using the red blood cell activity evaluation device based on a surface acoustic wave according to any one of claims 1 to 5, characterized by: ​ Step one, take 100 μL of the red blood cell solution to be tested, and add 300 μL of 0.9% NaCl solution, mix well, and set the working temperature of the temperature control layer to 37°C; Step two, load the mixed solution into a syringe, tighten the needle cap of the syringe, and centrifuge in a centrifuge at a speed of 3000 rpm for 5 min; Step three, unscrew the needle cap of the syringe, fix the syringe on the injection pump, connect the syringe needle and the silicone hose, and use the injection pump to push the mixed solution in the syringe into the microfluidic channel, the pushing speed of the injection pump is 35 μL / min, and the power signal generator is set to a power of 28 dBm; Step four, the amplitude phase measurement circuit records the acquisition amplitude curve and calculates the red blood cell activity, red blood cell activity L RBC is represented as: In the formula, T is the time required for the red blood cell solution to pass through the straight microchannel, and Tx is the time required for the active cells to pass through the straight microchannel.

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