CN111551701A - Flexible micro-column ring array for detecting clot contractility and preparation method and application thereof - Google Patents

Abstract

The invention discloses a flexible micro-column ring array for detecting clot contractility and a preparation method and application thereof. The micro-column ring array is composed of several micro-column rows arranged periodically, each of the micro-column rows has several micro-column rings, and each of the micro-column rings is surrounded by four or more than four rings. The micro-column ring is composed of circular pores with a certain diameter, and when the clot contractility is detected, whole blood is dropped into the circular pores of the micro-column ring for coagulation test. The invention utilizes the interfacial tension of the solid-liquid-gas system and the resistance of the micro-column to efficiently fix the whole blood sample in the micro-column ring; meanwhile, the micro-column is used as a force sensor to measure the size of the clot contraction force in real time, the detection result is highly sensitive, and the detection It has low cost and easy clinical transformation; in addition, there is no need to separate platelets from blood, and whole blood can be used for detection, fully considering the influence of other components in blood on platelet contraction, and the results are more objective and comprehensive.

Description

A flexible microcolumn ring array for detecting clot contractility and its preparation method and application

technical field

The invention belongs to the field of biomedical technology. Specifically, it relates to a flexible micro-column ring array for detecting clot contractility and its preparation method and application.

Background technique

Coagulation is a very important physiological process that must be properly regulated by the human body to maintain a delicate balance between bleeding and thrombosis. Insufficient coagulation function can lead to excessive blood loss during trauma, and excessive coagulation function is often accompanied by the risk of developing blood clots. Coagulation function test plays an important role in clinical practice. During cardiopulmonary bypass, surgeons need to detect blood coagulation function immediately to guide the administration of procoagulants and anticoagulants to prevent blood from coagulating during cardiopulmonary bypass [1]. In addition, blood loss caused by surgery, contact coagulation activation, blood dilution, etc., cause the loss of platelets and coagulation factors in the blood, which inevitably causes some patients to cause coagulation dysfunction after surgery [2]. Therefore, the treatment of coagulation disorders in the perioperative period should be carried out under the guidance of clinical coagulation tests to improve the pertinence of the treatment plan and reduce the demand for blood products. Moreover, in addition to perioperative care, coagulation testing has important applications in many other clinical scenarios, such as the treatment of sepsis [6], obstetric hemorrhage [7], thrombotic diseases, etc. [8] . Reliable coagulation function testing technology can provide valuable information for the treatment of such diseases and guide doctors' clinical decision-making, thereby improving the efficiency of treatment and reducing the cost of treatment.

Traditional coagulation testing methods include prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen (FIB). et al [9]. Although these tests have been widely used in clinical practice, they have certain shortcomings. For example, PT, aPTT, TT only examine the coagulation response speed on the time scale, and lack attention to the mechanical properties of the clot. On the other hand, the formation of thrombus is closely related to blood vessels, platelets, coagulation (anticoagulation) blood factors, blood viscosity and other factors, especially the changes in platelet function, and these four tests generally use plasma as the test specimen , so the effect of cellular components such as platelets on coagulation cannot be detected [10]. In addition, these tests end after fibrin has polymerized, so clot stability and fibrinolytic processes cannot be detected [3]. The methods for judging coagulation tests need to be updated.

In recent years, viscoelasticity detection systems for whole blood specimens have gained some clinical acceptance [11]. During blood coagulation, fibrinogen cross-linking causes blood to increase in viscosity and gradually become a gel-like solid. This kind of test method is to obtain a thromboelastogram by monitoring the viscoelastic changes of whole blood in the process of coagulation in real time, and then to calculate the reaction time and the maximum viscoelasticity of the clot, etc., to test the blood coagulation function more comprehensively and make up for the traditional test method. of insufficiency. The more successful commercial instruments include TEG (Haemonetics Company), ROTEM (Instrumentation Laboratory Company), Sonoclot Analyzer (Sienco Company) and so on. According to literature reports, the coagulation function test results obtained by TEG and ROTEM can reduce the blood transfusion needs of patients during and after cardiac surgery to a certain extent [12], Sonoclot also has a significant effect in the diagnosis of some thrombotic diseases [13] ]. In view of the potential of thrombus viscoelasticity testing in coagulation testing, many miniaturized blood viscoelasticity testing devices have also been reported for point-of-care testing (POCT) [14-20]. Although the thrombus viscoelasticity test is a description of the dynamic changes of platelet function, the monitoring of platelet function is not specific, and the cost is extremely expensive. blood transfusion regimen.

Furthermore, viscoelasticity tests of blood do not fully reflect the mechanical properties of the clot. Studies have confirmed that clot contraction is another important indicator of coagulation function, so measuring clot contractility is expected to become an important method for measuring coagulation function. As shown in Figure 1, Figure 1 is a schematic diagram of the generation of clot contraction force, which is roughly divided into four steps. First, endothelial cells are damaged, and collagen and von Willebrand Factor (vWF) are exposed to the blood; platelets in the blood release granules after contact with collagen and vWF, and platelets are activated and aggregated with each other; triggering a coagulation cascade Thrombin is produced, and fibrinogen is converted into fibrin under the action of thrombin, and fibrin traps white blood cells and red blood cells to form a clot. At the same time, platelets generate clot retraction force (CRF), which makes the clot shrink and harden. Therefore, the size of clot contractility and its dynamic changes can be used as test indicators of coagulation function.

Researchers have attached fluorescent beads and individual platelets to flexible substrates. Platelet contraction drives the deformation of the flexible substrate to change the position of the fluorescent beads. The position change of the fluorescent beads is monitored in real time with a fluorescence microscope, which is used to estimate the contractile force of a single platelet [21] (Figure 2a). In addition, fibrinogen and collagen were modified on the cantilever of atomic force microscope and polyacrylamide hydrogel substrate, so that platelets were attached between the substrate and the cantilever; the platelet contraction deformed the cantilever, and the deformation of the cantilever was used to calculate the single platelet contractility [22] (Fig. 2b), but this method has low platelet capture efficiency. On this basis, some people developed a platelet detection device with higher throughput [23] (Fig. 2c), using polyacrylamide hydrogel as the substrate, and modifying the regularly arranged fibrinogen microdot array on it. It is used to capture platelets; platelet contraction changes the position of the fibrin microdot array and uses it to infer platelet contractile force. In Figure 2d, platelets are coated on collagen-coated micropillars and whole blood is fed into the channel to create a fluid environment. When the platelets contract, the micropillars bend to produce displacement, which is used to calculate the platelet contractile force, and to study the effect of fluid shear force on the platelet contractile force in the whole blood fluid environment [24]. Figure 2e Capturing the flowing platelets with the collagen microtissue on top of the micropillar pair to form aggregates, the platelet shrinkage displaces the micropillar pair to bend. Used to calculate platelet contractility under fluid shear and to measure platelets using the underlying stretchable membrane Changes in stiffness of agglomerates before and after solidification [25].

However, the above-mentioned methods are complicated to process and require extremely precise inspection platforms and complex data acquisition equipment, such as fluorescence microscopes and atomic force microscopes. In addition, the above method requires the separation of platelets from blood, which complicates the operation.

Among them, the references are:

[1]. Sarkar, M.; Prabhu, V., Basics of cardiopulmonary bypass. Indian journal of anaesthesia 2017, 61(9), 760.

[2]. Ravn, H.B. In Hemostasis in pediatric cardiac surgery, 2017; Thieme Medical Publishers.

[3]. Shen, L.; Tabaie, S.; Ivascu, N., Viscoelastic testing inside and beyond the operating room. Journal of thoracic disease 2017, 9(Suppl 4), S299.

[4]. Muszynski, J.A.; Spinella, P.C.; Cholette, J.M.; Acker, J.P.; Hall, M.W.; Juffermans, N.P.; Kelly, D.P.; Blumberg, N.; Nicol, K.; Liedel, J., Transfusion-related immunomodulation : review of the literature and implications for pediatric critical illness. Transfusion 2017, 57(1), 195-206.

[5]. Kastrup, C.J.; Boedicker, J.Q.; Pomerantsev, A.P.; Moayeri, M.; Bian, Y.; Pompano, R.R.; Kline, T.R.; Sylvestre, P.; Shen, F.; Leppla, S.H.; Tang, W.J.; Ismagilov, R.F., Spatial localization of bacteria controls coagulation of human blood by 'quorum acting'. Nat Chem Biol 2008, 4(12), 742-50.

[6]. Wang Hong; Zhang Shuwen; Ren Aimin; Zhang Lixia; Wang Chao; Huang Ying; Su Yanli; 10), 804-806.

[7]. Yang Liyun; Le Aiping, Study on massive blood transfusion in obstetric hemorrhage patients. Chinese Journal of Blood Transfusion 2013, 26(9), 903-905.

K.；Saner,F.；Eggebrecht,H.；Peters,J.；Hartmann,M.,Comparison of thrombelastometry withsimplified acute physiology score II and sequential organ failure assessmentscores for the prediction of 30-day survival:a cohort study.Shock 2011,35(4),339-342.[8]. Adamzik, M.; Langemeier, T.; Frey, UH;

K.; Saner, F.; Eggebrecht, H.; Peters, J.; Hartmann, M., Comparison of thrombelastometry withsimplified acute physiology score II and sequential organ failure assessmentscores for the prediction of 30-day survival: a cohort study.Shock 2011, 35(4), 339-342.

[9]. Zhang Yingjie; Wang Huijun; Hou Rongwei; Tong Jiufen;

[10]. Harris, L.F.; Castro-López, V.; Killard, A.J., Coagulation monitoring devices: Past, present, and future at the point of care. TrAC Trends in Analytical Chemistry 2013, 50, 85-95.

[11]. McMichael, M.A.; Smith, S.A., Viscoelastic coagulation testing: technology, applications, and limitations. Veterinary Clinical Pathology 2011, 40(2), 140-153.

[12]. Ganter, M.T.; Hofer, C.K., Coagulation monitoring: current techniques and clinical use of viscoelastic point-of-care coagulation devices. Anesthesia & Analgesia 2008, 106(5), 1366-1375.

[13]. Wan, P.; Yu, M.; Qian, M.; Tong, H.; Su, L., Sonoclot coagulation analysis: a useful tool to predict mortality in overt disseminatedintravascular coagulation. Blood Coagul Fibrinolysis 2016, 27 ( 1), 77-83.

[14].Cakmak,O.;Ermek,E.;Kilinc,N.;Bulut,S.;Baris,I.;Kavakli,I.;Yaralioglu,G.;Urey,H.,A cartridge based sensor array platform for multiplecoagulation measurements from plasma.Lab on a Chip 2015, 15(1), 113-120.

[15]. Maji, D.; Suster, M.A.; Kucukal, E.; Sekhon, U.D.; Gupta, A.S.; Gurkan, U.A.; Stavrou, E.X.; Mohseni, P., ClotChip: A Microfluidic Dielectric Sensor for Point-of-Care Assessment of Hemostasis. IEEE transactions on biomedicalcircuits and systems 2017.

[16]. Müller, L.; Sinn, S.; Drechsel, H.; Ziegler, C.; Wendel, H.-P.; Northoff, H.; Gehring, F.K., Investigation of prothrombin time in human whole-blood samples with a quartz crystal biosensor. Analytical chemistry 2009, 82(2), 658-663.

[17]. Puckett, L.G.; Lewis, J.K.; Urbas, A.; Cui, X.; Gao, D.; ), 1737-1743.

[18]. Tripathi, M.M.; Hajjarian, Z.; Van Cott, E.M.; Nadkarni, S.K., Assessingblood coagulation status with laser speckle rheology. Biomedical opticsexpress 2014, 5(3), 817-831.

[19]. Viola, F.; Mauldin, F.W.; Lin-Schmidt, X.; Haverstick, D.M.; Lawrence, M.B.; Walker, W.F., A novel ultrasound-based method to evaluate hemostatic function of whole blood. Clinica Chimica Acta 2010, 411(1), 106-113.

[20]. Xu, W.; Appel, J.; Chae, J., Real-time monitoring of whole bloodcoagulation using a microfabricated contour-mode film bulk acousticresonator. Journal of Microelectromechanical Systems 2012, 21(2), 302-307 .

S.,Force fieldevolution during human blood platelet activation.J Cell Sci 2012,125(16),3914-3920.[21] Henriques, SS; Sandmann, R.; State, A.;

S., Force fieldevolution during human blood platelet activation. J Cell Sci 2012, 125(16), 3914-3920.

[22] Lam, W.A.; Chaudhuri, O.; Crow, A.; Webster, K.D.; Kita, A.; Huang, J.; Fletcher, D.A., Mechanics and contraction dynamics of single platelets and implications for clot stiffening. Nature materials 2011 , 10(1), 61-66.

[23] Myers, D.R.; Qiu, Y.; Fay, M.E.; Tennenbaum, M.; Chester, D.; Cuadrado, J.; Sakurai, Y.; Baek, J.; Tran, R.; Ciciliano, J.C.; Ahn, B.; Mannino, R.G.; Bunting, S.T.; Bennett, C.; Briones, M.; Fernandez-Nieves, A.;

[24] Chen, Z.; Lu, J.; Zhang, C.; Hsia, I.; Yu, X.; Marecki, L.; Marecki, E.; Asmani, M.; Jain, S.; Neelamegham,

[25] Ting, L.H.; Feghhi, S.; Taparia, N.; Smith, A.O.; Karchin, A.; Lim, E.; John, A.S.; Wang, X.; Rue, T.; White, N.J., Contractile platelet forces in platelet aggregates under microfluidic shear gradients reflect inhibition and bleeding risk. Nature communications 2019, 10(1), 1204.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a miniaturized, low-cost, and easy-to-operate flexible microcolumn ring array for detecting clot contractility, and a preparation method and application thereof.

The object of the present invention is achieved through the following technical solutions:

A flexible micro-column ring array for detecting clot contractility, the micro-column ring array is composed of several micro-column rows arranged periodically, and each micro-column row has several micro-column rings, Each of the micro-column rings is composed of four or more annular micro-columns; the micro-column ring has a circular hole with a certain diameter, and when the clot contractility is detected, whole blood is dropped into the micro-column. The coagulation test is performed in the circular aperture of the microcolumn ring.

The invention utilizes the interfacial tension of the solid-liquid-gas system and the resistance of the micro-column to efficiently fix the whole blood sample in the micro-column ring, preventing the whole blood from flowing out of the body; meanwhile, the micro-column is used as a force sensor to measure the size of the clot contraction force in real time; Each micro-column in the micro-column ring is a force sensor, and the detection result is highly sensitive; when blood is dropped into the micro-column ring, when the blood coagulates and contracts, the micro-columns around the micro-column ring are driven to bend to produce displacement, and finally the micro-column produces The displacement is converted into the clot contractile force. The flexible micro-column ring array is used to detect clot contractility with low cost, high detection sensitivity, and easy clinical transformation; in addition, there is no need to separate platelets from blood, and whole blood can be used for detection. The operation is simple and fully considered. The effects of other components in blood on platelet contraction are more objective and comprehensive.

In a preferred embodiment of the present invention, the diameter of the micro-column ring is 1-3 mm, the spacing between each micro-column ring is 1-5 mm; the column height of the micro-column is 1.2-1.6 mm, and the upper surface of the column is 1.2-1.6 mm. Diameter 0.2 ~ 0.4mm.

The diameter and height of the micro-column, as well as the diameter of the micro-column ring and the number of micro-columns will affect the detection results. According to the cantilever beam theory, the diameter and height of the micro-column determine the detection sensitivity. Within a certain range, the smaller the diameter and the larger the column height, the higher the detection sensitivity. The diameter of the micro-column ring also affects the amount of blood dripped, and the number of micro-columns also needs to be controlled within an appropriate range. Whole blood is fixed in the micro-column ring by surface tension. If the number of micro-columns is too small, it will Increase the influence of blood deformation on the test results; if the number of micro-columns is too large, it will cause mutual interference between the micro-columns and affect the test results. When designing the micro-column ring array, the above factors should be considered together, and the surface tension between the solid-liquid interface should be fully used to fix the whole blood test sample to reduce the influence of blood deformation on the test results.

In a preferred embodiment of the present invention, the micro-column ring array is any one of a circular array, an elliptical array or a polygonal array; the micro-columns are cylindrical, elliptical or polygonal cylinders. either.

In a preferred embodiment of the present invention, each of the micro-column rings is composed of 4-8 micro-columns enclosing a ring.

In a preferred embodiment of the present invention, the volume of the whole blood is 3-6 μL.

The invention utilizes the interfacial tension of the solid-liquid-gas system and the resistance of the micro-column, so that the blood sample can be efficiently fixed in the micro-column ring. Sample testing. Also, the volume of instilled whole blood volume can affect the test results. The more blood volume, the greater the influence of blood deformation caused by gravity on the test results, because the blood deformation will cause irregular bending of the micro-columns, which will interfere with the experimental results. Too little blood may result in insufficient contact between the whole blood test sample and the microcolumn to detect clot contractility. Experiments found that the volume of whole blood dripped is affected by the diameter of the micro-column ring. When the diameter of the micro-column ring is 1-3 mm, the appropriate value range of the whole blood volume is 3-6 μL.

The present invention also provides a flexible micro-pillar ring array chip for detecting clot contraction force, comprising a bottom flexible substrate, and the flexible micro-pillar ring array, wherein the flexible micro-pillar ring array is arranged on the bottom layer on a flexible substrate.

The present invention also provides a method for preparing the flexible micro-pillar ring array chip for detecting clot contractility, comprising the following steps:

S1. Use simulation software to simulate the scene of the micro-column bending under force, process and design the micro-column, determine the feasible size and actual spring coefficient of the micro-column, and the relevant parameters of the PDMS material;

S2. According to the design result of S1, a mold is obtained by cutting an acrylic plate with a laser, and finally, PDMS is used for reverse molding to produce the flexible micro-pillar ring array chip for detecting clot contraction force.

In the invention, the method of laser engraving and flexible material PDMS moulding is used to process the micro-pillar ring array or the micro-pillar ring array chip. The flexible substrate of the present invention and the micro-column ring array on the flexible substrate can be processed simultaneously. Both are obtained by pouring the flexible material PDMS. The PDMS polymer has good biocompatibility and is widely used in the field of biomaterials.

The preparation method of the invention is simple, the data collection is easy, and the defect of the multi-layer microlithography and soft lithography technology in the prior art that the processing of the micro-pillars requires complex processing methods and equipment is well overcome. The existing method requires precise processing instruments, and the processed structures are micro-column pairs, micro-column blocks, etc. The size of the obtained micro-columns is in the order of microns and the spring coefficient is small. The micro-column of the present invention adopts the method of laser engraving and PDMS injection to obtain a micro-column ring structure with a micro-column size of millimeter level, the spring coefficient is large, which can meet the needs of actual detection, the processing technology is simple, and complex processing equipment is not required. . In addition, unlike the reports in the existing literature that only use platelets as the detection sample, the present invention uses whole blood as the detection sample, fully considers the effects of other components in the blood on platelet contraction, and the detection results are more objective and comprehensive.

In a preferred embodiment of the present invention, the method for determining the feasible size of the micropillar described in S1 is as follows: changing the magnitude of the boundary load within the range of 30-50 μN, adjusting the size of the micropillar, and calculating the size of the micropillar by simulation. The bending displacement is used to determine the theoretically feasible micro-pillar size corresponding to the bending displacement, which is used to guide the design of the actual micro-pillar size.

In a preferred embodiment of the present invention, the method for determining the actual spring coefficient of the micro-column described in S1 is as follows: applying boundary loads of different sizes to the half sides of the micro-column, simulating and calculating the bending displacement of the micro-column, and calculating according to Hooke's law The boundary load and bending displacement are linearly fitted, and the obtained slope is the actual spring coefficient of the microcolumn.

In a preferred embodiment of the present invention, the method for obtaining a mold by laser cutting an acrylic sheet described in S2 is as follows: tear off the film on one side of the acrylic sheet, stick double-sided tape on the side, and keep the acrylic sheet on the other side. Plate film; when cutting, the side with double-sided tape will face the other side of the laser head, and the acrylic plate will be cut in the dot mode of the CAD software.

In a preferred embodiment of the present invention, the method of using PDMS for pouring described in S2 is as follows: mixing the PDMS matrix and the curing agent in a mass ratio of 10-20:1 and stirring evenly, vacuuming and degassing to obtain a mixed solution, and then adding Heating and curing at 55～65℃ for 6～10h to make PDMS solidify; after cooling, it can be obtained by cutting.

In a preferred embodiment of the present invention, the Young's modulus, Poisson's ratio, and density of the PDMS material are respectively 200-400 kPa, 0.4-0.5, and 900-1000 kg/m ³ .

The present invention also provides a blood coagulation testing system, including an experimental recording component and a temperature control component; wherein, the experimental recording component includes a microscope, a camera and a computer, and the microscope is connected with the computer through the camera to record the displacement of the top of the micro-column in real time Change; the temperature control assembly includes a temperature controller, a heating belt and a temperature control box, the heating belt is located around the temperature control box, the temperature controller is connected with the heating belt, and the flexible micro-column ring array is detected during detection. The chip can be placed in the temperature control box.

Correspondingly, the application of the flexible micro-column ring array, the flexible micro-column ring array chip, or the blood coagulation testing system in blood testing also falls within the protection scope of the present invention.

Compared with the prior art, the beneficial effects of the present invention are:

The invention provides a miniaturized, low-cost, convenient and flexible micro-column ring array for detecting clot contractility, and a preparation method and application thereof. The flexible micro-column ring array of the present invention has the characteristics of simple operation, simple processing, easy data acquisition and the like, and can achieve low-cost detection. In addition, the ingenious design of the micro-column ring enables the blood sample to be efficiently fixed in the micro-column ring by the combined force of the interfacial tension between the solid, gas and liquid of the micro-column, air and blood. The experimental operation is simple and fast, and the micro-column The small size of the ring allows for very low sample consumption, allowing for a small amount of detection. Moreover, it is designed as a microcolumn ring array, which can achieve high sensitivity, high throughput and multi-target detection. At the same time, the present invention uses whole blood as a sample, fully considers the influence of other blood components on the clot contractility, and can more comprehensively evaluate the clot contractility produced by platelets.

Description of drawings

Figure 1 is a schematic diagram of the generation of clot contractility.

Fig. 2 is the method for detecting clot contractility reported in the prior art literature; wherein, Fig. 2(a) is the contractile force of a single platelet detected by traction force microscopy; Fig. 2(b) is a single platelet captured by fibrinogen for detection Platelet contractility; Fig. 2(c) is a high-throughput platelet detection device; Fig. 2(d) is a study of the effect of fluid shear force on platelet contractility with a micro-column block; Fig. 2(e) is a micro-column The array pair acts as a force sensor to calculate platelet contractility under fluid shear using the deflection of the micropillars, and the underlying stretchable membrane to measure the change in stiffness of platelet aggregates before and after coagulation.

Fig. 3 is a schematic diagram of a single flexible micro-pillar ring and a flexible micro-pillar ring array chip, wherein Fig. 3(a) is a single flexible micro-pillar ring, and Fig. 3(b) is a flexible micro-pillar ring array chip.

FIG. 4 is a schematic flow chart of the flexible micropillar ring array of the present invention from design, manufacture to use.

Figure 5 is the mechanical model of the micropillar bending; Figure 5(a) is the deformation and stress distribution of the micropillar in the numerical simulation; Figure 5(b) is the calculation result of the spring coefficient of the micropillar.

FIG. 6 is a schematic diagram of the processing of the flexible micropillar ring array and its chip.

Fig. 7 is a blood coagulation test system in practical application; Fig. 7(a) is a blood coagulation test system, and Fig. 7(b) is a blood coagulation test chip.

Fig. 8 is the image processing flow of blood coagulation test.

Fig. 9 is the validity verification of the flexible micropillar ring array and its chip of the present invention; Fig. 9(a) is a graph of the experimental result of clot contractility; Fig. 9(b) is a curve of clot contractility.

Figure 10 shows the test results of clot contractility in patients with normal and weak coagulation function.

Figure 11 is a schematic diagram of the structure of the blood coagulation testing system, wherein 1-microscope, 2-camera, 3-computer, 6-temperature controller, 5-heating belt, 4-temperature control box, 7-flexible micro-column ring array chip.

Detailed ways

The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Embodiment 1 A flexible micropillar ring array and its chip for detecting clot contractility

As shown in Figure 3, a flexible micro-column ring array for detecting clot contractility, wherein the micro-column ring array is composed of several micro-column rows arranged periodically, and each micro-column row has several Micro-column ring, each micro-column ring consists of four annular micro-columns; the micro-column ring has a circular hole with a certain diameter, when the clot contractility is detected, whole blood is dropped into the micro-column ring. Coagulation tests are performed in circular pores. The diameter of the micro-column ring is 1 mm, and the spacing of each micro-column ring is 1 mm; the column height of the micro-column is 1.2 mm, and the diameter of the upper surface of the column is 0.2 mm. The micropillar ring array is a circular array; the micropillars are cylindrical.

In addition, as shown in (b) of FIG. 3 , this embodiment further provides a flexible micro-column ring array chip for detecting clot contractility, including a bottom flexible substrate and the above-mentioned flexible micro-column ring array , the flexible micro-pillar ring array is arranged on the underlying flexible substrate. In this embodiment, 12 micro-column rings are arranged in 4*3 to form a flexible micro-column ring array, that is, a blood coagulation test chip. The flexible micro-column ring array is composed of two parts, one is the bottom flexible substrate, and the other is several micro-column ring arrays on the bottom flexible substrate. Both are obtained by simultaneous PDMS injection molding.

Example 2 A flexible micropillar ring array and its chip for detecting clot contractility

As shown in Figure 3, a flexible micro-column ring array for detecting clot contractility, wherein the micro-column ring array is composed of several micro-column rows arranged periodically, and each micro-column row has several Micro-column ring, each micro-column ring consists of four annular micro-columns; the micro-column ring has a circular hole with a certain diameter, when the clot contractility is detected, whole blood is dropped into the micro-column ring. Coagulation tests are performed in circular pores. The diameter of the micro-column ring is 3 mm, and the spacing of each micro-column ring is 5 mm; the column height of the micro-column is 1.6 mm, and the diameter of the upper surface of the column is 0.4 mm. The micro-column ring array is a circular array, an elliptical array or a polygonal array; the micro-columns are cylindrical, elliptical or polygonal.

In addition, as shown in (b) of FIG. 3 , this embodiment further provides a flexible micro-column ring array chip for detecting clot contractility, including a bottom flexible substrate and the above-mentioned flexible micro-column ring array , the flexible micro-pillar ring array is arranged on the underlying flexible substrate. In this embodiment, 12 micro-column rings are arranged in 4*3 to form a flexible micro-column ring array, that is, a blood coagulation test chip. The flexible micro-column ring array is composed of two parts, one is the bottom flexible substrate, and the other is several micro-column ring arrays on the bottom flexible substrate. Both are obtained by simultaneous PDMS injection molding.

Embodiments 1 and 2 of the present invention utilize the interfacial tension of the solid-liquid-gas system and the resistance of the micro-column to efficiently fix the whole blood sample in the micro-column ring, preventing the outflow of the whole blood; meanwhile, the micro-column is used as a force sensor to measure real-time The size of the clot contraction force; each micro-column in the micro-column ring is a force sensor, and the detection results are highly sensitive; blood is dropped into the micro-column ring, and when the blood coagulates and contracts, it drives the micro-columns around the micro-column ring to bend Displacement is generated, and finally the displacement generated by the micropillars is converted into clot contractile force. The flexible micro-column ring array is used to detect clot contractility with low cost, high detection sensitivity, and easy clinical transformation; in addition, there is no need to separate platelets from blood, and whole blood can be used for detection. The operation is simple and fully considered. The effects of other components in blood on platelet contraction are more objective and comprehensive.

Embodiment 3 A kind of flexible micro-pillar ring array for detecting clot contractility and preparation method of its chip

A method for preparing the flexible micropillar ring array chip for detecting clot contractility, comprising the following steps:

S1. Use Comsol simulation software to simulate the scene of micro-column bending under force, process and design the micro-column, determine the feasible size and actual spring coefficient of the micro-column, and the relevant parameters of the PDMS material;

S2. According to the design result of S1, a mold is obtained by cutting an acrylic plate with a laser, and finally, PDMS is used for reverse molding to produce the flexible micro-pillar ring array chip for detecting clot contraction force.

In the invention, the method of laser engraving and flexible material PDMS moulding is used to process the micro-pillar ring array or the micro-pillar ring array chip. Specifically:

1. Theoretical Simulation

The Comsol simulation software was used to simulate the bending of the micro-column, and the feasible size of the micro-column was preliminarily determined, and the spring coefficient of the micro-column was calculated for the calculation of the clot contraction force.

Utilize solid mechanics models in Comsol simulation software. The bottom end of the micro-column is fixed, and a certain boundary load is applied to the half side of the micro-column to simulate the clot shrinkage force on the half-side of the micro-column. The Young's modulus, Poisson's ratio and density of the fixed PDMS material are 300kPa and 0.5 respectively. , 970kg/m ³ , the bending degree of the micro-pillar is only affected by the boundary load and the size of the micro-pillar. Use Comsol simulation software to determine the feasible size of micro-pillar in theory and calculate the spring coefficient of the micro-pillar. The specific method is:

(1) As shown in Figure 5(a), the boundary load is appropriately changed within a reasonable range of 30-50 μN, and the size of the micro-column is adjusted. The ideal bending displacement of the micro-column is simulated and calculated, so as to determine the corresponding displacement of the micro-column. The theoretically feasible micro-column size below is used to guide the design of the actual micro-column size; the actual diameter and height of the obtained micro-column are 167 μm and 1.332 mm, respectively;

(2) As shown in Figure 5(b), the spring coefficient of the micro-column is calculated by the Comsol simulation software, which is used for the calculation of the clot contraction force, and the boundary loads of different sizes are applied to the half sides of the micro-column. According to Hooke's law, the slope obtained by linear fitting is the actual spring coefficient of the micro-column.

The main determinant of the volume of whole blood instilled is the diameter of the microcolumn ring, and the height of the microcolumn also affects the volume of whole blood instilled. The standard for the appropriate volume of whole blood dripped into the micro-column ring is that the side of each micro-column facing the center of the micro-column ring can fully contact the blood sample, so the larger the diameter of the micro-column ring and the height of the micro-column, the greater the drop in the micro-column ring. The volume of whole blood injected is also larger. At the same time, the volume of whole blood will also affect the test results. The more blood volume, the greater the influence of blood deformation caused by gravity on the test results, because the blood deformation will cause irregular bending of the micro-columns, which will interfere with the experimental results. Too little blood may result in insufficient contact between the whole blood test sample and the microcolumn to detect clot contractility. For example, when the diameter of the micro-column ring is 2 mm and the height of the micro-column is 1.4 mm, the volume of whole blood is suitable in the range of 4-5 μL.

The interfacial tension of the solid-liquid-gas system and the resistance of the microcolumn make the blood sample immobilized in the microcolumn ring efficiently. Specifically, it refers to the interfacial tension force of the three interfaces between the whole blood body and the micro-column solid, the whole blood body and the solid substrate, and the whole blood body and the air, and the resistance of the micro-column to prevent the outflow of the whole blood. Therefore, the number of micro-columns and the diameter of the micro-columns affect to a certain extent whether the whole blood droplet can be efficiently fixed in the micro-column ring, so as to minimize the deformation of the whole blood droplet. Generally speaking, the larger the number of micro-columns in the micro-column ring, the larger the diameter of the micro-columns, and the larger the contact surface between the whole blood droplet and the micro-column, the more conducive to the fixation of the whole blood droplet. However, the number of micro-pillars is not as large as possible, and the diameter of the micro-pillars is not as large as possible. The more the number of micro-pillars and the larger the diameter, the interference between the micro-pillars will lead to irregular bending of the micro-pillars; More critically, if the diameter of the microcolumn is too large or too small, it will affect the detection sensitivity of the whole blood clot contractility.

In the present invention, the diameter of the micro-column ring is designed to be 1-3 mm, and the spacing of each micro-column ring is designed to be 1-5 mm; the column height of the micro-column is designed to be 1.2-1.6 mm, and the diameter of the upper surface of the column is 0.2-0.4 mm. Under the condition that the blood sample can be fully contacted with the micro-columns around the micro-column ring, a small amount of sample detection can be achieved.

2. Device processing

We use the methods of laser cutting and PDMS injection molding to obtain the micro-column ring device.

The first is CAD design drawings for laser cutting. The diameter and height of the micro-column ring determine the amount of blood dripped into the micro-column ring. 12 micro-column ring devices as shown in Fig. 3(a) can be processed on one chip as a blood coagulation test chip, and the interval between each micro-column ring device is designed to be 1 mm to realize high-throughput detection. Multiple groups of such blood coagulation testing chips can be processed at one time. As shown in Figure 3(b), 12 groups of such blood coagulation testing chips can be processed at one time, and the interval between each blood coagulation testing device is designed to be 3 mm.

FIG. 6 is a schematic diagram of the processing of the flexible micropillar ring array and its chip. Cut the acrylic plate with a laser engraving machine to obtain a mold, and use PDMS to reverse the mold to finally obtain a flexible micro-pillar ring array chip (ie, a blood coagulation test chip).

Among them, the specific method of cutting the acrylic plate by the laser engraving machine to obtain the mold is as follows: tear off the film on one side of the acrylic plate, stick double-sided tape on this side, and keep the acrylic plate film on the other side to protect the mold from being dirty. And when cutting, the side with double-sided tape faces the other side of the laser head, which is convenient for PDMS demolding. The acrylic plate is cut in the dot pattern of the CAD software, and three dot parameters determine the final size of the micro-column, including the diameter and height of the micro-column. For example, in the three dotting parameters, the number of dots and the interval time are fixed to 2 and 10ms, respectively, and the single-dot processing time is changed to 12ms, the diameter and height of the micropillars are 0.292mm and 1.398mm, respectively. After cutting a certain number of coagulation test chip molds, such as the number of 12 groups of blood coagulation test chip molds in Figure 3(b), properly clean the laser head and remove the powder produced by the previous cutting to avoid contamination of the micro-columns obtained by subsequent processing. and affect subsequent image processing.

The specific method of PDMS injection mold is: paste the mold obtained by laser engraving machine on a petri dish with a diameter of 90mm, for example, according to the design in Figure 3(b), mix 15-20g of PDMS matrix and its curing agent at a ratio of 20:1 Stir evenly, vacuum and degas, and then pour the mixture into the petri dish with the mold attached, so that the processed substrate is about 1.5mm, the thickness is suitable, and the PDMS material will not be wasted. The petri dish was then placed in a 60°C oven with a flat substrate, and baked for about 10 hours to cure. Finally, the cured PDMS is carefully peeled off, and a single coagulation test chip is obtained by cutting, which can be used for the whole blood clot contractility test.

Embodiment 4 A kind of coagulation test system for detecting clot contractility

The flexible micro-column ring array chips of the above-mentioned Example 1, Example 2 and Example 3 were used to build a blood coagulation test system, which was used to record the displacement changes of the top of the micro-column during blood coagulation in real time, and to control the temperature of the blood coagulation test.

As shown in Figures 7 and 11, a blood coagulation testing system includes an experiment recording component and a temperature control component; wherein, the experiment recording component includes a stereo microscope 1, a camera 2 and a computer 3, and the stereo microscope 1 communicates with the computer through the camera 2 3 is connected, and the displacement change of the top of the micro-column is recorded in real time; the temperature control assembly includes a temperature controller 6, a heating belt 5 and a temperature control box 4, the heating belt 5 is located around the temperature control box 4, and the temperature controller 6 is connected with the heating belt 5 , during detection, put the flexible micro-column ring array chip 7 into the temperature control box 4, start the microscope 1 to shoot, and record the displacement change of the top of the micro-column.

As shown in FIG. 4 , it is a schematic diagram of the process flow from design, manufacture to use of the flexible micro-pillar ring array of the present invention. After setting up the coagulation test system, drop a small amount of whole blood into the micro-column ring, the blood coagulation shrinks and the micro-column is bent and displaced. The displacement of the micro-column is calculated by the simulation method, and then the clot contraction force is calculated by multiplying the displacement by the spring coefficient according to Hooke's law.

Example 5 Coagulation test

First, collect blood samples of patients for coagulation function testing from the Laboratory Department of Shenzhen University General Hospital, draw 200 μL of uncentrifuged whole blood samples from the hospital blue tube blood collection tube at one time, and ensure that the experiment is completed within 6 hours after sample collection. The blue tube blood collection tube is a special tube for blood coagulation test in the hospital. Sodium citrate is added in the tube in advance to prevent blood coagulation. The strength of the patient's coagulation function can be determined in advance through the four-item coagulation report of the coagulation function test in the hospital, namely: thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (aPTT) And the test results of prothrombin activity (Pa) were compared with the normal range to judge the coagulation function of the patient.

1. The specific process of coagulation test is roughly as follows:

1. Turn on the temperature controller, so that the temperature in the box is about 37℃;

2. Use a pipette to take a certain volume of patient blood sample into a 1.5mL small test tube, add CaCl ₂ solution with a concentration of 0.2mol/L in a volume ratio of 17:1, and use a pipette to suck and release two to three times. CaCl ₂ is mixed with whole blood evenly; CaCl ₂ is used to neutralize the effect of sodium citrate, an anticoagulant added during blood collection, to make blood coagulate;

3. Put the flexible micro-column ring array chip (i.e. blood coagulation test chip) of Example 3 into a petri dish with a diameter of 30mm;

4. Take an appropriate volume of the mixed solution of whole blood and CaCl ₂ and quickly drop them into the 12 micro-column rings in the coagulation test chip;

5. Drop an appropriate amount of water droplets into the petri dish to reduce water evaporation, and cover the petri dish;

6. Put the petri dish into the temperature control box, cover the temperature control box, and start the microscope to take a picture at regular intervals.

7. The coagulation test is over.

Second, the construction of image processing methods

An image processing method was constructed to analyze the real-time coagulation test images captured by the microscope to obtain the clot contractility curve.

As shown in Figure 8, firstly, a single micro-column is cut out, and grayscale processing is performed on it to convert the RGB three channels into a single channel; then the grayscale image is binarized, so that the grayscale values are only 0 and 0. 255, even if the image is either black or white; look for connected domains, connect areas with the same gray value and adjacent positions, and then fit the connected domains at the top of the micro-pillars with circumscribed circles; the program monitors the circumscribed circles in real time The coordinates of the center of the circle change to obtain a distance curve, in which the abscissa frame is converted into time, and the ordinate pixel is converted into displacement, and then according to Hooke's law, the micro-column displacement is multiplied by the simulation. The spring coefficient of the micro-column can be calculated The size of the clot contraction force.

3. Validation of the device

After the device is completed and the related methods are constructed, a blank control is designed in the present invention to verify the effectiveness of the device, that is, to verify whether the device can successfully detect the clot contractility.

1. Blank control experiment

As shown in Figure 9, the diameter of the micro-column used in this experiment is 292 μm, and the height of the column is 1.398 mm; the diameter of the micro-column ring is 2 mm, the number of micro-columns is 4, and the amount of blood dripped into each micro-column ring device is 4.5 μL.

The experiment was divided into two groups. One group added CaCl ₂ solution with a concentration of 0.2mol/L in a volume ratio of 17:1 to neutralize the effect of sodium citrate, an anticoagulant added during blood collection, to make blood coagulate; the other group No reagents are added, because the blood collected contains the anticoagulant sodium citrate, and the blood will never clot.

According to the cross mark in Figure 9(a), compared with the group without CaCl ₂ , it is compared whether the top of the micro-column in the group with CaCl ₂ is bent toward the center of the micro-column ring.

It can be seen from Figure 9(a) that the micropillars in the group with CaCl ₂ are obviously bent towards the center of the micropillar ring, while the micropillars in the group without CaCl ₂ do not move at all, and the actual detected displacement of the top of the micropillars is in 40～80μm. Figure 9(b) is a graph of clot contractility, and there is a significant difference in the clot contractility between the CaCl ₂ group and the non-CaCl ₂ group. The results of Figure 9(a) and Figure 9(b) show that the flexible micro-column ring array and flexible micro-column ring array chip designed by the present invention can successfully detect the clot contraction force, that is, the effectiveness of the device has been verified.

2. Differential test for normal and abnormal coagulation function

Figure 10 shows the test results of clot contractility in patients with normal and weak coagulation function. The status of normal coagulation function and weak coagulation function can be known from the test results of the hospital in advance.

The diameter of the microcolumn, the height of the column, the diameter of the microcolumn ring, the number of the microcolumn, and the sample volume detected are the same as in Fig. 9 .

The weak coagulation function of the patient samples was manifested in clinically high prothrombin activity (Pa) and thrombin time (TT) (the lower curve in Figure 10). Figure 10 shows that there are obvious differences in the clot contractility curves obtained by the two groups of experiments, that is, the clot contractility of patients with weak coagulation function (the lower curve in Figure 10) is significantly lower than that of patients with normal coagulation function. (the upper curve in Fig. 10 ), which further illustrates that the design of the present invention can successfully detect the clot contractility, and even distinguish patient samples with normal and abnormal coagulation function.

In conclusion, the present invention provides a miniaturized, low-cost, and easy-to-operate flexible microcolumn ring array for detecting clot contractility, and a preparation method and application thereof. The flexible micro-column ring array of the present invention has the characteristics of simple operation, simple processing, easy data acquisition and the like, and can achieve low-cost detection. In addition, the ingenious design of the micro-column ring enables the blood sample to be efficiently fixed in the micro-column ring by the combined force of the interfacial tension between the solid, gas and liquid of the micro-column, air and blood. The experimental operation is simple and fast, and the micro-column The small size of the ring allows for very low sample consumption, allowing for a small amount of detection. Moreover, it is designed as a microcolumn ring array, which can achieve high sensitivity, high throughput and multi-target detection. At the same time, the present invention uses whole blood as a sample, fully considers the influence of other blood components on the clot contractility, and can more comprehensively evaluate the clot contractility produced by platelets.

The above is only used to illustrate the technical solution of the present invention and not to limit it. Other modifications or equivalent replacements made by those of ordinary skill in the art to the technical solution of the present invention, as long as they do not depart from the spirit and scope of the technical solution of the present invention, should be Included within the scope of the claims of the present invention.

Claims (10)

1. a flexible micro-column ring array for detecting clot contractility, wherein the micro-column ring array is composed of several periodically arranged micro-column rows, and each of the micro-column rows has Several micro-column rings, each of which is composed of four or more annular micro-columns; the micro-column ring has circular pores with a certain diameter, and when detecting the clot contraction force, The coagulation test was performed by dripping whole blood into the circular aperture of the microcolumn ring. 2 . The flexible micro-column ring array according to claim 1 , wherein the diameter of the micro-column ring is 1-3 mm, and the spacing of each micro-column ring is 1-5 mm; the column height of the micro-column is 1-5 mm . It is 1.2-1.6mm, and the diameter of the upper surface of the column is 0.2-0.4mm. 3. The flexible micro-pillar ring array according to claim 2, wherein the micro-pillar ring array is any one of a circular array, an elliptical array or a polygonal array; the micro-pillars are cylindrical, Either an elliptical cylinder or a polygonal cylinder. 4 . The flexible micro-column ring array according to claim 3 , wherein each of the micro-column rings is composed of 4-8 micro-columns enclosing a ring. 5 . 5 . A flexible micro-pillar ring array chip for detecting clot contraction force, characterized in that it comprises a flexible substrate at the bottom layer, and the flexible micro-pillar ring array according to any one of claims 1 to 4 , wherein the flexible micro-pillar ring array An array of pillar rings is disposed on the underlying flexible substrate. 6. A method for preparing the flexible micro-column ring array chip for detecting clot contraction force according to claim 5, characterized in that, comprising the following steps: S1. Use the simulation software to simulate the scene of the micro-pillar being subjected to force and bending, process and design the micro-pillar, determine the feasible size and actual spring coefficient of the micro-pillar, and the relevant parameters of the polydimethylsiloxane (PDMS) material; S2. According to the design result of S1, a mold is obtained by cutting an acrylic plate with a laser, and finally PDMS is used for reverse molding to produce the flexible micro-pillar ring array chip for detecting clot contraction force. 7 . The method according to claim 6 , wherein the method for determining the feasible size of the micropillar in S1 is: changing the size of the boundary load within the range of 30-50 μN, and adjusting the size of the micropillar, simulating the Calculate the bending displacement of the micro-pillar, determine the theoretically feasible micro-pillar size corresponding to the bending displacement, and use it to guide the design of the actual micro-pillar size; The method for determining the actual spring coefficient of the micro-column described in S1 is as follows: applying boundary loads of different sizes to the half sides of the micro-column, calculating the bending displacement of the micro-column by simulation, and linearly fitting the boundary load and bending displacement according to Hooke's law, The obtained slope is the actual spring coefficient of the micropillar; The method for obtaining a mold by laser cutting an acrylic sheet described in S2 is as follows: tear off the film on one side of the acrylic sheet, stick double-sided tape on the side, and keep the acrylic sheet film on the other side; when cutting, there will be double-sided tape One side of the laser head is facing the other side of the laser head, and the acrylic plate is cut in the dot mode of the CAD software; The method of using PDMS for mold injection described in S2 is as follows: mix the PDMS matrix and the curing agent in a mass ratio of 10-20:1 and stir evenly, vacuum and degas to obtain a mixed solution, and then heat and solidify at 55-65 ° C for 6-10 hours Allow PDMS to solidify; cut after cooling. The method according to claim 6, wherein the Young's modulus, Poisson's ratio and density of the PDMS material are respectively 200-400 kPa, 0.4-0.5, 900-1000 kg/m ³ . 9. A blood coagulation testing system, characterized in that it comprises an experimental recording component and a temperature control component; wherein, the experimental recording component comprises a microscope, a video camera and a computer, and the microscope is connected with the computer through the video camera, and records the real-time recording of the micro-column top. Displacement changes; the temperature control assembly includes a temperature controller, a heating belt and a temperature control box, the heating belt is located around the temperature control box, the temperature controller is connected with the heating belt, and the flexible micro-column ring is connected during detection. The array chip can be put into the temperature control box. 10 . The application of the flexible micro-column ring array according to any one of claims 1 to 4 , the flexible micro-column ring array chip according to claim 5 , or the blood coagulation testing system according to claim 9 in blood testing. 11 .

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