WO2022105869A1 - 一种异质结构的结构色微纤维及其制备方法和心肌细胞检测方法 - Google Patents

一种异质结构的结构色微纤维及其制备方法和心肌细胞检测方法 Download PDF

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WO2022105869A1
WO2022105869A1 PCT/CN2021/131723 CN2021131723W WO2022105869A1 WO 2022105869 A1 WO2022105869 A1 WO 2022105869A1 CN 2021131723 W CN2021131723 W CN 2021131723W WO 2022105869 A1 WO2022105869 A1 WO 2022105869A1
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structural color
microfibers
cardiomyocyte
cardiomyocytes
microfiber
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French (fr)
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赵远锦
陈卓玥
王月桐
池俊杰
张大淦
商珞然
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南京鼓楼医院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B01J19/12—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electromagnetic waves
    • B01J19/122—Incoherent waves
    • B01J19/123—Ultraviolet light
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F1/00—General methods for the manufacture of artificial filaments or the like
    • D01F1/02—Addition of substances to the spinning solution or to the melt
    • D01F1/10—Other agents for modifying properties
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F6/00—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof
    • D01F6/44—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from mixtures of polymers obtained by reactions only involving carbon-to-carbon unsaturated bonds as major constituent with other polymers or low-molecular-weight compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
    • G01B11/00—Measuring arrangements characterised by the use of optical techniques
    • G01B11/16—Measuring arrangements characterised by the use of optical techniques for measuring the deformation in a solid, e.g. optical strain gauge

Definitions

  • the invention belongs to the field of biomedical materials, and in particular relates to a heterostructured structural color microfiber, a preparation method thereof, and a cardiomyocyte detection method.
  • the heart is one of the most important organs in the human body. It provides sufficient blood flow to organs and tissues throughout the body and maintains the normal metabolism and function of organs and tissue cells.
  • the cyclical contractions produced by cardiomyocytes are an important prerequisite for the functioning of the heart, and if cardiomyocytes are damaged, heart defects can result.
  • the research and development, screening and evaluation of cardiomyocyte drugs have become one of the urgent problems in the biomedical field.
  • a sensing platform that can solve real-time monitoring and synchronous feedback of cardiomyocytes is also a challenge in this field.
  • Photonic crystal materials are a class of periodic micro-nano-structured materials with two or more refractive indices, because they can control the movement of photons within them and reflect electromagnetic waves of a certain wavelength, so they have the unique characteristics of photonic band gaps and structural colors. nature. By adjusting the structural composition or refractive index of photonic crystal materials, the control of its structural color can be achieved.
  • the present invention provides a microfluidic chip-based structural color microfiber with an axial heterostructure and a preparation method thereof.
  • the cardiomyocyte detection method is characterized in that, through the periodic contraction of cardiomyocytes, the color area of the microfiber structure is pulled to produce deformation and color changes, so as to realize the visual detection and sensing of cardiomyocytes.
  • the microfiber can be applied in technical fields such as cardiomyocyte physiological signal conversion, visual detection and the like.
  • the present invention provides a method for preparing a heterostructured structural color microfiber, which has the following characteristics: a microfluidic chip is used to construct a microfluidic chip for a heterostructured structural color microfiber preparation platform; As the fiber component, the prepolymerization solution is divided into two categories. One kind of prepolymerization solution is a biocompatible hydrogel solution, and the other kind of prepolymerization solution is at least one concentration of silica non-closely packed colloidal crystals.
  • microfibers If the microfluidic speed corresponding to one of the components is increased, and the passage time is prolonged, the length of the fiber where the corresponding component is located will increase accordingly; if the diameter of the collection tube capillary is increased or decreased, the fiber diameter will increase accordingly. or decrease; increase or decrease the number of liquid injection ports and the number of prepolymerized liquid components, and the components contained in the structural color microfibers of the heterostructure will increase or decrease accordingly.
  • the present invention provides a method for preparing a heterostructured structural color microfiber, which may also have the following characteristics: wherein, the biocompatible hydrogel solution is a methyl group with a concentration of 10-30% by mass by volume. Acrylated gelatin solution.
  • the present invention provides a method for preparing a heterostructured structural color microfiber, which may also have the following characteristics: wherein the silica non-closely packed colloidal crystal pregel is sulfonate modified silica particles Dispersed in an aqueous solution of one or more materials of acrylamide (AM), isopropyl acrylamide (NIPAM), polyacrylamide (PAM), polyethylene glycol diacrylate (PEGDA), which contains acrylamide A cross-linking agent is also added to the aqueous solution of one or two or more materials of polyacrylamide and isopropylacrylamide; the mass volume ratio of the sulfonic acid modified silica particles is 5%-60% (w/v ). Different concentrations of sulfonate-modified silica particles show different structural colors, and multi-component color coding can be achieved in multi-component heterostructured structural color microfibers.
  • AM acrylamide
  • NIPAM isopropyl acrylamide
  • PAM polyacrylamide
  • the present invention provides a method for preparing a heterostructured structural color microfiber, which may also have the following characteristics: wherein, the method for constructing a microfluidic chip is: according to the required diameter of the structural color microfiber, selecting the same pipe diameter
  • the capillary is used as the collection tube; according to the required number of components, set the same number of capillary injection ports, and assemble the microfluidic chip with the collection tube on the glass slide. Both the collection tube and the injection port are fixed on the glass slide.
  • the ports are connected to the collection tube; the biocompatible hydrogel solution and the silica non-close-packed colloidal crystal pregel are injected into the liquid injection port through a syringe and a hose, respectively, connected to the microfluidic chip, and passed through The microfluidic chip was programmed to alternately pump biocompatible hydrogel solutions and silica non-close-packed colloidal crystal pregels.
  • the present invention also provides the heterostructured structural color microfibers prepared by the above method, with a diameter of 150-600 ⁇ m and a length of 500-3000 mm.
  • the present invention also provides a method for detecting cardiomyocytes of the heterostructured structural color microfibers, which has the following characteristics: the heterostructured structural color microfibers are used as a cardiomyocyte culture substrate, and the biocompatible hydrogel
  • the part formed after solution polymerization is the cardiomyocyte growth area, and the cardiomyocyte grows on the cardiomyocyte growth area.
  • the part formed by the pregelation of silica non-closely packed colloidal crystals has a structural color and is sensitive to stretching.
  • Detection area When the cardiomyocytes resume their voluntary beating, their periodic contraction pulls the structural color detection area, and the structural color detection area produces structural color changes that are synchronized with the myocardial contraction cycle, thereby detecting cardiomyocytes.
  • the present invention provides a method for detecting cardiomyocytes of heterostructured structural color microfibers, which may also have the following features: a method for detecting cardiomyocytes by single-end stretching: respectively fix the heterostructured structural color microfibers in the middle phase
  • a method for detecting cardiomyocytes by single-end stretching respectively fix the heterostructured structural color microfibers in the middle phase
  • the present invention provides a method for detecting cardiomyocytes of structural color microfibers with heterostructures, which may also have the following features: a method for detecting cardiomyocytes stretched at both ends: the structural color microfibers with heterostructures are respectively fixed by The distal end of two cardiomyocyte growth regions (biocompatible hydrogel portion) separated by a structural color detection region (silica non-close-packed colloidal crystal portion), where the distal end refers to a distance away from the structural color detection region One end of the interface; cardiomyocytes grow in the two segments of cardiomyocyte growth area (biocompatible hydrogel part), and in the case where the structural color microfibrils of the heterostructure are fixed at two places, the structural color detection area (dioxide) The myocardial cell contraction on both sides of the silicon non-close-packed colloidal crystal part) will pull it from left and right directions in a similar "tug-of-war" manner to produce deformation and structural color changes, and convert myocardial mechanical signals into optical signals to detect cardiomyocytes
  • the present invention provides a method for detecting cardiomyocytes of heterostructured structural color microfibers, which can also have the following characteristics: the heterostructured structural color microfibers are used as a cardiomyocyte culture substrate, and the specific culture process is: After sterilizing the prepared structural color microfibers of the heterogeneous structure, place them in a clean cell culture dish; after the cardiomyocytes are fully dispersed with the culture medium, a cell suspension is formed, and the cell suspension is added dropwise to the cells with the heterogeneous structure.
  • the present invention uses structural color microfibers with axial heterostructures as the cardiomyocyte culture and sensing substrate, and has the characteristics of convenient, sensitive, real-time, and visual detection and sensing.
  • the structural color microfiber proposed in the present invention is an axially heterogeneous fiber, which separates the optical sensing area from the cell growth area; the cells do not adhere to the optical sensing area, so the physiological activities of the cells will not affect the optical sensing area.
  • the surface structure of the area ensures the stability, sensitivity and accuracy of the optical signal.
  • the present invention uses a microfluidic chip as a preparation platform for structural color microfibers with a heterogeneous structure, the preparation method is simple, the operation is convenient, the reusability is reusable, and the morphology and components of the prepared microfibers are highly controllable.
  • each component material can be directly prepared by one-step ultraviolet polymerization prepolymerization solution, which has the advantages of easier preparation, simple operation, and more controllable optical properties.
  • the cardiomyocyte detection method based on the heterostructured structural color microfibers designed by the present invention can be used for the screening and evaluation of myocardial drugs.
  • the cardiomyocyte detection method of the present invention is a method based on the axial deformation of microfibers, That is, when cardiomyocytes contract, the structural color detection area with optical sensing properties will be pulled to deform (stretched and elongated), and the spacing of colloidal particles in non-closely packed colloidal crystals will be changed, resulting in structural color and corresponding spectral changes.
  • the optical signal conversion method based on axial stretching has better repeatability, and has higher sensing stability and sensitivity to the smaller myocardial cell contractility.
  • different structural colors can be used as multivariate codes to achieve simultaneous testing and evaluation of multiple groups of drugs.
  • Figure 1 is a schematic diagram of the preparation process of two-component heterostructure structural color microfibers; wherein 1 is the injection port, 2 is the pregel of silica non-closely packed colloidal crystals, 3 is the biocompatible hydrogel solution, and 4 is the collection tube, and 5 is the ultraviolet lamp and ultraviolet light;
  • Figure 2 is a picture of bicomponent heterostructure structural color microfibers
  • Figure 3 is a schematic diagram of myocardial detection of bi-component heterostructure structural color microfibers in single-end stretching mode; 6 is a fixture, 7 is the structural color detection area before stretching and its partial magnification, and 8 is stretching 9 is the stretched structural color detection area and its local magnified image, 10 is the stretched cardiomyocyte growth area and its partially magnified cell map;
  • Figure 4 is a picture of a half-cycle of stretching and contraction of a cardiomyocyte single-end stretched bi-component heterostructure structural color microfiber
  • Figure 5 is a picture of multi-component heterostructure structural color microfibers
  • Figure 6 is a picture of the stretching and contraction half cycle of the multi-component heterostructure structural color microfibers at both ends of the cardiomyocyte;
  • Figure 7 is a schematic diagram of myocardial detection of multi-component heterostructure structural color microfibers in two-end stretching mode; 11 is a fixture, 12 is a structural color detection area with a lower silica concentration, and 13 is a silica concentration The medium structural color detection area, 14 is the structural color detection area with higher silica concentration, and 15 is the myocardial cell growth area;
  • Figure 8 shows the spectral changes of structural color under different culture conditions, wherein (a) is common medium, (b) is isoproterenol, and (c) is esmolol.
  • This embodiment provides a method for preparing a bi-component heterostructured structural color microfiber and a method for detecting cardiomyocytes based on single-end stretching.
  • a hydrophobic capillary with an inner diameter of 200 ⁇ m is used as a collection tube.
  • the capillary tube with an inner diameter of 300 ⁇ m is heated and elongated in the outer flame of the spray gun to form a tapered front end, and two sections of tapered front end capillary tubes of suitable length are cut to be used as liquid injection ports. Assembled into a microfluidic chip.
  • aqueous gelatin methacrylate (GelMA) solution disperse sulfonate-modified silica nanoparticles (about 145 nm in diameter) at a concentration of 12% (w/v) in 15% (w/v)
  • Polyacrylamide (PAM):N,N'-methylenebisacrylamide (Bis, cross-linking agent) is an aqueous solution of 29:1; the above two solutions are added with 1% (v/v)
  • the photoinitiator (2-hydroxy-2 methyl Propiophenone) is formulated into a prepolymerization solution.
  • the two prepolymer solutions were added to the syringe, connected to the injection port of the microfluidic chip through a PE hose, and the syringe was mounted on the peristaltic pump.
  • the two prepolymerization solutions were alternately pumped at a flow rate of 0.5mL/h, and the collection tube was exposed to ultraviolet light to obtain polymerized bi-component heterostructure structural color microfibers at the end of the collection tube, as shown in Figure 2 ( d) shown.
  • the concentration of the methacrylated gelatin solution can also be any concentration in the range of 10-30%; the dispersion concentration of the sulfonic acid group-modified silica particles can also be any concentration in the range of 5%-60%.
  • Different concentrations of acid-modified silica particles show different structural colors, for example, when the dispersion concentration of sulfonate-modified silica nanoparticles is 35%, 25% and 20%, the obtained two-component heterostructures Structural color microfibers are shown in Figure 2 (a)-(c).
  • the polyacrylamide aqueous solution containing the crosslinking agent Bis can also be replaced with an aqueous solution of one or more materials selected from acrylamide (AM), isopropylacrylamide (NIPAM), and polyethylene glycol diacrylate (PEGDA).
  • AM acrylamide
  • NIPAM isopropylacrylamide
  • PEGDA polyethylene glycol diacrylate
  • the aqueous solution containing acrylamide and/or isopropylacrylamide material also needs to add a crosslinking agent.
  • Cardiomyocyte detection method based on single-end stretched two-component heterostructure structural color microfibers The cardiomyocytes of neonatal rat were extracted and prepared into a cell suspension with a density of 10 6 cells/mL. The prepared two-component heterostructure structural color microfibers were soaked in 75% alcohol and fully sterilized by ultraviolet irradiation, and then placed in a cell flask culture dish, and a cardiomyocyte suspension was added. Incubate for 1 day in a 37°C, 5% CO2 incubator.
  • FIG. 3 is a picture of a half-cycle of stretching and contraction of a two-component heterostructured structural color microfiber in single-end stretching of cardiomyocytes.
  • This embodiment provides a method for preparing a bi-component heterostructured structural color microfiber and a method for detecting cardiomyocytes based on single-end stretching.
  • Preparation method use a hydrophobic capillary with an inner diameter of 300 ⁇ m as a collection tube.
  • the capillary with an inner diameter of 500 ⁇ m is heated and elongated in the outer flame of the spray gun to form a tapered front end, and four sections of tapered front end capillaries of suitable length are intercepted as liquid injection ports. Assembled into a microfluidic chip.
  • the four prepolymer solutions were added to the syringe, which was connected to the injection port of the microfluidic chip through a hose, and the syringe was mounted on the peristaltic pump.
  • the GelMA aqueous solution and a concentration of silica non-close-packed colloidal crystal pregel were alternately pumped, and the collection tube was exposed to ultraviolet light to obtain the polymerized multicomponent isoform at the end of the collection tube.
  • Texture Structural Color Microfibers were used to obtain the polymerized multicomponent isoform at the end of the collection tube.
  • multi-component heterostructure structural color microfibers with GelMA-red-GelMA-red, GelMA-red-GelMA-green, and GelMA-red-GelMA-blue can be prepared, as shown in Figure 5. shown in (a)-(c).
  • Cardiomyocyte detection method based on multi-component heterostructured structurally colored microfibrils stretched at both ends: resuscitation of cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs), configured to a density of 5 *105 cells/mL cell suspension.
  • iPSC-CMs induced pluripotent stem cells
  • FIG. 7 is a picture of the stretching and contraction half cycle of the multi-component heterostructure structural color microfibers at both ends of the cardiomyocyte.
  • isoproterenol has a positive effect on the periodic contraction of myocardial cells, and has a positive effect on the recovery of myocardial cells, while esmolol has a positive effect on myocardial cells. It has less effect on contractility and mainly affects the frequency of contractions.

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Abstract

一种异质结构的结构色微纤维及其制备方法和心肌细胞检测方法，其中制备方法为：采用毛细管构建微流控芯片用于异质结构的结构色微纤维制备平台，配置预聚液作为纤维组分，预聚液分为两类，一类预聚液为生物相容性水凝胶溶液(3)，另一类预聚液为至少一种浓度的二氧化硅非密堆积胶体晶体预凝胶(2)，各预聚液中均加入有光引发剂；两类预聚液经由微流控芯片程序化交替注入收集管(4)中，通过紫外光(5)持续照射收集管(4)部分，获得异质结构的结构色微纤维。制备可控、成本低廉、可实现细胞生理可视化定量传感。

Description

一种异质结构的结构色微纤维及其制备方法和心肌细胞检测方法 技术领域
本发明属于生物医药材料领域，尤其涉及一种异质结构的结构色微纤维及其制备方法和心肌细胞检测方法。
背景技术
细胞产生的机械力在调节从细胞到组织水平的生物学功能中具有重要作用。细胞机械力的异常通常与器官组织的功能失调和解剖状态异常有关，因此细胞机械力已逐渐被视为疾病评估的要素。为了有效地测量细胞机械力，研究人员已经提出并开发了各种机械力传感技术，例如原子力显微镜、牵引力显微镜、弹性体微柱阵列分析等。这些微量力学传感技术虽然在生物力学过程和亚细胞结构观察方面具有巨大优势，但是大多都依赖于复杂的设备和繁琐的模拟计算，费用高昂，耗时较长。此外，其中的一些技术甚至可能在测量过程中对细胞造成结构损坏，导致测量结果的可信度下降。因此，可用于细胞力学的简便有效的动态检测方法仍有待开发。
心脏是人体最重要的器官之一，其为全身器官、组织提供充足的血流量，维持器官、组织细胞的正常代谢和功能。心肌细胞产生的周期性收缩是心脏发挥功能的重要前提，如果心肌细胞有损，则会导致心脏功能缺陷。近年来，随着冠心病及先心病对人类威胁日益加重，针对心肌细胞药物的研发、筛选和评估成为生物医学领域急需解决的问题之一。同时，能够解决心肌细胞实时监测、同步反馈的传感平台也是该领域面临的挑战。
光子晶体材料是一类具有两种或两种以上折射率的周期性微纳结构材料，因 其可以调控光子在其内部的运动、反射一定波长的电磁波，从而具有光子带隙和结构色的独特性质。通过调控光子晶体材料的结构构成或折射率大小，可以实现对其结构色的调控。
发明内容
为了解决心肌细胞传感依赖大型复杂仪器、心脏新药开发缺乏有效体外模型的缺点，本发明提供了一种基于微流控芯片的、具有轴向异质结构的结构色微纤维及其制备方法和心肌细胞检测方法，所述心肌细胞检测方法的特征在于通过心肌细胞周期性收缩拉动微纤维结构色区域产生形变和颜色变化，实现心肌细胞的可视化检测传感。该微纤维可应用于心肌细胞生理信号转化、可视化检测等技术领域中。
为实现上述目的，本发明提供一种异质结构的结构色微纤维的制备方法，具有这样的特征：采用毛细管构建微流控芯片用于异质结构的结构色微纤维制备平台，配置预聚液作为纤维组分，预聚液分为两类，一类预聚液为生物相容性水凝胶溶液，另一类预聚液为至少一种浓度的二氧化硅非密堆积胶体晶体预凝胶，各预聚液中均加入有光引发剂；两类预聚液经由微流控芯片程序化交替注入收集管中，通过紫外光持续照射收集管部分，聚合后获得异质结构的结构色微纤维。
其中通过调整微流控流速、预聚液通入时间、收集管毛细管管径、注液口数量及预聚液组分数量，可以获得不同长度、组分、直径、结构的异质结构结构色微纤维。将其中一种组分对应的微流控速调快，通入时间加长，对应组分所在纤维中的长度会相应增长；增大或减小收集管毛细管管径，纤维直径会相应地增大或减小；增加或减少注液口数量及预聚液组分数量，异质结构的结构色微纤维所 包含组分会相应的增加或减少。
进一步，本发明提供一种异质结构的结构色微纤维的制备方法，还可以具有这样的特征：其中，所述生物相容性水凝胶溶液为10～30％质量体积比浓度的甲基丙烯酸酯化明胶溶液。
进一步，本发明提供一种异质结构的结构色微纤维的制备方法，还可以具有这样的特征：其中，所述二氧化硅非密堆积胶体晶体预凝胶为磺酸根修饰的二氧化硅粒子分散在丙烯酰胺(AM)、异丙基丙烯酰胺(NIPAM)、聚丙烯酰胺(PAM)、聚乙二醇双丙烯酸酯(PEGDA)的一种或两种以上材料的水溶液中，其中含有丙烯酰胺、聚丙烯酰胺、异丙基丙烯酰胺的一个或两种以上材料的水溶液中还添加有交联剂；磺酸根修饰的二氧化硅粒子的质量体积比浓度为5％-60％(w/v)。磺酸根修饰的二氧化硅粒子浓度不同，所展现的结构色不同，在多组分异质结构结构色微纤维中可以实现多元颜色编码。
进一步，本发明提供一种异质结构的结构色微纤维的制备方法，还可以具有这样的特征：其中，构建微流控芯片的方法为：根据所需结构色微纤维直径，选择同样管径的毛细管作为收集管；根据所需组分数量，设置相同数量的毛细管注液口，并在玻片上与收集管组装成微流控芯片，收集管和注液口均固定在玻片上，注液口均与收集管连接相通；所述生物相容性水凝胶溶液和二氧化硅非密堆积胶体晶体预凝胶，分别经由注射器、软管注入注液口，接入微流控芯片，通过程序化交替地向微流控芯片中泵入生物相容性水凝胶溶液和二氧化硅非密堆积胶体晶体预凝胶。
本发明还提供由上述方法制备的异质结构的结构色微纤维，直径为150-600 μm，长度为500-3000mm。
本发明还提供上述异质结构的结构色微纤维的心肌细胞检测方法，具有这样的特征：将所述异质结构的结构色微纤维用作心肌细胞培养基底，其生物相容性水凝胶溶液聚合后形成的部分为心肌细胞生长区域，心肌细胞在心肌细胞生长区域上生长，二氧化硅非密堆积胶体晶体预凝胶聚合后形成的部分具有结构色，且对拉伸敏感为结构色检测区域；当心肌细胞恢复自主跳动后，其周期性收缩拉动结构色检测区域，结构色检测区域产生与心肌收缩周期同步的结构色变化，从而检测心肌细胞。
进一步，本发明提供一种异质结构的结构色微纤维的心肌细胞检测方法，还可以具有这样的特征：单端拉伸的心肌细胞检测方法：分别固定异质结构的结构色微纤维中相邻的结构色检测区域(二氧化硅非密堆积胶体晶体部分)远端与心肌细胞生长区域(生物相容性水凝胶部分)远端，其中远端指远离两区域交界面的一端；心肌细胞只生长在心肌细胞生长区域(生物相容性水凝胶部分)，在异质结构的结构色微纤维两端被固定的情况下，心肌细胞收缩将经由两个区域的交界面将力学信号传导到结构色检测区域(二氧化硅非密堆积胶体晶体部分)，进而使其产生形变和结构色变化，将心肌力学信号转为光学信号，从而检测心肌细胞。
进一步，本发明提供一种异质结构的结构色微纤维的心肌细胞检测方法，还可以具有这样的特征：两端拉伸的心肌细胞检测方法：分别固定异质结构的结构色微纤维中由一段结构色检测区域(二氧化硅非密堆积胶体晶体部分)分隔开的两个心肌细胞生长区域(生物相容性水凝胶部分)的远端，其中远端指远离与结 构色检测区域交界面的一端；心肌细胞生长在两段心肌细胞生长区域(生物相容性水凝胶部分)，在异质结构的结构色微纤维两处被固定的情况下，结构色检测区域(二氧化硅非密堆积胶体晶体部分)两侧的心肌细胞收缩将通过类似“拔河”的方式从左右两个方向拉动其产生形变和结构色变化，将心肌力学信号转为光学信号，从而检测心肌细胞。
进一步，本发明提供一种异质结构的结构色微纤维的心肌细胞检测方法，还可以具有这样的特征：所述异质结构的结构色微纤维用作心肌细胞培养基底，具体培养过程为：将制备得到的所述异质结构的结构色微纤维灭菌后，置于干净的细胞培养皿中；心肌细胞用培养基充分分散后形成细胞悬液，将细胞悬液滴加入置有异质结构的结构色微纤维的细胞培养皿中，放于37℃、5％CO 2培养箱中培养1天；待细胞黏附于异质结构的结构色微纤维的心肌细胞生长区域后，将异质结构的结构色微纤维转移至新的培养环境，并将其固定；每天更换37℃预热的培养基，观察结构色检测区域。
当心肌恢复自主跳动，其周期性收缩会拉动异质结构的结构色微纤维中结构色检测区域(二氧化硅非密堆积胶体晶体部分)并导致其发生形变和结构色变化，从而实现心肌细胞的动态检测。通过对异质结构的结构色微纤维的结构色检测区域(二氧化硅非密堆积胶体晶体部分)进行光谱统计，可以得到心肌细胞收缩力大小和跳动频率的相关信息。
本发明的有益效果在于：
一、本发明以轴向异质结构的结构色微纤维为心肌细胞培养和传感基底，具有便捷、灵敏、实时、可视化检测传感的特点。具体的，本发明提出的结构色微 纤维是一种轴向异质的纤维，将光学传感区与细胞生长区分开；细胞不黏附在光学传感区域，因此细胞的生理活动不会影响该区域的表面结构，保证了光学信号的稳定性、灵敏性和准确性。
二、本发明以微流控芯片为异质结构的结构色微纤维的制备平台，制备方法简单、操作方便、可重复利用，且所制备的微纤维形貌、组分高度可控。具体的，各组分材料可直接通过紫外聚合预聚液一步制得，具有更易制备、操作简单、光学特性更可控等优点。
三、本发明设计的基于异质结构的结构色微纤维的心肌细胞检测方法可以用于心肌药物的筛选和评估，具体的，本发明的心肌细胞检测方法是基于微纤维轴向形变的方法，即心肌细胞收缩时，将拉动具有光学传感特性的结构色检测区域产生形变(被拉伸变长)，改变非密堆积胶体晶体中胶体粒子的间距，进而产生结构色及对应光谱变化。且基于轴向拉伸的光学信号转换方法具有更好的重复性，对更微小的心肌细胞收缩力具有更高的传感稳定性和灵敏性。此外，还可通过不同结构色作为多元编码，实现多组药物的同时测试和评估。
附图说明
图1是双组分异质结构结构色微纤维制备过程示意图；其中1为注液口，2为二氧化硅非密堆积胶体晶体预凝胶，3为生物相容性水凝胶溶液，4为收集管，5为紫外灯和紫外光；
图2是双组分异质结构结构色微纤维的图片；
图3是单端拉伸模式下双组分异质结构结构色微纤维的心肌检测示意图；其中6为固定器，7为拉伸前的结构色检测区域及其局部放大图，8为拉伸前的心肌细 胞生长区域及其局部放大细胞图，9为拉伸后的结构色检测区域及其局部放大图，10为拉伸后的心肌细胞生长区域及其局部放大细胞图；
图4是心肌细胞单端拉伸双组分异质结构结构色微纤维拉伸和收缩半个循环的图片；
图5是多组分异质结构结构色微纤维的图片；
图6是心肌细胞两端拉伸多组分异质结构结构色微纤维拉伸和收缩半个循环的图片；
图7是两端拉伸模式下多组分异质结构结构色微纤维的心肌检测示意图；其中11为固定器，12为二氧化硅浓度较低的结构色检测区域，13为二氧化硅浓度中等的结构色检测区域，14为二氧化硅浓度较高的结构色检测区域，15为心肌细胞生长区域；
图8为不同培养条件下结构色光谱变化，其中(a)为普通培养基，(b)为异丙肾上腺素，(c)为艾司洛尔。
具体实施方式
以下结合具体实施例对本发明作进一步说明。
实施例1
本实施例提供一种双组分异质结构结构色微纤维的制备方法及其基于单端拉伸的心肌细胞检测方法。
制备方法过程如图1所示：用内径200μm疏水的毛细管作为收集管。将内径300μm的毛细管在喷枪外焰中烧热并拉长，形成锥形前端，截取两段合适长 度的锥形前端毛细管用作注液口，注液口锥形前端均与收集管连接相通，组装成微流控芯片。
配置15％(w/v)的甲基丙烯酸酯化明胶(GelMA)水溶液；将磺酸根修饰的二氧化硅纳米粒子(粒径约145nm)按12％(w/v)的浓度分散在15％(w/v)聚丙烯酰胺(PAM)∶N,N’-亚甲基双丙烯酰胺(Bis、交联剂)为29∶1的水溶液；上述两种溶液分别加入1％(v/v)光引发剂(2-羟基-2甲基苯丙酮)配成预聚液。
将两种预聚液加入到注射器中，通过PE软管连接到微流控芯片的注液口，并将注射器安装在蠕动泵上。以0.5mL/h的流速交替泵入两种预聚液，将收集管暴露在紫外光下，即可在收集管末端得到聚合的双组分异质结构结构色微纤维，如图2中(d)所示。
其中，甲基丙烯酸酯化明胶溶液的浓度还可以为10～30％范围中的任意浓度；磺酸根修饰的二氧化硅粒子的分散浓度也可以为5％-60％范围中的任意浓度，磺酸根修饰的二氧化硅粒子浓度不同，所展现的结构色不同，例如，当磺酸根修饰的二氧化硅纳米粒子的分散浓度为35％、25％和20％所得到的双组分异质结构结构色微纤维如图2中(a)-(c)所示。
含交联剂Bis的聚丙烯酰胺水溶液还可以替换为丙烯酰胺(AM)、异丙基丙烯酰胺(NIPAM)、聚乙二醇双丙烯酸酯(PEGDA)中的一种或两种以上材料的水溶液，其中含有丙烯酰胺和/或异丙基丙烯酰胺材料的水溶液中还需要添加有交联剂。
基于单端拉伸的双组分异质结构结构色微纤维的心肌细胞检测方法：提取新 生乳大鼠心肌细胞，配置成密度为10 6个/mL的细胞悬液。制备所得的双组分异质结构结构色微纤维经75％酒精浸泡、紫外照射充分灭菌后，将其放置在细胞瓶培养皿中，并加入心肌细胞悬液。放于37℃、5％CO 2培养箱中培养1天。待细胞贴壁后，用平头镊子轻轻夹起微纤维结构色一端，将其转移至新的培养环境中，并将两端固定，如图3所示；每天更换37℃预热的培养基。当心肌细胞恢复周期性收缩后，异质结构结构色微纤维的结构色检测区域会在心肌细胞的带动下产生同步的周期性拉伸和收缩，并产生颜色变化，即可实时反馈心肌细胞生理状态。图4为心肌细胞单端拉伸双组分异质结构结构色微纤维拉伸和收缩半个循环的图片。
实施例2
本实施例提供一种双组分异质结构结构色微纤维的制备方法及其基于单端拉伸的心肌细胞检测方法。
制备方法：用内径300μm疏水的毛细管作为收集管。将内径500μm的毛细管在喷枪外焰中烧热并拉长，形成锥形前端，截取四段合适长度的锥形前端毛细管用作注液口，注液口锥形前端均与收集管连接相通，组装成微流控芯片。
配置20％(w/v)的GelMA水溶液；将聚乙二醇双丙烯酸酯(PEGDA)与AM(含1/30的交联剂Bis)按2∶15的比例配制成复合水凝胶前聚体溶液，将磺酸根修饰的二氧化硅纳米粒子(粒径约145nm)分别按15％，23％，35％(w/v)的浓度分散在15％(w/v)的复合水凝胶前聚体溶液中，其分别显示红、绿、蓝结构色；上述四种溶液分别加入1％(v/v)光引发剂(2-羟基-2甲基苯丙酮)配 成预聚液。
将四种预聚液加入到注射器中，通过软管连接到微流控芯片的注液口，并将注射器安装在蠕动泵上。以0.25mL/h的流速交替泵入GelMA水溶液和一种浓度的二氧化硅非密堆积胶体晶体预凝胶，将收集管暴露在紫外下，即可在收集管末端得到聚合的多组分异质结构结构色微纤维。通过控制通入组分，可以制备得到具有GelMA-红-GelMA-红、GelMA-红-GelMA-绿、GelMA-红-GelMA-蓝的多组分异质结构结构色微纤维，依次如图5中(a)-(c)所示。
基于两端拉伸的多组分异质结构结构色微纤维的心肌细胞检测方法：复苏由诱导多能干细胞分化的心肌细胞(iPSC-CMs)，配置成密度为5*10 5个/mL的细胞悬液。制备所得多组分异质结构结构色微纤维充分灭菌后，将其放置在不同的细胞瓶培养皿中，并加入细胞悬液。放于37℃、5％CO 2培养箱中培养1天。待细胞贴壁后，用平头镊子轻轻夹起微纤维游离的结构色一端，将其转移至新的培养环境中，并将纤维红色部分两边GelMA段的远离红色部分的两端固定，如图7所示。当心肌细胞恢复周期性收缩后，异质结构微纤维的结构色区域会在心肌细胞的带动下产生类似“拔河”的同步的周期性拉伸和收缩，产生颜色变化。图6为心肌细胞两端拉伸多组分异质结构结构色微纤维拉伸和收缩半个循环的图片。
如图7所示，通过红-红、红-绿、红-蓝三种颜色编码，分别对应普通培养基、加入异丙肾上腺素的培养基和加入艾司洛尔的培养基，以这三种多组分异质结构结构色微纤维中夹在两段GelMA段(心肌细胞生长区域)中的红色区域作为传感区域(结构色检测区域)，通过观察、记录、分析其光谱位移，得到药物对iPSC-CMs的作用，实现心肌监测和药物筛选。图8为不同培养条件下结构色光 谱变化，由图可知，异丙肾上腺素对心肌细胞周期性收缩有正向作用，对心肌细胞的恢复有积极影响，而艾司洛尔对心肌产生的作用在收缩力上影响较小，主要影响收缩频率。

Claims (10)

  1. 一种异质结构的结构色微纤维的制备方法，其特征在于：
    采用毛细管构建微流控芯片用于异质结构的结构色微纤维制备平台，配置预聚液作为纤维组分，预聚液分为两类，一类预聚液为生物相容性水凝胶溶液，另一类预聚液为至少一种浓度的二氧化硅非密堆积胶体晶体预凝胶，各预聚液中均加入有光引发剂；
    两类预聚液经由微流控芯片程序化交替注入收集管中，通过紫外光持续照射收集管部分，获得异质结构的结构色微纤维。
  2. 根据权利要求1所述的异质结构的结构色微纤维的制备方法，其特征在于：
    其中，所述生物相容性水凝胶溶液为10～30％质量体积比浓度的甲基丙烯酸酯化明胶溶液。
  3. 根据权利要求1所述的异质结构的结构色微纤维的制备方法，其特征在于：
    其中，所述二氧化硅非密堆积胶体晶体预凝胶为磺酸根修饰的二氧化硅粒子分散在丙烯酰胺、异丙基丙烯酰胺、聚丙烯酰胺、聚乙二醇双丙烯酸酯的一种或两种以上材料的水溶液中，其中含有丙烯酰胺、聚丙烯酰胺、异丙基丙烯酰胺的一个或两种以上材料的水溶液中还添加有交联剂；
    磺酸根修饰的二氧化硅粒子的质量体积比浓度为5％-60％。
  4. 根据权利要求1所述的异质结构的结构色微纤维的制备方法，其特征在 于：
    其中，构建微流控芯片的方法为：根据所需结构色微纤维直径，选择同样管径的毛细管作为收集管；根据所需组分数量，设置相同数量的毛细管注液口，并在玻片上与收集管组装成微流控芯片，收集管和注液口均固定在玻片上，注液口均与收集管连接相通；
    所述生物相容性水凝胶溶液和二氧化硅非密堆积胶体晶体预凝胶，分别经由注射器、软管注入注液口，接入微流控芯片，通过程序化交替地向微流控芯片中泵入生物相容性水凝胶溶液和二氧化硅非密堆积胶体晶体预凝胶。
  5. 权利要求1-4任意一项所述方法制备的异质结构的结构色微纤维。
  6. 根据权利要求5所述的异质结构的结构色微纤维，其特征在于：
    直径为150-600μm，长度为500-3000mm。
  7. 如权利要求5所述的异质结构的结构色微纤维的心肌细胞检测方法，其特征在于：
    将所述异质结构的结构色微纤维用作心肌细胞培养基底，其生物相容性水凝胶溶液形成的部分为心肌细胞生长区域，心肌细胞在心肌细胞生长区域上生长，二氧化硅非密堆积胶体晶体预凝胶形成的部分为结构色检测区域；
    当心肌细胞恢复自主跳动后，其周期性收缩拉动结构色检测区域，结构色检测区域产生与心肌收缩周期同步的结构色变化，从而检测心肌细胞。
  8. 根据权利要求7所述的异质结构的结构色微纤维的心肌细胞检测方法，其特征在于：
    单端拉伸的心肌细胞检测方法：分别固定异质结构的结构色微纤维中相邻的结构色检测区域远端与心肌细胞生长区域远端；
    心肌细胞只生长在心肌细胞生长区域，在异质结构的结构色微纤维两端被固定的情况下，心肌细胞收缩将经由两个区域的交界面将力学信号传导到结构色检测区域，进而使其产生形变和结构色变化，从而检测心肌细胞。
  9. 根据权利要求7所述的异质结构的结构色微纤维的心肌细胞检测方法，其特征在于：
    两端拉伸的心肌细胞检测方法：分别固定异质结构的结构色微纤维中由一段结构色检测区域分隔开的两个心肌细胞生长区域的远端；
    心肌细胞生长在两段心肌细胞生长区域，在异质结构的结构色微纤维两处被固定的情况下，结构色检测区域两侧的心肌细胞收缩将从左右两个方向拉动其产生形变和结构色变化，从而检测心肌细胞。
  10. 根据权利要求7所述的异质结构的结构色微纤维的心肌细胞检测方法，其特征在于：
    所述异质结构的结构色微纤维用作心肌细胞培养基底，具体培养过程为：
    将制备得到的所述异质结构的结构色微纤维灭菌后，置于干净的细胞培养皿中；
    心肌细胞用培养基充分分散后形成细胞悬液，将细胞悬液滴加入置有异质结 构的结构色微纤维的细胞培养皿中，放于37℃、5％CO 2培养箱中培养1天；
    待细胞黏附于异质结构的结构色微纤维的心肌细胞生长区域后，将异质结构的结构色微纤维转移至新的培养环境，并将其固定；
    每天更换37℃预热的培养基，观察结构色检测区域。
PCT/CN2021/131723 2020-11-20 2021-11-19 一种异质结构的结构色微纤维及其制备方法和心肌细胞检测方法 WO2022105869A1 (zh)

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