Preparation method of optical cuvette attached with two-dimensional collagen nanofiber array film layer

Abstract

The invention discloses a preparation method of an optical cuvette attached with a two-dimensional collagen nanofiber array film layer, which comprises the following steps: firstly preparing a mica sheet substrate, preparing a required buffer solution and a monomer collagen solution, then uniformly dropwise adding the prepared monomer collagen solution on the surface of the mica sheet substrate, and preparing a two-dimensional collagen nanofiber array film layer on the surface of the mica sheet substrate by using an atomic force microscope, so as to obtain the optical cuvette attached with the two-dimensional collagen nanofiber array film layer. The method comprises the following steps: carrying out fine self-adaptive assembly on monomer collagen in a knocking mode, and finally preparing the cuvette from a mica sheet subjected to supervised self-adaptive assembly treatment on the monomer collagen. According to the scheme, the protein nanofibers are manufactured in different areas of the surface of the mica sheet in a knocking mode of an atomic force microscope, fibers with the direction capable of being accurately controlled can be generated under the appropriate acting force of a probe, and the protein nanofiber array in the cuvette prepared through the scheme shows the excellent polarization characteristic and can be used for preparing the protein nanofibers in the cuvette. If the nano-material is used as a mask, a novel biosensor can be innovatively synthesized by combining an electrochemical method, which indicates that the nano-material has wide popularization and application prospects in a plurality of fields.

Description

Technical Field

[0001] The invention belongs to the field of cuvettes, and in particular relates to a method for preparing an optical cuvette with a two-dimensional collagen nanofiber array membrane layer attached thereto. Background Art

[0002] The collagen monomer molecules adaptively assembled on the surface of mica sheets have typical epitaxial characteristics. They can recognize each other with mica crystals at the mesoscopic scale. The crystal mineralization process is a common phenomenon in nature. The adaptively assembled collagen fiber bundles can provide a skeleton for mineralization and correspondingly regulate the opening and closing of the mica crystal mineralization process. Therefore, on the contrary, we can use the recognition effect between collagen monomer molecules and mica crystals to control the adaptive assembly of collagen molecules on the crystal surface in liquid buffer solution. Careful processing can ensure that the fiber direction is consistent and exhibits excellent polarization characteristics. If it is used as a mask, it can be combined with electrochemical methods to innovatively synthesize new biosensors, indicating that it has broad prospects for promotion and application in many fields. Summary of the invention

[0003] In view of the above problems, the purpose of the present invention is to provide a method for preparing an optical cuvette with an attached two-dimensional collagen nanofiber array membrane layer. The optical cuvette with an attached two-dimensional collagen nanofiber array membrane layer is prepared using an atomic force microscope. The operation is simple and the direction of the generated collagen nanofiber array structure can be precisely controlled.

[0004] The specific technical solution for achieving the purpose of the present invention is:

[0005] A method for preparing an optical cuvette with a two-dimensional collagen nanofiber array membrane layer attached thereto comprises the following steps:

[0006] Step 1, preparing a mica sheet substrate;

[0007] Step 2, preparing the required buffer solution and monomer collagen solution;

[0008] Step 3, evenly dripping the prepared monomer collagen solution onto the surface of the mica substrate, and using an atomic force microscope to perform fine adaptive assembly of the monomer collagen protein in a knocking mode;

[0009] Step 4: Prepare a cuvette using the mica sheet that has been subjected to the monomeric collagen supervised adaptive assembly process.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] (1) The scheme of the present invention aims to produce protein nanofibers by using the tapping mode of atomic force microscopy on different areas of the surface of mica sheets. Under the appropriate probe force, fibers with precisely controlled directions can be produced. Specifically, when the probe force is significantly greater than the interaction force between the adsorbed molecules and the matrix or molecules, the probe is able to sweep the protein molecules out of the scanning area, and the generated fiber orientation will be perpendicular to the line scanning direction of the probe. In addition, the size of a single fiber is determined by the probe force and the scanning line spacing;

[0012] (2) The solution of the present invention is easy to operate and can achieve precise control of the orientation of the structure of the generated collagen nanofiber array. Experimental data have confirmed that the cuvette prepared by the present invention exhibits excellent hydrophilic properties and is therefore very suitable for application scenarios such as cell culture;

[0013] (3) The protein nanofiber array on the mica sheet in the cuvette of the present invention is carefully processed to ensure that the fiber direction is consistent and exhibits excellent polarization properties. If it is used as a mask, it can be combined with electrochemical methods to innovatively synthesize new biosensors, indicating that it has broad prospects for promotion and application in multiple fields.

[0014] The present invention is further described below in conjunction with specific implementation modes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The present invention is a schematic flow chart of a method for preparing an optical cuvette with a two-dimensional collagen nanofiber array membrane layer attached thereto.

[0016] Figure 2 This is a 3D height model diagram of the two-dimensional collagen nanofiber array membrane layer optical cuvette at a scale of 10 um using an atomic force microscope, which is prepared at room temperature (natural cooling) in an embodiment of the present invention.

[0017] Figure 3 This is a height map of the two-dimensional collagen nanofiber array membrane layer optical cuvette at a scale of 10 um under an atomic force microscope, which was prepared at room temperature (natural cooling) in an embodiment of the present invention.

[0018] Figure 4 This is a 3D height model diagram of the two-dimensional collagen nanofiber array membrane layer optical cuvette at a 3um scale using an atomic force microscope, which is prepared at room temperature (natural cooling) in an embodiment of the present invention.

[0019] Figure 5 This is a height map of the two-dimensional collagen nanofiber array membrane layer optical cuvette at a 3 um scale under an atomic force microscope, which was prepared at room temperature (natural cooling) in an embodiment of the present invention.

[0020] Figure 6 This is a 3D height model diagram of the two-dimensional collagen nanofiber array membrane layer optical cuvette at the 1um scale of an atomic force microscope made at room temperature (natural cooling) in an embodiment of the present invention.

[0021] Figure 7 This is a height map of the two-dimensional collagen nanofiber array membrane layer optical cuvette at 1 um scale under atomic force microscopy made at room temperature (natural cooling) in an embodiment of the present invention.

[0022] Figure 8 This is a 3D height model diagram of the two-dimensional collagen nanofiber array membrane layer optical cuvette at an atomic force microscope 10um scale obtained at room temperature (after drying) in an embodiment of the present invention.

[0023] Fig. 9 This is a height map of the two-dimensional collagen nanofiber array membrane layer optical cuvette produced at room temperature (after drying) in an embodiment of the present invention at a scale of 10 um using an atomic force microscope.

[0024] Fig.10 This is a 3D height model diagram of the two-dimensional collagen nanofiber array membrane layer optical cuvette at an atomic force microscope 5um scale obtained at room temperature (after drying) in an embodiment of the present invention.

[0025] Fig.11 This is a height map of the two-dimensional collagen nanofiber array membrane optical cuvette at a 5 um scale using an atomic force microscope, obtained at room temperature (after drying) in an embodiment of the present invention.

[0026] Fig.12 This is a 3D height model diagram at the 1um scale of an atomic force microscope of a two-dimensional collagen nanofiber array membrane optical cuvette prepared at room temperature (after drying) in an embodiment of the present invention.

[0027] Fig.13 This is a height map of the two-dimensional collagen nanofiber array membrane layer optical cuvette at a 1 um scale under an atomic force microscope obtained at room temperature (after drying) in an embodiment of the present invention.

[0028] Fig.14 The schematic diagram is a structural diagram of the two-dimensional collagen nanofiber array membrane layer optical cuvette prepared in the present invention. DETAILED DESCRIPTION

[0029] In order to clearly describe the technical solution and the effects achieved by the present invention, the technical solution of the present invention is clearly explained below in combination with the accompanying drawings and specific embodiments of the present invention, so that technical personnel in the field can implement the invention without performing creative work. The structure shown in the accompanying drawings is not the entire actual structure but only a part of the actual structure. It should be noted that all other embodiments made by ordinary technical personnel in the field on the basis of the embodiments of the present invention without performing creative work should fall within the protection scope of the present invention.

[0030] The following examples are only for better explanation of the present invention, but the content of the present invention is not limited to the application in the examples. Therefore, those skilled in the art can make non-essential modifications to the implementation scheme based on the above invention content and apply it to other embodiments, which is still within the protection scope of the present invention. In addition, the experimental methods without specific conditions in the following examples should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the expressions in the text are not specially explained, they are only used for distinction and have no other meanings.

[0031] Example

[0032] Combination Figure 1 A method for preparing an optical cuvette with a two-dimensional collagen nanofiber array membrane layer attached thereto comprises the following steps:

[0033] Step 1: Prepare mica sheet substrate:

[0034] Cut a mica sheet of a certain size into four square pieces, for example, cut a 2cm*2cm mica sheet into four square pieces of about 1cm*1cm, and stick a double-sided tape of about 5cm in length in the center of a glass slide. After tearing off the protective layer of the double-sided tape, stick the mica sheet to the exposed side of the double-sided tape, and align the double-sided tape on both sides. Place two mica sheets on a glass slide;

[0035] Use single-sided tape to stick a corner of the mica sheet, and tear off 1-3 layers of the surface of the mica sheet neatly to expose a fresh and clean crystal surface. At the same time, wash the crystal surface with fresh deionized water. Each two mica sheets on the same slide are a group.

[0036] Step 2: Prepare the required buffer and monomer collagen solution:

[0037] Step 2-1, preparing a buffer solution: first, prepare a 1 mol / L Na2HPO4 solution and a 1 mol / L NaH2PO4 solution respectively, then, measure 77.4 ml from the prepared Na2HPO4 solution and measure 22.6 ml from the NaH2PO4 solution, mix the two solutions thoroughly, and then adjust the volume to 100 ml to form a phosphate buffer solution with a pH value of 7.4;

[0038] Step 2-2, diluting the original protein solution to obtain an experimental solution;

[0039] For example, the concentration of the original protein solution is ρ = 5 mg / mL, and the concentration of the required experimental solution is ρ = 10 μg / mL. If a 1.5 mL test tube is used, the following formula is used:

[0040]

[0041] Then we can calculate:

[0042] Set the pipette with a range of 10-100ul to 50ul, and accurately measure the rat tail protein solution three times in a row until the total volume reaches 150ul (the total amount of protein contained in this solution is 750ug).

[0043] Step 2-3: Mix the buffer solution with the monomer collagen solution to obtain a protein experimental solution:

[0044] Subsequently, the protein solution measured above was fully mixed with an appropriate amount of buffer solution to finally prepare a protein experimental solution with a total volume of 75 ml. After calculation, the protein concentration in the experimental solution was 10 ug / ml.

[0045] Step 3: Evenly drip the prepared monomer collagen solution onto the surface of the mica substrate, and use an atomic force microscope to perform fine adaptive assembly of the monomer collagen protein in a knocking mode:

[0046] During the experiment, firstly, 25 microliters of the protein experimental solution was accurately measured with a pipette and dropped onto the surface of the prepared mica substrate. Then, a glass slide was placed in the center of the culture dish. The glass slide and the sample on it were incubated for 12 hours at room temperature.

[0047] During the static incubation of the glass slide and the sample thereon, deionized water is dripped onto both sides of the glass slide in order to maintain the moisture of the glass slide and prevent it from being over-dried and affecting the experimental results.

[0048] After the incubation period, the samples were dried. According to the established experimental procedures, we used two different methods to dry the samples for control experiments. The first method was to let the samples dry naturally at room temperature; the second method was to use a blower to gently dry the side of the sample at a low setting, and after the drying process was completed, wait for the sample to cool down to room temperature naturally, and then put it back into the culture dish;

[0049] During the characterization observation stage, the probe of an atomic force microscope was used to apply force to the collagen molecules attached to the surface of the crystal in tapping mode, so that the collagen molecules formed a two-dimensional fiber array with a consistent overall orientation.

[0050] Wherein, the overall orientation of the two-dimensional fiber array is perpendicular to the scanning direction of the probe.

[0051] The force applied by the probe of the atomic force microscope to the collagen molecules attached to the surface of the crystal plane in the knocking mode includes a vertical force and a horizontal force;

[0052] The vertical force applied by the probe should be kept constant, which is achieved by real-time detection of the piezoelectric sensor on the atomic force microscope probe and position control of the probe cantilever;

[0053] The horizontal force should be greater than the binding force between the collagen molecules and the crystal surface.

[0054] During the formation of the fiber array:

[0055] The probe exerts force on the collagen molecules attached to the crystal surface and pushes the collagen molecules. At this time, the force on the needle and the bending degree of the cantilever will increase. When the vertical force is greater than the threshold set by the piezoelectric sensor, the atomic force microscope will reduce the vertical force by lifting the cantilever.

[0056] If the vertical force is less than the threshold set by the piezoelectric sensor, the atomic force microscope lowers the cantilever to lower the probe, forming a new round of pushing.

[0057] During the tapping and scanning process of the probe, the collagen molecules are pushed to form an aggregate of several sample molecules. The sample molecules in the aggregate are randomly combined to form a protein nanofiber structure with random and repeated bending characteristics.

[0058] Step 4: Prepare a cuvette using the mica sheet that has been subjected to the monomeric collagen supervised adaptive assembly process.

[0059] In this study, a Bruker Multimode 8 atomic force microscope and a BRUKER cantilever probe (SNL-10, the probe is made of silicon with gold and chromium plating, and the force constants of the cantilever and the tip are both 0.35N / m. The atomic force microscope was set to tapping mode, with an amplitude of about 75nm and a drive frequency of about 50-80kHz. Atomic force microscopy was used for characterization at 10um, 3um, and 1um scales.

[0060] Figures 2 to 14This is a schematic diagram of the preparation results of the two-dimensional collagen nanofiber array membrane layer optical cuvette in this embodiment. It can be seen that the protein nanofiber array on the mica sheet in the cuvette prepared in this scheme has been carefully processed to ensure that the fiber direction is consistent, exhibits excellent polarization characteristics, and has certain application prospects.

[0061] The above embodiments show and describe the basic principles and main features of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for preparing an optical cuvette with a two-dimensional collagen nanofiber array membrane layer, characterized in that: The following steps are involved: Step 1, preparing a mica sheet substrate; Step 2, preparing the required buffer solution and monomer collagen solution; Step 3, evenly dripping the prepared monomer collagen solution onto the surface of the mica substrate, and using an atomic force microscope to perform fine adaptive assembly of the monomer collagen protein in a knocking mode; Step 4: Prepare a cuvette using the mica sheet that has been subjected to the monomeric collagen supervised adaptive assembly process.

2. The method for preparing an optical cuvette with a two-dimensional collagen nanofiber array membrane layer attached according to claim 1, characterized in that: The step 1 of preparing the mica sheet substrate is specifically as follows: Cut a mica sheet of a certain size into four square pieces, and stick double-sided tape in the center of a glass slide. After tearing off the protective layer of the double-sided tape, stick the mica sheet to the exposed side of the double-sided tape, and align the double-sided tape on both sides. Place two mica sheets on a glass slide. Use single-sided tape to stick a corner of the mica sheet, and tear off 1-3 layers of the surface of the mica sheet neatly to expose a fresh and clean crystal surface. At the same time, wash the crystal surface with fresh deionized water. Each two mica sheets on the same slide are a group.

3. The method for preparing an optical cuvette with a two-dimensional collagen nanofiber array membrane layer attached according to claim 1, characterized in that: The required buffer solution and monomer collagen solution are prepared in step 2, specifically: Step 2-1, preparing a buffer solution: first, prepare a 1 mol / L Na2HPO4 solution and a 1 mol / L NaH2PO4 solution respectively, then, measure 77.4 ml from the prepared Na2HPO4 solution and measure 22.6 ml from the NaH2PO4 solution, mix the two solutions thoroughly, and then adjust the volume to 100 ml to form a phosphate buffer solution with a pH value of 7.4; Step 2-2, diluting the original protein solution to obtain an experimental solution; Step 2-3: Mix the buffer solution and the monomer collagen solution to obtain a protein experimental solution.

4. The method for preparing an optical cuvette with a two-dimensional collagen nanofiber array membrane layer attached according to claim 1, characterized in that: The step 3 of finely adaptively assembling the monomeric collagen protein is specifically as follows: First, use a pipette to accurately measure 25 microliters of the protein experimental solution and drop it onto the surface of the prepared mica substrate. Then, place a glass slide in the center of the culture dish and incubate the glass slide and the sample on it for 12 hours at room temperature. After the incubation period, the samples were dried; During the characterization observation stage, the probe of an atomic force microscope was used to apply force to the collagen molecules attached to the surface of the crystal in tapping mode, so that the collagen molecules formed a two-dimensional fiber array with a consistent overall orientation.

5. The method for preparing an optical cuvette with a two-dimensional collagen nanofiber array membrane layer attached according to claim 4, characterized in that: The overall orientation of the two-dimensional fiber array is perpendicular to the scanning direction of the probe.

6. The method for preparing an optical cuvette with a two-dimensional collagen nanofiber array membrane layer attached according to claim 4, characterized in that: During the static incubation of the glass slide and the sample thereon, deionized water is dripped onto both sides of the glass slide in order to maintain the moisture of the glass slide and prevent it from being over-dried and affecting the experimental results.

7. The method for preparing an optical cuvette with a two-dimensional collagen nanofiber array membrane layer attached according to claim 4, characterized in that: The force applied by the probe of the atomic force microscope to the collagen molecules attached to the surface of the crystal plane in the knocking mode includes a vertical force and a horizontal force; The vertical force applied by the probe should be kept constant, which is achieved by real-time detection of the piezoelectric sensor on the atomic force microscope probe and position control of the probe cantilever; The horizontal force should be greater than the binding force between the collagen molecules and the crystal surface.

8. The method for preparing an optical cuvette with a two-dimensional collagen nanofiber array membrane layer attached according to claim 7, characterized in that: During the formation of the fiber array: The probe exerts force on the collagen molecules attached to the crystal surface and pushes the collagen molecules. At this time, the force on the needle and the bending degree of the cantilever will increase. When the vertical force is greater than the threshold set by the piezoelectric sensor, the atomic force microscope will reduce the vertical force by lifting the cantilever. If the vertical force is less than the threshold set by the piezoelectric sensor, the atomic force microscope lowers the cantilever to lower the probe, forming a new round of pushing.

9. The method for preparing an optical cuvette with a two-dimensional collagen nanofiber array membrane layer attached according to claim 8, characterized in that: During the tapping and scanning process of the probe, the collagen molecules are pushed to form an aggregate of several sample molecules. The sample molecules in the aggregate are randomly combined to form a protein nanofiber structure with random and repeated bending characteristics.

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