Method for preparing non-close-packed colloidal crystals based on pdms films

Abstract

The application discloses a preparation method of a non-close-packed colloidal crystal based on a PDMS film. The method comprises the following steps: providing a polystyrene microsphere solution which is surface-modified with DNA probes; forming a single-layer polystyrene microsphere array which is surface-modified with DNA probes on a water surface by using the polystyrene microsphere solution which is surface-modified with DNA probes; performing hydrophilic treatment on a PDMS film to obtain a hydrophilic PDMS film; transferring the single-layer polystyrene microsphere array which is surface-modified with DNA probes onto the hydrophilic PDMS film as it is, and drying the array to obtain a close-packed colloidal crystal based on the PDMS film; stretching the close-packed colloidal crystal based on the PDMS film to a predetermined stretching increment of the PDMS film; and transferring the close-packed colloidal crystal on the stretched PDMS film to another non-stretched PDMS film to obtain a non-close-packed colloidal crystal based on the PDMS film.

Description

Technical Field

[0001] This invention belongs to the field of materials science, specifically relating to a method for preparing non-close-packed colloidal crystals based on PDMS thin films. Background Technology

[0002] Non-close-packed colloidal crystals have extremely high resolution and are in high demand and have promising applications in fields such as fluorescence recognition and nanostructure design.

[0003] If non-close-packed colloidal crystals are prepared using polystyrene microspheres with DNA probes on their surface, the use of chemical reagents will, to some extent, damage the morphology of the microspheres and destroy the activity of the DNA probes. Manual stretching is possible, but multiple transfers will cause some damage to the DNA probes, weaken the fluorescence properties, and there is uncertainty in the error of manual stretching. High-temperature treatment is also required to increase the interaction force between PDMS and microspheres, which will also destroy the DNA probes. Using larger-sized microspheres to assemble closely packed colloidal crystals and then etching to obtain non-close-packed colloidal crystals of the corresponding target size has limitations such as the difficulty of assembling large-sized colloidal crystals, the difficulty of exploring etching parameters, and the large limitation of spacing. Summary of the Invention

[0004] The purpose of this invention is to provide a simple, automated, and highly controllable method for preparing non-close-packed colloidal crystals based on PDMS thin films.

[0005] The method for preparing non-close-packed colloidal crystals based on PDMS thin films provided by this invention includes the following steps: A solution of polystyrene microspheres with DNA probes modified on the surface is provided; The solution of polystyrene microspheres with DNA probes on the surface is used to form a monolayer array of polystyrene microspheres with DNA probes on the water surface; The first PDMS film was subjected to hydrophilic treatment to obtain a hydrophilic PDMS film; The polystyrene microsphere array with DNA probes on its monolayer surface is transferred to the hydrophilic PDMS film as is, and dried to obtain a closely packed colloidal crystal based on the first PDMS film. The close-packed colloidal crystals based on the first PDMS film are stretched to a predetermined stretching increment from the first PDMS film. The closely packed colloidal crystals on the stretched first PDMS film are transferred onto the unstretched second PDMS film to obtain non-closely packed colloidal crystals based on the PDMS film.

[0006] In one embodiment, the polystyrene microspheres with the DNA probe modified on their surface are dispersed in an aqueous ethanol solution by ultrasonic treatment to obtain a polystyrene microsphere solution with the DNA probe modified on its surface.

[0007] In one embodiment, the relative tack of the first PDMS film is lower than that of the second PDMS film.

[0008] In one embodiment, the first PDMS film is a PDMS film with a relative viscosity of 100 to 300.

[0009] In one embodiment, the unstretched second PDMS film is a PDMS film with a relative viscosity of 1000-2500.

[0010] The non-close-packed colloidal crystals based on PDMS thin films prepared by the preparation method of PDMS thin films are also within the scope of protection of this invention.

[0011] The present invention provides a method for preparing non-close-packed colloidal crystals based on PDMS films. This method assembles closely packed colloidal crystals with DNA probes modified on their surfaces onto a PDMS film substrate with a relative viscosity of 100-300. By adjusting the film size, stretching distance, and crystal phase of the colloidal crystals, a periodic array of non-close-packed colloidal crystals with a relative viscosity of 1000-2500 as the substrate is obtained. These crystals can be directly subjected to fluorescence testing and water rinsing, and have the advantages of low shedding and few defects.

[0012] The method for preparing non-close-packed colloidal crystals based on PDMS films of the present invention is carried out at room temperature, which reduces the damage to DNA probes and polystyrene microspheres. The prepared non-close-packed colloidal crystals carrying DNA probes can be applied to fields such as high-resolution fluorescence periodic array detection. Attached Figure Description

[0013] Figure 1 This is a flowchart illustrating a method for preparing non-close-packed colloidal crystals based on PDMS thin films, as described in an embodiment of the present invention.

[0014] Figure 2 This is a schematic diagram of an automated four-way stretching device according to an embodiment of the present invention; wherein, A, B, and C are the schematic diagram, front view, and top view of the stretching device, respectively, and D is an enlarged detail view of the components of the stretching device.

[0015] Figure 3 These are electron microscope images of colloidal crystals according to embodiments of the present invention; wherein, A and B are electron microscope images of closely packed colloidal crystals based on PDMS thin films; C and D are electron microscope images of non-closely packed colloidal crystals based on PDMS thin films obtained therefrom.

[0016] Figure 4 The fluorescence color development results of a non-close-packed colloidal crystal based on a PDMS thin film after incubation with a probe solution are shown in an embodiment of the present invention. Detailed Implementation

[0017] the term PDMS: Polydimethylsiloxane, is a high molecular weight polymer with the chemical formula (C2H6OSi). n Depending on their relative molecular mass, the appearance ranges from colorless and transparent volatile liquids to extremely viscous liquids or silica gel. They are odorless, highly transparent, and possess heat resistance, cold resistance, low viscosity variation with temperature, water resistance, low surface tension, and thermal conductivity (0.134-0.159 W / (m·K)). They also have 100% light transmittance. Dimethyl silicone oil is non-toxic, odorless, physiologically inert, and has good chemical stability.

[0018] DNA: Deoxyribonucleic acid is one of the four biological macromolecules contained in biological cells, namely nucleic acid.

[0019] The preparation method of the non-close-packed colloidal crystals of the present invention involves techniques such as close-packed colloidal crystal assembly, hydrophilic treatment, stretching transfer, and PDMS gradient design. The flowchart of the preparation process of the non-close-packed colloidal crystals of the present invention is shown below. Figure 1 As shown.

[0020] The method for preparing non-close-packed colloidal crystals based on PDMS thin films provided by this invention includes the following steps: A solution of polystyrene microspheres with DNA probes modified on the surface is provided; The solution of polystyrene microspheres with DNA probes on the surface is used to form a monolayer array of polystyrene microspheres with DNA probes on the water surface; Hydrophilic PDMS films are obtained by hydrophilic treatment of PDMS films; The polystyrene microsphere array with DNA probes on its monolayer surface was transferred onto the hydrophilic PDMS film as is, and then dried to obtain a close-packed colloidal crystal based on the PDMS film. Stretch the closely packed colloidal crystals based on the PDMS film to a predetermined stretching increment of the PDMS film; Closely packed colloidal crystals on a stretched PDMS film are transferred onto another unstretched PDMS film to obtain non-closely packed colloidal crystals based on PDMS film.

[0021] The method for preparing non-close-packed colloidal crystals based on PDMS films of the present invention is carried out at room temperature, is simple to operate, and has high controllability. By controlling the size of the microspheres and the stretching distance, non-close-packed colloidal crystals with different periods and spacings can be prepared. The prepared non-close-packed colloidal crystals are low in cost, can highly protect the activity of DNA probes, have a large effective area, and good fluorescence effect, making them more suitable for practical applications.

[0022] Figure 2 This is a schematic diagram of an automated four-way stretching device. The slides, motors, drivers, power converters, and LCD controllers of the automated four-way stretching device are not shown. The main operating system of the automated four-way stretching device consists of four slides, four motors, four drivers, one 24V 20A DC power supply, and one LCD controller. It can perform functions such as synchronous speed control, start and end point setting, and dwell time setting. It also includes a 50cm*50cm*1cm black fiberglass board with specified perforations and four commercially available film clamps. The assembly system includes fiberglass board 1, first magnesium-aluminum alloy sheet metal part 2, second magnesium-aluminum alloy sheet metal part 3, PLA printed part 4, central force plate 5, slides 6, and pressure clamps 7. It supports and integrates the main operating system, allowing the film stretching to be transferred to the main operating system, thereby realizing the operating function and ensuring the stable and effective operation of the system.

[0023] The system includes: a fiberglass board 1, measuring 50 cm x 50 cm x 1 cm, with spaced holes to support and fix four slides 6; a first magnesium-aluminum alloy sheet metal part 2, measuring 10 cm x 4.5 cm x 1 cm, with four holes in the center, which is fixed to the bottom of the slide 6 with nuts via M4 threaded posts; tightening the nuts ensures a tight connection between the first magnesium-aluminum alloy sheet metal part 2 and the slide; two first magnesium-aluminum alloy sheet metal parts 2 are used to fix both ends of each slide 6 to keep the slide level; and a central support plate 5, measuring 4.5 cm x 4.5 cm x 2 cm, whose central holes are connected and fixed to the central holes of the fiberglass board 1 via rubber pads and M6 screws. The slide table, to which the first magnesium-aluminum alloy sheet metal part 2 is fixed, is tightly attached to the central force plate 5 in four vertical directions. The first magnesium-aluminum alloy sheet metal part 2 has an M6 hole communicating with the fiberglass board 1. The fiberglass board 1 and the first magnesium-aluminum alloy sheet metal part 2 are fixed at the bottom of the fiberglass board 1 with rubber pads and M6 nuts, thus preventing the slide table from sliding. The second magnesium-aluminum alloy sheet metal part 3 is 8.8 cm * 4.2 cm * 1 cm. The recessed design on one side of the second magnesium-aluminum alloy sheet metal part 3 is designed to be higher than the other side of the slide table to prevent collision. The part longer than the slider is used to support the pressure clamp 7 with threaded teeth. The second magnesium-aluminum alloy sheet metal part 3 and the slider are fixedly connected to the slider of the slide table with M4 threads. The PLA printed part 4, 5.8 * 4.2 * 4 cm, is fixed to the pressure clamp with M6 threads and fixed to the second magnesium-aluminum alloy sheet metal part 3.

[0024] The pressure clamp 7 with threaded teeth (commercial, purchased from Taobao store "Yongfa Mechanics Experimental Instruments", model ZQ-H26B) is coated with friction rubber to fix the PDMS film and prevent it from breaking. The clamp spacing is adjustable by 1 cm, and the clamp dimensions are 2*5 cm. Each slide (commercial, from "Ouli Transmission Co., Ltd.", model SGX-1610-100) has one stepper motor on one side controlled by one driver (commercial, from "Ouli Transmission Co., Ltd.", model DM542). Four drivers are integrated into one LCD controller (commercial, from "Ouli Transmission Co., Ltd.", model YJ004), powered by a 24 V 20 A DC power supply. The slide dimensions are 22*4.3*5.7 cm, with an effective stroke of 10 cm and an adjustable speed of 1~100 mm / s. It can perform synchronous speed control, start and end point setting, and dwell time setting functions.

[0025] This invention relates to a method for preparing non-close-packed colloidal crystals from polystyrene microspheres modified with DNA probes or other bioactive components. All operations are performed at room temperature, minimizing DNA probe damage through a single transfer process. An automated mechanical stretching system is used for the transfer, resulting in a more intelligent and stable process. This method offers advantages such as simplified operation, avoidance of high-temperature damage, reduced manual stretching errors, and the ability to handle large-sized microspheres. It yields non-close-packed colloidal crystals with arbitrary spacing and periodicity. Furthermore, using a relatively viscous PDMS film as the final substrate does not affect fluorescence properties, resulting in significant fluorescence effects. The method also increases reusability, allowing for multiple rinsing and soaking, reducing detachment rates. This has profound implications for applications in high-resolution fluorescence array colorimetry.

[0026] Example: At room temperature, a solution of 2.5 wt% 5 μm polystyrene microspheres (the diameter of the polystyrene microspheres was 0.5~3 μm, purchased from Tianjin Bestlite Chromatography Technology Development Center (CAS No.: 7440-57-5), with surface modified with various DNA probes) was sonicated for 30 min. After sonication, 1 mL was pipetted into a 2 mL centrifuge tube, and deionized water was added to the 1.5 mL mark. The tube was then sonicated for 5 min in an ultrasonic cleaner, followed by centrifugation at 2000 rpm for 5 min. After centrifugation, the supernatant was aspirated with a 0.2~3 mL pipette. This centrifugation and sonication process was repeated 6 times. The temperature during the centrifugation and sonication process did not exceed 40℃. No more deionized water was added to the centrifuge tube mark. 0.15 mL of water and 0.3 mL of anhydrous ethanol were added to the precipitate. After sonication for 30 min, a 5 wt% polystyrene microsphere solution was obtained.

[0027] Take a new circular plastic culture dish with a diameter of 90 mm, rinse with deionized water for 1 min, add 5 mL of anhydrous ethanol and sonicate, then rinse with anhydrous ethanol for 10 s, place in a drying oven at 40℃ for 10 min, and after drying, place in a plasma cleaner with a power of 50 W, a time of 40 s, an air flow rate of 50 sccm, an air pressure of 0.024 kPa, and a temperature not exceeding 40℃. After the process, a hydrophilic plastic culture dish is obtained. Place it against a black background, and place a 2cm*2cm glass block with a thickness of 0.3 cm on one side of the culture dish. Quickly add deionized water to cover the bottom of the raised culture dish.

[0028] Aspirate the 5 wt% polystyrene microsphere solution into a disposable syringe. The needle should be 1-2 cm above the bottom of the raised culture dish. Slowly drip the solution onto the raised part of the culture dish. After the water surface disperses and then closes again, add one drop of microsphere solution within 2 seconds. Repeat this drip-disperse-close action until the polystyrene microsphere solution is exhausted. Then, use tweezers to pick up a very small amount of oil and insert it into the water at the raised part of the culture dish. Next, place an 80-140 mm diameter Teflon ring around the area where the microspheres are located to separate the microspheres from the culture dish wall. Remove the glass pad and slowly add deionized water between the Teflon ring and the culture dish until it is 3 mm from the highest edge of the culture dish. Then, use a 5 mL pipette to slowly aspirate the aqueous solution containing the polystyrene microspheres. Repeat the water injection and aspiration action 3 times until the aqueous solution is clear, obtaining a monolayer of tightly packed colloidal crystals on the water surface.

[0029] A circular PDMS film with a relative viscosity of 300, an elongation of 300%, a thickness of 500 μm, and a diameter of 70 mm (purchased from Hangzhou Guige New Material Technology Co., Ltd. (batch number 2023-3-28)) was taken, with the film on both sides torn off, and placed on a silicon wafer with a size of 100 mm. Then, it was placed in a plasma cleaner for surface hydrophilic treatment. The power was 50 W, the time was 20 s, the air flow rate was 50 sccm, the air pressure was 0.024 KPa, and the temperature did not exceed 40℃. After the treatment, a hydrophilic PDMS film was obtained.

[0030] Place the hydrophilic side of the PDMS film on top, slowly insert it into the Teflon ring without damaging the polystyrene microsphere array, press the PDMS film down with tweezers to make it adhere to the bottom of the culture dish, slowly absorb the aqueous solution between the Teflon ring and the culture dish until the polystyrene microsphere array slowly and without breaking falls onto the hydrophilic PDMS, and then air dry in the room for 3 hours to obtain tightly packed colloidal crystals.

[0031] The obtained close-packed colloidal crystals were observed under a microscope. Figure 3These are electron microscope images of colloidal crystals. Images A and B are electron microscope images of closely packed colloidal crystals based on PDMS films; images C and D are electron microscope images of non-close-packed colloidal crystals based on PDMS films. A large, regular crystalline phase was observed under the microscope. Its row and vertical orientations were marked. The closely packed colloidal crystals faced upwards. The PDMS film was placed horizontally and vertically at the center of an automated four-way stretching device and clamped. After fixing, stretching began at a speed of 1 mm / s. The 4 cm * 4 cm square PDMS at the center was the effective stretching area. The stretching distance from the four sliders to the surrounding area was 7 cm, with a stretching increment of 1.75 times. Stretching stopped at the 7 cm mark, yielding non-close-packed colloidal crystals. Tear open one side of a square PDMS film with a relative viscosity of 2000 and a side length of 5cm. Slowly attach it to the non-close-packed colloidal crystal, aligning it with the four stretching directions. Clamp and press it firmly with a flat clamp. Then, slowly and steadily lift the top PDMS film with tweezers to obtain a non-close-packed colloidal crystal based on the PDMS film. Figure 3 The microsphere arrays contained in A and C have an area of ​​0.92 mm * 0.73 mm (magnification 5x). Figure 3 The microsphere arrays contained in B and D have an area of ​​0.18 mm * 0.14 mm (magnification 50x).

[0032] Take a non-close-packed colloidal crystal based on a PDMS film and add 100 μL of a hybridization probe solution (oligonucleotide chain with FAM group modified at the end) prepared with 1 μM PBST buffer (8 mM Na2HPO4, 2 mM KH2PO4, 10 mM KCl, 140 mM NaCl, 0.05% (V / V) Tween-20, pH 7.2~7.4). This allows the oligonucleotide chain modified with FAM group in the hybridization probe solution to be complementary to the oligonucleotide chain on the polystyrene microspheres. After incubation at room temperature for 30 min, the microspheres are washed three times by centrifugation with PBST buffer.

[0033] like Figure 4 As shown, after incubation with the probe solution, the fluorescence color development was normal, which proves that the oligonucleotide single strands modified on the surface of the polystyrene microspheres are functional. The method of the present invention will not cause abnormal DNA probe activity during the formation of non-close-packed colloidal crystals.

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the technical solution of the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing non-close-packed colloidal crystals based on PDMS thin films, comprising the following steps: A solution of polystyrene microspheres with surface-modified DNA probes is provided; The solution of polystyrene microspheres with DNA probes on the surface is used to form a monolayer array of polystyrene microspheres with DNA probes on the water surface; The unstretched first PDMS film was subjected to hydrophilic treatment to obtain a hydrophilic PDMS film; The polystyrene microsphere array with DNA probes on its monolayer surface is transferred to the hydrophilic PDMS film as is, and dried to obtain a closely packed colloidal crystal based on the first PDMS film. The close-packed colloidal crystals based on the first PDMS film are stretched to a predetermined stretching increment from the first PDMS film. The closely packed colloidal crystals on the stretched first PDMS film are transferred onto the unstretched second PDMS film to obtain non-closely packed colloidal crystals based on PDMS film. in, The relative viscosity of the unstretched first PDMS film is lower than that of the unstretched second PDMS film.

2. The method according to claim 1, characterized in that, The polystyrene microspheres with DNA probes on their surface were dispersed in an ethanol-water solution by ultrasonic treatment to obtain a solution of the polystyrene microspheres with DNA probes on their surface.

3. The method according to claim 1, characterized in that, The unstretched first PDMS film is a PDMS film with a relative viscosity of 100~300.

4. The method according to claim 1, characterized in that, The unstretched second PDMS film is a PDMS film with a relative viscosity of 1000~2500.

5. A non-close-packed colloidal crystal based on a PDMS thin film prepared by the method of any one of claims 1 to 4.

6. The use of the non-close-packed colloidal crystal based on PDMS thin film as described in claim 5 in high-resolution fluorescence periodic array detection.

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