CN1212543C - Method for Immobilizing Biomacromolecules Patterned on Polymer Surfaces Using Microdelivery Technology - Google Patents

Abstract

The present invention discloses a method for patternizing and fixing biologic macromolecules on the active surfaces of polymers by microtransfer technology, which comprises the following steps that firstly, biologic macromolecules are selectively loaded in the microwell of a polydimethylsiloxane stamp; one layer of condensed water is formed on the surface of the stamp, the biologic macromolecules are dissolved again by utilizing the original water in the microwell and water transversely diffused in contact areas after the stamp and the modified polymer film are pressed, and thus, the biologic macromolecules in a solution form chemically react with surface base groups so as to realize that biologic macromolecules are patternized and fixed on the active surfaces of the polymers. The present invention has the advantages of simple and flexible technology, favorable repeatability, high contrast degree and firmness of the obtained micropatterns, and low requirements to the surface property and the microwell depth of the stamp, and the preparation method is a simple cheap effective method for patternizing and fixing the biologic macromolecules, particularly applicable to biomolecule / active surface system with low reactivity. The present invention has a favorable application prospect in the fields of biologic device construction based on the cells, tissue engineering, basic research of cell biology, etc.

Description

Method for Immobilizing Biomacromolecules Patterned on Polymer Surfaces Using Microdelivery Technology

Technical field

The invention relates to a method for patterning and immobilizing biomacromolecules on the active surface of a polymer using micro-delivery technology. Specifically, the protein biomacromolecule solution "contained" in the microwells on the surface of the polydimethylsiloxane (PDMS) stamp surface array is transferred to the active surface of the polymer so that the reaction with the surface active groups can be carried out in a solution state. Therefore, it is more convenient to realize the patterned immobilization of protein biomacromolecules on the polymer surface.

Background technique

With the development of modern biotechnology, patterned immobilization of biological macromolecules has attracted extensive attention. Patterning and immobilizing biomacromolecules and cells on the surface of polymer materials has great practical value for specific biological detection, construction of biosensors based on living cells, and repair of artificial neural networks. At present, microcontact printing technology can realize the patterned immobilization of biomacromolecules on the surface of gold, glass, silicon, and polymers through physical adsorption. But there are disadvantages of easy desorption and poor stability. How to achieve firm immobilization of biomacromolecular patterns is a problem that needs to be solved at present.

Polymer materials are ideal substrates for patterned immobilization of biomacromolecules due to their good physical and chemical properties. Various reactive groups can be conveniently introduced on the surface of the polymer to achieve firm immobilization of biomacromolecules through covalent bonding. Traditional reactive microprinting technology can conveniently realize the patterned firm immobilization of small biological molecules. However, in order to avoid "pollution" caused by the lateral diffusion of solvents, it is generally required to dry the template before pad printing or retain a very small amount of solvent, or require a short reaction time. This is unfavorable for the reaction of non-liquid biomacromolecules with surface active groups, especially for biomacromolecules/active surface systems with low reactivity.

Contents of Invention

The purpose of the present invention is to provide a method for patterning and immobilizing biomacromolecules on the surface of a polymer using micro-delivery technology.

The method of the present invention is first to selectively "load" biological macromolecules into the micro-wells of polydimethylsiloxane (PDMS) stamps, and then form a layer of condensed water on its surface, utilize the stamps and modified polymer film After compaction, the original water in the microwell and the water diffused laterally in the contact area redissolve the biomacromolecules, thereby chemically reacting with the surface groups in the form of a solution, and realizing the patterned immobilization of biomacromolecules on the active surface of the polymer. Specifically include the following steps:

1) Modify the polymer with conventional chemical modification or plasma modification methods, and introduce hydroxyl, carboxyl, and amino groups that can react with biomacromolecules on the surface of the polymer;

2) Prepare a PDMS stamp with microwells on the surface, and dip-coat the biomacromolecule solution with a concentration of 0.1-20 mg/ml on the side with microwells, and dry it slowly with nitrogen flow;

3) Take a piece of the above-mentioned polymer film and emboss it on the surface of the stamp for 0.1-10 hours, with a pressure of 10-200g/cm ² , to selectively remove the biological macromolecules adsorbed in the area between the micro-wells;

4) Keep the stamp at a temperature of -18 to 0°C for 0.1 to 5 minutes, then place it in an environment with a relative humidity of 50 to 99% at a temperature of 20 to 37°C for 0.1 to 5 minutes to form a layer on its surface condensed droplets;

5) Immediately impress the stamp on the surface of the modified polymer obtained in step 1) at 0-100°C for 0.1-10 hours at a pressure of 10-200g/cm ² , preferably at a temperature of 4-50°C;

6) Take off the seal, rinse with a buffer solution or an aqueous solution with a corresponding pH value of the biomacromolecule, soak for 24 hours, and then blow dry to obtain a micropattern of the biomacromolecule.

In the present invention, the polymer is a polymer that can produce active groups that can react with biomacromolecules on its surface through chemical modification or plasma modification, such as poly (L-lactic acid) (PLLA), poly (D-lactic acid) (PDLA), polycaprolactone (PCL), poly(D,L-lactic-co-glycolic acid) (PLGA), polyurethane (PU), polyethylene terephthalate (PET ), polypropylene (PP), polystyrene (PS), etc.

The polymer film embossed on the surface of the stamp in step 3) is the same polymer as in step 1), which can be an unmodified polymer or a modified polymer. The selective removal of biomacromolecules refers to the transfer of biomacromolecules in the area where the protrusions of the stamp are in contact with the imprinted polymer film while retaining the biomacromolecules in the microwells.

In the invention, the preparation of PDMS stamps with micro-wells on the surface is to first construct a bottom mold containing micron-scale columnar protrusions through conventional "photolithography" technology, and then pour PDMS prepolymer on the surface of the bottom mold, heat and cross-link and solidify. Made from. Generally, UV-type photoresist is spin-coated on a flat and clean glass or silicon surface, and then exposed to ultraviolet light under a photomask with a characteristic pattern and developed to obtain an initial template containing micron-scale columnar protrusions, and then use this as the base Mold, then pour the mixture of PDMS prepolymer and crosslinking agent on it, heat and cure it and lift it up. Generally, the curing temperature is 40-100°C and the time is 6-24 hours.

In the present invention, the biomacromolecules refer to protein biomacromolecules and their derivatives, including albumin, fibronectin, laminin, polylysine, collagen and gelatin. Step 2) said biomacromolecule solution refers to buffer solution or aqueous solution of its corresponding pH value, as the PBS solution of the phosphate buffer saline (PBS) solution of albumin, gelatin, the 0.3% of collagen or chitosan Acetic acid solution, etc.

For the micropattern of the biomacromolecule obtained by the method of the present invention, its existence can be detected with a laser confocal microscope (CLSM) or a fluorescent microscope. Rhodamine et al.

With the method of the invention, biomacromolecule micropatterns of corresponding size and spacing can be obtained by adjusting the diameter and spacing of the microwells in the PDMS stamp. The quality of biomacromolecule micropatterns can be controlled by adjusting the amount of condensed water, and the amount of condensed water can be adjusted from two factors: condensation time and relative humidity.

The process of the invention is simple and flexible, has good repeatability, and the obtained micropattern has high contrast and fastness, and is a simple and cheap effective method for patterning and immobilizing biomacromolecules. The "drying-transfer-condensation" method is used to conveniently realize the "containment" of the biomacromolecule solution in the microwell. Each microwell becomes a microreactor, enabling the reaction between biomacromolecules and surface groups to proceed at a rate comparable to that of interfacial grafting reactions in solution, while avoiding pattern “contamination” by lateral diffusion of solvents. ". Compared with the traditional reactive micro-printing technology, the present invention has lower requirements on the hydrophilicity and hydrophobicity of the seal surface and the depth of micro-wells. PDMS seals do not need to be treated with plasma hydrophilicity, and the depth of micro-wells does not need to be too deep. Conventional "photolithography" Technology can be made. The invention is especially suitable for patterning and immobilizing biomacromolecules on the surface of polymers with general reactivity. It has good application prospects in the fields of specific biological detection, biosensor construction based on living cells, and repair of artificial neural networks.

Description of drawings

Fig. 1 is a flow chart of patterning and immobilizing biomacromolecules on the active surface of modified polymers by micro-delivery technology. 1 in the figure indicates that 0.1-20 mg/ml biomacromolecule solution is dip-coated on the side of the PDMS stamp with micro-wells; 2 indicates that the stamp is slowly dried with a nitrogen stream; 3 indicates that a polymer film is embossed on the surface of the stamp to selectively remove Biomacromolecules in the area between the microwells; 4 indicates the formation of condensed water on the surface of the stamp; 5 indicates the imprinting of the stamp on the surface of the modified polymer. 6 indicates that the stamp is lifted and the patterned polymer film immobilized with biomacromolecules is rinsed.

Fig. 2 is an atomic force microscope photo of a PDMS stamp containing arrayed microwells on the surface used in the invention.

Fig. 3 (a) is the laser confocal microscope (CLSM) picture of the albumin (BSA) patterned immobilization on the polycaprolactone surface (PCL-CHO) of aldehyde by the method of the present invention, albumin is due to isosulfur Fluorescein cyanate (FITC) was labeled to display bright colors; (b) is the corresponding fluorescence intensity analysis of (a).

Fig. 4 is the CLSM photo of the PDMS stamp before and after patterning and immobilizing albumin on the surface of aldehydated polycaprolactone (PCL-CHO) by the method of the present invention, wherein (a) is before patterning and immobilization, and (b) is the pattern after fixation. Albumin shows bright color due to labeling with FITC.

Figure 5(a) is the CLSM image of albumin (BSA) immobilized on the surface of PCL-CHO using the existing reactive microprinting technology, albumin shows bright color due to FITC labeling; (b) is (a) Corresponding fluorescence intensity analysis.

Fig. 6 is a pattern of defects caused by unsuitable amount of condensed water when albumin is patterned and immobilized on the surface of formylated polycaprolactone (PCL-CHO) by the method of the present invention, wherein (a) is a circular ring obtained when condensed water is insufficient Shaped pattern, (b) is the pattern "contamination" caused by excessive condensed water.

Detailed ways

The present invention is described in detail below with specific examples.

Example 1

The method for patterning and immobilizing biomacromolecules on the active surface of polymers using micro-transfer technology of the present invention comprises the following steps:

1. Prepare the PCL film with free aldehyde groups on the surface: the PCL film is placed in the isopropanol solution of hexamethylenediamine with a concentration of 10% (wt), and it is aminated for 10 minutes at 37° C. Lots of free amine groups. Then put the PCL membrane rich in amine groups on the surface into 2% glutaraldehyde solution, and conduct a coupling reaction at 37° C. for 30 minutes to prepare the PCL membrane rich in free aldehyde groups on the surface. At this time, the film appears light red or orange red due to the aldylation reaction.

2. Preparation of PDMS stamps with arrayed microwells on the surface: first prepare an original bottom mold with conventional "photolithography" technology, that is, spin-coat BP218 on the surface of a clean 3× ^3cm2 glass slide at a speed of 2000 rpm The UV positive photoresist is then baked in an oven at 90°C for 30 minutes, then rotated for 150 seconds under a 1000-watt UV lamp and a photomask, and then developed in 2% NaOH solution for 45 seconds. Uniformity. Since the photomask used contains the blind holes of the microarray, the surface of the finally prepared bottom mold contains the corresponding protruding columns of the microarray. On the surface of the original bottom mold, PDMS prepolymer was poured, and the prepolymer had been added with a cross-linking agent at a mass ratio of 10:1. After 12 hours of cross-linking and curing in an oven at 60°C, the PDMS soft stamp was obtained, and the array microwells of corresponding sizes on the surface could be observed with an atomic force microscope (see Figure 2).

3. Selective loading of albumin (BSA) in microwells: make 1.0 mg/ml PBS buffer solution with fluorescein isothiocyanate-labeled albumin (FITC-BSA), add one drop to the above-mentioned On the side of PDMS with micro-wells, use a glass rod to drive back and forth evenly, and blow dry slowly with nitrogen flow. Put a piece of PCL film lightly on the FITC-BSA-coated side of the PDMS stamp, and press it with a pressure of 100g/ ^cm2 , and keep it at room temperature for 1 hour. Lift the PCL membrane, and the BSA adsorbed in the area between the microwells is transferred away, leaving only the BSA contained in the microwells of the array.

4. Formation of condensed water: put the PDMS stamp filled with BSA in a refrigerator at -12°C to cool for 2 minutes, and then let it stand in an atmospheric environment (25°C) with a relative humidity of 80-90% for 1 minute, that is, A thin layer of condensation forms on its surface.

5. Put the stamp upside down on the aldehyde-based PCL film quickly, and press it tightly with a pressure of 100g/cm ² , and keep it at room temperature for 3 hours. After the seal and the aldehyde-based PCL membrane are pressed tightly, the original water in the micro-well and the water diffused laterally in the contact area redissolve the biomacromolecules and chemically react with the surface groups in the form of a solution. Each micro-well becomes a micro-reactor .

6. Gently lift the PDMS stamp, rinse the patterned PCL membrane immobilized with biomacromolecules with a large amount of PBS buffer, rinse in PBS for 24 hours, and blow dry with nitrogen.

Observation results with a laser confocal microscope (CLSM): Fig. 3a is the microarray pattern of BSA obtained by the above method on the surface of the aldehyde-based PCL film, and b is the corresponding fluorescence intensity analysis of the pattern, illustrating the use of the microtransfer method

High pattern contrast was obtained, confirming the effectiveness of the microtransfer method. Figure 4a and b are the BSA patterns in the PDMS stamp before and after micro-transfer, respectively, confirming that BSA has been completely transferred from the PDMS micro-well to the surface of the PCL membrane.

Example 2

This example is a comparative example with the present invention. The other steps were the same as in Example 1, but the existing reactive microcontact printing technology was used to pattern and immobilize albumin. That is, dip-coat the PBS buffer solution of FITC-BSA with a concentration of 1.0 mg/ml on the side of the PDMS stamp with microwells, and slowly dry it with nitrogen flow, and quickly transfer it on the aldehyde-formed PCL membrane. Pressure, time, temperature and subsequent flushing are all the same as in Example 1. CLSM observation found that the BSA pattern obtained by this method (Figure 5a) is much darker than the BSA pattern obtained by the present invention, and the corresponding fluorescence intensity analysis shows that the pattern contrast is also much lower (Figure 5b).

Example 3

The other steps are the same as in Example 1, but the PDMS stamp is left standing for 0.1 minute or 3 minutes in an atmospheric environment (25°C) with a relative humidity of 80-90%, and its influence on the pattern quality is observed. It was found that the former formed a donut-shaped pattern due to insufficient condensed water (Fig. 6a), while the latter caused pattern "contamination" due to excessive condensed water (Fig. 6b). This shows that when using the microtransfer method to pattern and immobilize biomacromolecules, the amount of condensed water formed on the PDMS surface has a great influence on the quality of the final pattern, and too low or too high will lead to pattern defects.

Claims (5)

1. With little transmission technology in the macromolecular method of polymer surfaces patterning fixed biologically, it is characterized in that may further comprise the steps:

   1) polymer-modified with conventional chemical modification or plasma modification method, polymer surfaces introduce can with hydroxyl, carboxyl, the amino group of biomacromolecule reaction;

   2) the preparation surface contains the dimethyl silicone polymer seal of little well, and at it biological macromolecule solns of one side dip-coating 0.1～20mg/ml concentration of little well is arranged, and slowly dries up with nitrogen stream;

   3) get above-mentioned thin polymer film a slice and impressed in the seal surface pressure 10～200g/cm 0.1～10 hour ², remove the biomacromolecule that is adsorbed on zone between little well with selectivity;

   4) seal was kept 0.1～5 minute under-18～0 ℃ of temperature, again under 20～37 ℃ of temperature, relative humidity is to leave standstill 0.1～5 minute in 50～99% the environment, forms one deck condensation droplet on its surface;

   5) under 0～100 ℃, immediately this seal is impressed in the reforming polymer surface of step 1) gained 0.1～10 hour pressure 10～200g/cm ²

   6) uncover seal, the damping fluid or the aqueous solution of the corresponding pH value of usefulness biomacromolecule are washed, and soak to dry up after 24 hours, promptly obtain little pattern of biomacromolecule.
2. 2. press the little transmission technology of the described usefulness of claim 1 in the macromolecular method of polymer surfaces patterning fixed biologically, it is characterized in that said polymkeric substance is poly (l-lactic acid), poly-D-lactic acid, polycaprolactone, poly-D, L-lactic acid-be total to-glycollic acid, polyurethane, polyethylene terephthalate, polypropylene, polystyrene.
3. 3. press the little transmission technology of the described usefulness of claim 1 in the macromolecular method of polymer surfaces patterning fixed biologically, it is characterized in that the surface contains the dimethyl silicone polymer seal of little well, be earlier by conventional bed die that contains the micron order columnar protrusions of " photoetching " technique construction, again at this bed die surface casting dimethyl silicone polymer prepolymer, uncover after heat cross-linking solidifies and to make.
4. 4. press the little transmission technology of the described usefulness of claim 1 in the macromolecular method of polymer surfaces patterning fixed biologically, it is characterized in that said biomacromolecule is meant protide biomacromolecule and derivant thereof, comprises albumin, fibronectin, laminin, poly-D-lysine, collagen and gelatin etc.
5. By the little transmission technology of the described usefulness of claim 1 in the macromolecular method of polymer surfaces patterning fixed biologically, it is characterized in that step 2) said biological macromolecule solns is meant the buffer soln or the aqueous solution of its corresponding pH value.

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