A method for rapidly preparing and on-line characterizing an amino-silane coating in an aqueous phase system

Abstract

The present invention discloses a method for rapidly preparing and on-line characterizing an amino-silane coating in an aqueous phase system. Under negative pressure conditions, an amino-silane buffer solution is introduced into a fused silica capillary. The process of forming Si-O-Si bonds by the bonding of Si-OR in the amino-silane molecule and Si-OH on the surface of the microchannel is utilized to achieve the preparation and real-time characterization of the amino-silane coating: the formation of the amino-silane bonded coating is characterized in real time by the change of the surface electrical signal with time during the formation of the Si-O-Si bond. The stability of the amino-silane bonded coating in the aqueous phase system is characterized in real time by the change of the chemical bond caused by the hydrolysis and cleavage of the Si-O-Si bond in the pure buffer system; the content of amino groups on the capillary surface is indirectly characterized by the change of the fluorescence intensity of the green fluorescent protein bonded and fixed by the amino-silane coating.

Description

Technical Field

[0001] The present invention relates to a method for rapidly preparing and on-line characterizing an amino-silane coating in an aqueous phase system, belonging to the technical field of sensing detection. Background Art

[0002] The formation of Si-O-Si bonds between amino-silanes and surfaces containing Si-OH is the most commonly used surface amino modification method. Among them, 3-aminopropyltriethoxysilane (APTES) and 3-aminopropyltrimethoxysilane (APTMS) are the most commonly used amino-silane molecules. After the APTES or APTMS molecules are bonded to the silicon hydroxyl surface to form Si-O-Si bonds, they are fixed on the surface, and finally a modified coating with exposed amino groups is formed. The use of this amino coating can achieve the fixation of biomolecules such as DNA molecules, antibodies, enzymes, and metal nanoparticles on the surface, and further complete related detections and applications.

[0003] Currently, the methods for preparing and characterizing amino-silane coatings on surfaces containing Si-OH such as glass or quartz mainly include: (1) Coating immersion method with a 3% APTES 95% ethanol solution under nitrogen protection (static reaction for 2 h, curing at 115°C for 2 h, and characterizing the formation of the coating using an atomic force microscope); (2) Preparation method by soaking in a 10% APTES ethanol solution at room temperature (static reaction for 30 min, and directly observing the channel roughness using a microscope to judge the formation of the coating); (3) Room temperature immersion method with anhydrous toluene solutions of different concentrations of APTES under nitrogen protection (static for 12 h, curing at 100°C for 1 h, and measuring the thickness of the coating using ellipsometry to judge the formation of the coating); (4) Immersion method with a 0.05% (v / v) APTES aqueous solution (static reaction for 5 min, characterized by scanning electron microscopy); In addition, there are many other coating preparation methods for APTES based on organic systems and aqueous phase systems. However, the above methods all have the problem of a long coating time in the organic phase and cannot achieve the on-line characterization of such coatings. At the same time, whether in the aqueous phase or the organic phase, the characterization methods of such coatings are mainly based on changes in surface roughness, coating thickness, and surface hydrophilicity. Therefore, realizing the preparation, on-line characterization, and stability determination of such coatings in the aqueous phase system is of great significance for their subsequent applications in the aqueous phase system. Summary of the Invention

[0004] The object of the present invention is to provide a method for rapidly preparing and on-line characterizing an amino-silane coating in an aqueous phase system.

[0005] A method for rapidly preparing and on-line characterizing an amino-silane coating in an aqueous phase system, under negative pressure conditions, an amino-silane buffer solution is introduced into a microchannel, and the Si-OR in the amino-silane molecule and the Si-OH on the surface of the microchannel are bonded to form a Si-O-Si bond, thereby realizing the preparation and real-time characterization of the amino-silane coating.

[0006] The buffer solution is a phosphate buffer solution, the concentration of the buffer solution is 1 mM to 10 mM, and the pH is 7.0; the concentration of the amino-silane in the buffer solution is 0.01 μM to 3.0 mM.

[0007] The microchannel is a fused silica capillary, and the inner diameter of the channel is 50 to 100 μm.

[0008] The amino-silane is all silane compounds containing amino groups.

[0009] The negative pressure condition is a negative pressure of 30 to 70 kPa.

[0010] The method for preparing and on-line characterizing the amino-silane bonded coating by using the surface charge measurement means adopted in the present invention, and the chemical bonding coating characterization principle is as Figure 1 shown. Figure 1 In A, for the preparation of the amino-silane coating, a Si-O-Si bond is formed through the bonding between the Si-O-R bond in the amino-silane molecule and the Si-OH bond on the surface of the channel, and the amino-silane is fixed on the surface of the microchannel ( Figure 1 A). Since the surface of the fused silica capillary is negatively charged, and the positive charge is exposed on the outer layer after the amino-silane is fixed, as the bonding proceeds, the surface charge of the microchannel changes from negative to positive, and when the bonding is saturated, the surface charge becomes stable. When a blank buffer is introduced into the microchannel, the Si-O-Si bond of the formed amino-silane coating will undergo partial hydrolysis, thereby exposing Si-OH again, and the positive charge on the channel surface gradually decreases. Therefore, by measuring the change of the surface electrical signal of the microchannel with time during the preparation of the amino-silane bonded coating, the formation of the amino-silane bonded coating and the stability of the amino-silane coating can be on-line and real-time characterized ( Figure 1 B).

[0011] Green fluorescent protein is introduced into the microchannel bonded with amino-silane, and the change of the fluorescence intensity of the green fluorescent protein bonded and fixed by the amino-silane coating is used to indirectly characterize the content of amino groups on the capillary surface. It is also possible to use an inverted fluorescence microscope to take images of the capillary fixed with green fluorescent protein, and then use Image J software to measure the gray value of the image, and use the average gray value to represent the content of the green fluorescent protein fixed on the capillary surface, thereby indirectly characterizing the content of amino groups on the capillary surface. Description of the Drawings

[0012] Figure 1 This is the schematic diagram for the in - line characterization of the silicon - oxygen chemical bond on the surface of silanol groups in the present invention.

[0013] Figure 2 This is the diagram of the APTES - bonded coating prepared in Example 1 and its characterization results.

[0014] Figure 3 This is the diagram of the APTMS - bonded coating prepared in Example 2 and its characterization results.

[0015] Figure 4 This is the diagram of the APTMS - bonded coating prepared in Example 3 and its characterization results.

[0016] Figure 5 This is the diagram of the result of fixing green fluorescent protein on the APTMS - bonded coating prepared in Example 4.

[0017] Figure 6 This is the diagram of the result of fixing green fluorescent protein on the APTMS - bonded coating prepared in Example 5. Detailed implementation manners

[0018] Taking 3 - aminopropyltriethoxysilane (APTES) and 3 - aminopropyltrimethoxysilane (APTMS) as examples, the preparation and in - line characterization process of the amino - silane coating of the present invention will be further described below in conjunction with the accompanying drawings.

[0019] Example 1

[0020] A fused - silica capillary (microchannel) with an inner diameter of 50 μm was rinsed with 1 M NaOH and ultrapure water for 5 min in sequence, and then the determination of the formation of Si - O - Si chemical bonds between 3 - aminopropyltriethoxysilane (APTES) and the Si - OH on the capillary surface with three different hydrolysis times was carried out. Specifically as follows:

[0021] (1) In - line characterization of the formation of Si - O - Si bonds between APTES and the surface of the fused - silica capillary with 0 min hydrolysis: Under the condition of 60 kPa, a solution of 0.5 μM APTES (APTES was dispersed in 10 mM phosphate buffer and immediately introduced into the microchannel, and the hydrolysis time of APTES was 0 min) was introduced into the fused capillary for 500 s to realize the determination of the in - line characterization of the formation of Si - O - Si bonds between APTES with 0 min hydrolysis and the Si - OH on the capillary surface.

[0022] (2)In-situ characterization of the formation of Si-O-Si bonds between hydrolyzed APTES for 10 min and the surface of fused silica capillary: Under the condition of 60 kPa, 500 s of 0.5 μM APTES solution (APTES was dispersed in 10 mM phosphate buffer, and after standing for 10 min, it was introduced into the microchannel, with the hydrolysis time of APTES being 10 min) was introduced into the fused capillary to achieve the in-situ characterization of the formation of Si-O-Si bonds by the bonding of hydrolyzed APTES for 10 min with the Si-OH groups on the capillary surface;

[0023] (3)In-situ characterization of the formation of Si-O-Si bonds between hydrolyzed APTES for 20 min and the surface of fused silica capillary: Under the condition of 60 kPa, 500 s of 0.5 μM APTES solution (APTES was dispersed in 10 mM phosphate buffer, and after standing for 20 min, it was introduced into the microchannel, with the hydrolysis time of APTES being 20 min) was introduced into the fused capillary to achieve the in-situ characterization of the formation of Si-O-Si bonds by the bonding of hydrolyzed APTES for 20 min with the Si-OH groups on the capillary surface.

[0024] Figure 2 Figure showing the prepared APTES-bonded coating and the characterization results. Figure 2 The results show that the APTES hydrolyzed for 0 min completely reacted with the Si-OH on the capillary surface within 60 s, indicating that the formation of Si-O-Si is very fast. While the APTES hydrolyzed for 10 min and 20 min completely reacted with the Si-OH on the capillary surface at about 300 s, indicating that the formation of Si-O-Si bonds between molecules in the aqueous phase system of APTES will affect its interaction with the Si-OH on the capillary surface.

[0025] Example 2

[0026] A fused silica capillary (microchannel) with an inner diameter of 50 μm was rinsed with 1 M NaOH and ultrapure water for 5 min in sequence, and then the stability of the formation of Si-O-Si chemical bonds by the interaction of 3-aminopropyltrimethoxysilane (APTMS) with different hydrolysis times with the Si-OH on the capillary surface was measured (that is, after the amino-silane coating was formed on the capillary surface, the influence of the hydrolysis and fracture of Si-O-Si bonds in the coating structure on its stability). Specifically as follows:

[0027] (1)Online determination of hydrolysis of Si-O-Si bonds formed on the surface of fused silica capillary by APTMS for 5 min: Under the condition of 70 kPa, a solution of 0.5 μM APTMS (APTMS was dispersed in 10 mM phosphate buffer and left for 5 min before being introduced into the capillary for measurement) was introduced into the fused capillary for 500 s to achieve the online characterization of the formation of Si-O-Si bonds by the bonding of APTMS hydrolyzed for 5 min with the Si-OH groups on the capillary surface; then, 10 mM phosphate buffer was introduced into the capillary for 500 s to online characterize the hydrolysis of the Si-O-Si bonds formed under the above conditions.

[0028] (2)Online determination of hydrolysis of Si-O-Si bonds formed on the surface of fused silica capillary by APTMS for 22 min: Under the condition of 70 kPa negative pressure, a solution of 0.5 μM APTMS (APTMS was dispersed in 10 mM phosphate buffer and left for 22 min, and the hydrolysis time of APTMS was 22 min) was introduced into the fused capillary for 500 s to achieve the online characterization of the formation of Si-O-Si bonds by the bonding of APTMS hydrolyzed for 22 min with the Si-OH groups on the capillary surface; then, 10 mM phosphate buffer was introduced into the capillary for 500 s to online characterize the hydrolysis of the Si-O-Si bonds formed under the above conditions.

[0029] (3)Online determination of hydrolysis of Si-O-Si bonds formed on the surface of fused silica capillary by APTMS for 39 min: At 70 kPa, a solution of 0.5 μM APTMS (APTMS was dispersed in 10 mM phosphate buffer and left for 39 min, and the hydrolysis time of APTES was 39 min) was introduced into the fused capillary for 500 s to achieve the online characterization of the formation of Si-O-Si bonds by the bonding of APTMS hydrolyzed for 39 min with the Si-OH groups on the capillary surface; then, 10 mM phosphate buffer was introduced into the capillary for 500 s to online characterize the hydrolysis of the Si-O-Si bonds formed under the above conditions.

[0030] Figure 3 It is the figure of the prepared APTMS bonded coating and the characterization results. Figure 3 The results show that the hydrolysis rate of the Si-O-Si bonds formed by APTMS hydrolyzed for 5 min on the capillary surface is 1.68×10 -4 s -1 ; the hydrolysis rate of the Si-O-Si bonds formed by APTMS hydrolyzed for 22 min on the capillary surface is 2.77×10 -4 s -1 ; the hydrolysis rate of the Si-O-Si bonds formed by APTMS hydrolyzed for 39 min on the capillary surface is 4.08×10 -4 s-1 It is noted that during the hydrolysis of APTMS in an aqueous system, the Si-O-Si bonds formed between molecules will affect the stability of the Si-O-Si bonds formed with Si-OH on the capillary surface. The longer the hydrolysis time of APTMS, the poorer the stability of the Si-O-Si bonds formed with the capillary surface.

[0031] Example 3

[0032] A fused silica capillary (microchannel) with an inner diameter of 50 μm was rinsed successively with 1 M NaOH and ultrapure water for 5 min. Then, the formation of Si-O-Si bonds between different concentrations of aminosilane and Si-OH on the capillary surface was measured, as well as the hydrolysis of Si-O-Si bonds and its effect on stability, to verify the influence of concentration on the formation of Si-O-Si bonds. The specific steps are as follows:

[0033] (1) Measurement of the interaction between different concentrations of APTMS and Si-OH on the quartz capillary surface. At 70 kPa, different concentrations of APTMS (0.1 μM, 0.7 μM, 1.0 μM, with a hydrolysis time of 5 min) were introduced into the treated fused silica capillary for 500 s to obtain the on-line characterization results of the interaction between APTMS and Si-OH on the capillary surface. Then, 10 mM phosphate buffer was introduced into the above capillary to measure the results of the change in surface charge caused by the hydrolysis of the Si-O-Si bonds formed between different concentrations of APTMS and Si-OH on the capillary surface. As Figure 4 shown in A, the rates of formation of Si-O-Si bonds formed by different concentrations of APTMS are 0.0203 s -1 (0.1 μM), 0.02589 s -1 (0.7 μM), 0.02546 s -1 (1.0 μM), indicating that as the concentration of APTMS increases, its interaction with Si-OH on the capillary surface accelerates, but not significantly. The hydrolysis results of the Si-O-Si bonds formed between APTMS and the capillary surface show that the Si-O-Si bonds formed by APTMS with a higher concentration have better stability with the capillary surface, which are 4.07 × 10 -4 s -1 (0.1 μM), 2.09 × 10 -5 s -1 (0.7 μM), 3.53 × 10 -5 s -1 (1.0 μM). The above results show that when the concentration of APTMS is 0.7 μM, the rate of formation of Si-O-Si bonds is the fastest and the stability is the highest.

[0034] (2)Determination of the interaction between APTES and Si-OH on the surface of quartz capillary. At 70 kpa, different concentrations of APTMS (concentrations were 0.5 μM, 1.5 μM, 3.0 μM respectively, hydrolysis time was 5 min) were introduced into the treated fused quartz capillary for 500 s to obtain the online characterization results of the interaction between APTES and Si-OH on the capillary surface; then, 10 mM phosphate buffer was introduced into the above capillary to measure the results of the change in surface charge caused by the hydrolysis of the Si-O-Si bond formed between different concentrations of APTES and Si-OH on the capillary surface. As Figure 4 shown in B, the rates of the Si-O-Si bonds formed by different concentrations of APTES were 0.00883 s -1 (0.1 μM), 0.01753 s -1 (0.7 μM), 0.01877 s -1 (1.0 μM), indicating that as the concentration of APTES increases, its interaction with Si-OH on the capillary surface speeds up. The hydrolysis results of the Si-O-Si bonds formed between APTES and the capillary surface show that the Si-O-Si bonds formed by higher-concentration APTES with the capillary surface have better stability, which are 6.59×10 -4 s -1 (0.5 μM), 5.41×10 -4 s -1 (1.5 μM), 3.56×10 -4 s -1 (3.0 μM). The above results show that as the concentration of APTES increases, the stability of the Si-O-Si bonds formed on the capillary surface increases. By comparing the results of APTMS and APTES, it is found that APTMS is more likely to form Si-O-Si bonds with the Si-OH surface on the capillary, and the formed Si-O-Si bonds have higher stability.

[0035] Example 4

[0036] The role of the amino silane in forming Si-O-Si bonds with the capillary surface is to modify the amino group on the capillary surface. To verify the formation of the amino group, the influence of hydrolysis time on the interaction between APTMS and the capillary was used to prepare capillaries with different amino densities, and then green fluorescent protein was introduced into the capillary to achieve the immobilization of green fluorescent protein on the capillary surface. The capillary with immobilized green fluorescent protein was photographed using a fluorescence microscope to indirectly characterize the content of amino groups on the capillary surface. Specifically as follows:

[0037] The fused silica capillary (microchannel) with an inner diameter of 50 μm was rinsed with 1 M NaOH and ultrapure water for 5 min, respectively, and then three groups of 3-aminopropyltrimethoxysilane (APTMS) with different hydrolysis times were subjected to the reaction between the capillary surface Si-OCH 3 Determination of the Si-O-Si chemical bond formed by action.

[0038] 70 kPa, 0.5 μM APTMS (APTMS was dispersed in 10 mM phosphate buffer and placed in the capillary for 0 min, 10 min, and 20 min before being introduced into the capillary for measurement) solution was introduced into the molten capillary for 500 s to achieve the hydrolysis time of APTMS and Si-OCH on the capillary surface. 3 Determination of online characterization of Si-O-Si bonds generated by bonding, such as Figure 5 As shown in A; then 100 μg / mL of green fluorescent protein was introduced into the capillary for 500 s, and the excess green fluorescent protein was washed away with blank buffer. The image of the capillary fixed with green fluorescent protein was taken using an inverted fluorescence microscope (excitation wavelength 470-495 nm, 20X, time 5 s, gain 2 times), and then the gray value of the image was determined using Image J software. The average gray value was used to represent the content of green fluorescent protein fixed on the capillary surface (MGV), as shown in Figure 5 As shown in B.

[0039] The results show that the surface charge contents of APTMS with different hydrolysis time after 500s of capillary action are 0.224, 0.1224, and -0.03221 (the negative sign indicates that aminosilane does not completely cover the surface of the molten capillary), and the average gray values ​​after immobilization of green fluorescent protein are 64.74, 62.41, and 40.71. The results of MGV show that the capillary with a large surface charge content has more green fluorescent protein immobilized, which further proves that the amino group is exposed on the outermost layer after the aminosilane interacts with the capillary surface.

[0040] Example 5

[0041] In order to verify that the charge change used in this experiment to determine the interaction between aminosilane and Si-OH on the capillary surface is the formation of Si-O-Si bonds (after the Si-O-Si bonds are formed, the amino groups are exposed on the surface), different concentrations of APTMS and capillary action are used to prepare capillaries with different amino density, and then green fluorescent protein is introduced into the capillary to achieve the fixation of green fluorescent protein on the capillary surface. The capillary fixed with green fluorescent protein is photographed using a fluorescence microscope, and the content of amino groups on the capillary surface is indirectly characterized based on the change in fluorescence intensity. The details are as follows:

[0042] A fused silica capillary (microchannel) with an inner diameter of 50 μm was rinsed with 1 M NaOH and ultrapure water for 5 min in sequence, and then three groups of measurements were carried out on the formation of Si-O-Si chemical bonds by the reaction of 3-aminopropyltrimethoxysilane (APTMS) with different concentrations with the Si-OH on the capillary surface.

[0043] At 70 kpa, solutions of APTMS with different concentrations (APTMS was dispersed in 10 mM phosphate buffer with concentrations of 0.05 μM, 0.1 μM, and 1.0 μM respectively) were introduced into the fused capillary for 500 s to achieve the on-line characterization measurement of the formation of Si-O-Si bonds by the bonding of APTMS with different concentrations to the Si-OCH on the capillary surface, as 3 shown in Figure 6 A; then, a solution of green fluorescent protein at 100 μg / mL was introduced into the capillary for 500 s, and the excess green fluorescent protein was rinsed off with blank buffer. The image of the capillary fixed with green fluorescent protein was taken using an inverted fluorescence microscope (excitation wavelength 470 - 495 nm, 20X, time 5 s, gain 1 times), and then the gray value of the image was measured using Image J software. The average gray value was used to represent the content of green fluorescent protein fixed on the capillary surface (MGV), as Figure 6 shown in

[0044] The experimental results showed that after the reaction of APTMS with different hydrolysis times with the capillary for 500 s, the surface charge contents were 0.2691, 0.0471, and -0.0222 respectively (the negative sign indicates that the amino silane did not completely cover the surface of the fused capillary), and the average gray values after fixing the green fluorescent protein were 37.68, 27.28, and 20.23 respectively. The results of MGV indicated that the capillary with a larger surface charge content fixed more green fluorescent protein, further proving that the amino group was exposed on the outermost layer after the reaction of the amino silane with the capillary surface.

[0045] In the above embodiments, the concentration of the background buffer was 10 mM and pH = 7.0.

Claims

1. A method for rapid preparation and online characterization of aminosilane coatings in aqueous phosphate buffer, characterized in that: Under negative pressure conditions, an aminosilane buffer solution is introduced into the microchannel, and the surface electrical signal changes over time during the process of Si-OR in the aminosilane molecule bonding with Si-OH on the microchannel surface to form Si-O-Si bonds, so as to achieve online real-time characterization of the formation of the aminosilane bonded coating and the stability of the aminosilane coating; The negative pressure condition is 30-70 kPa; The aminosilane is 3-aminopropyltriethoxysilane and 3-aminopropyltrimethoxysilane; The buffer solution is a phosphate buffer solution, the concentration of the buffer solution is 1 mM to 10 mM, and the pH is 7.0; the concentration of aminosilane in the buffer solution is 0.01 μM to 3.0 mM.

2. The method for rapidly preparing and online characterizing an aminosilane coating in an aqueous system according to claim 1, characterized in that: The microchannel is a fused silica capillary, and the inner diameter of the channel is 50-100 μm.

3. The method for rapid preparation and online characterization of aminosilane coatings in an aqueous system according to claim 1, characterized in that: A phosphate buffer solution was introduced into the microchannel bonded with aminosilane, and the Si-O-Si breakage caused by the hydrolysis of Si-O-Si bonds in a pure buffer system was used to achieve online real-time characterization of the stability of the aminosilane coating.

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