Titration method for measuring the content of hydroxyl groups on the surface of silica microspheres

Abstract

The present invention relates to the field of biochip technology, specifically a method for measuring the content of hydroxyl groups on the surface of silica microspheres by titration, wherein an excess of 2,4-toluene diisocyanate and diethylamine are added to silica microsphere powder, and the diethylamine is titrated, and the content of hydroxyl groups connected to the surface of the silica microspheres is deduced from the final consumed hydrochloric acid content. The present invention introduces titration into the quality inspection of microsphere incoming materials for the first time, quantifies the surface hydroxyl groups of unmodified microspheres, and helps better control the quality of incoming materials and evaluate the effects of subsequent reactions.

Description

Technical Field

[0001] The invention relates to the technical field of biochips, in particular to a method for measuring the content of hydroxyl groups on the surface of silicon dioxide microspheres by titration. Background Art

[0002] High-density gene chips are a low-cost, large-scale detection site, uniform format, and fast analysis method for gene detection. High-density biochips first need to use silica microspheres as fixed carriers for oligonucleotide probes, and use silicon plates as the main carrier matrix of the gene chip. The surface of untreated silica microspheres is connected to a certain number of hydroxyl groups. Since the number of surface amino groups will affect the number of probes connected to the surface of the microspheres in the subsequent reaction steps, the number of probes will affect the accuracy of the test results. Therefore, it is necessary to detect the number of surface hydroxyl groups. The unclear number of hydroxyl groups on the surface of the microspheres seriously affects the control of the quality of the incoming materials and the evaluation of the amination effect in the next reaction.

[0003] A method for measuring the surface hydroxyl content of silica microspheres has not been reported yet. Summary of the invention

[0004] The purpose of the present invention is to provide a method for measuring the surface hydroxyl content of silica microspheres, and to quantify the surface hydroxyl content of unmodified microspheres by titration. By this method, the surface hydroxyl content of unmodified microspheres is quantified, which helps to better control the quality of incoming materials and evaluate the effect of subsequent reactions.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The present invention introduces the titration method into the quality inspection of microsphere raw materials for the first time, and quantifies the surface hydroxyl groups of unmodified microspheres, which helps to better control the quality of raw materials and evaluate the effects of subsequent reactions.

[0007] The content of hydroxyl groups attached to untreated silica microspheres is not easy to detect directly, but 2,4-toluene diisocyanate can react with hydroxyl groups, and the reaction stoichiometric ratio is determined. 2,4-toluene diisocyanate can react with diethylamine, and the reaction stoichiometric ratio is determined. Diethylamine can be titrated with hydrochloric acid when bromocresol green is used as an indicator. Therefore, the method of adding excess 2,4-toluene diisocyanate and diethylamine to the untreated silica microsphere powder and titrating the diethylamine is adopted. The content of hydroxyl groups attached to the silica microspheres is calculated from the final hydrochloric acid content consumed. The ratio of the amount of hydroxyl groups to the amount of hydrochloric acid consumed in the titration is 1:1.

[0008] The experimental principle is based on the following three reaction equations ( Figure 1 ):

[0009] ① One mole of 2,4-toluene diisocyanate can react with two moles of silanol groups to form a carbonate silicon ester bond.

[0010] ② One mole of 2,4-toluene diisocyanate can react with two moles of diethylamine to form two moles of amide bonds;

[0011] ③One mole of diethylamine can combine with one mole of hydrogen ions to produce positively charged ions.

[0012] It can be seen from the above reaction formula that the ratio of the amount of hydrochloric acid consumed in the back titration method to the amount of hydroxyl groups attached to the untreated silica microspheres is 1:1. Therefore, the hydroxyl content can be calculated from the amount of hydrochloric acid consumed.

[0013] In a first aspect of the present invention, a method for measuring the hydroxyl content on the surface of silica microspheres is provided, wherein an excess of 2,4-toluene diisocyanate and diethylamine are added to silica microsphere powder, and the diethylamine is titrated, and the hydroxyl content on the surface of the silica microspheres is calculated from the final hydrochloric acid content consumed. The ratio of the amount of hydroxyl to the amount of hydrochloric acid consumed in the titration is 1:1.

[0014] Further, the method comprises the following steps:

[0015] A. Weigh 100 mg of silica microspheres into a 1.5 mL centrifuge tube, add 1 mL of 2,4-toluene diisocyanate solution and 100 μL of N,N-dimethylcyclohexylamine solution, and shake for 3-5 hours;

[0016] B. Transfer the shaken mixed microsphere suspension to a 25 mL conical flask, add 1 mL ethylenediamine solution, 2 mL xylene, 2 mL isopropanol and 3 drops of bromocresol green indicator, shake evenly, titrate with 0.3 mmol / L hydrochloric acid, and record the amount of hydrochloric acid consumed;

[0017] C. Calculation of hydroxyl content: The amount of hydrochloric acid consumed in the back titration method is the amount of hydroxyl groups connected to the silica microspheres, and the hydroxyl content on the surface of the silica microspheres is calculated from this.

[0018] Furthermore, the 2,4-toluene diisocyanate solution is a 1.22 mg / mL 2,4-toluene diisocyanate xylene solution. The preparation method is: take 50 μL of 2,4-toluene diisocyanate and quantify it to 50 mL with xylene.

[0019] Further, the N,N-dimethylcyclohexylamine solution is 4.26*10 -3mg / mL N,N-dimethylcyclohexylamine in xylene. The preparation method is as follows: take 25μL N,N-dimethylcyclohexylamine, add 975μL xylene to dilute to 1mL to make a stock solution; take 10μL of the stock solution and dilute to 50mL with xylene.

[0020] Furthermore, the ethylenediamine solution is a xylene solution of 1.0295 mg / mL diethylamine. The preparation method is: take 72.5 μL of diethylamine and dilute to 50 mL with xylene.

[0021] Furthermore, the bromocresol green indicator is a 1 g / L bromocresol green indicator, and its preparation method is: weigh 0.05 g bromocresol green, dissolve it in ethanol, transfer it to a 50 mL volumetric flask, and make up to volume.

[0022] Furthermore, the silica microspheres may be selected from acidified silica microspheres. The surface of untreated silica microspheres is connected with hydroxyl groups, but long-term storage and other reasons may cause the hydroxyl groups on the surface of the microspheres to become inactivated, so acidification treatment is required to make the hydroxyl groups active again. If acidification is not performed, it will not affect the quantification itself, but since the number of hydroxyl groups directly affects the number of connected probes and affects the accuracy of the results, the more probes there are, the more accurate the results are, that is, the more hydroxyl groups there are, the more accurate the results are. Therefore, in gene chip detection, in order to make the microspheres have more hydroxyl groups on the surface, the microspheres that have been stored for a long time will be acidified before use.

[0023] In a preferred embodiment of the present invention, the acidification treatment method is:

[0024] Weigh 500 mg of silica microspheres and add them to a 4 mL glass bottle. Add 1.33 mL of concentrated sulfuric acid and 600 μL of hydrogen peroxide. After ultrasonic dispersion, place in a 90 ° C water bath for heating and acidification for 1-4 hours. Add 10 mL of sterile water to a 15 mL centrifuge tube, add the acidified microsphere suspension to the centrifuge tube, centrifuge to remove the supernatant, add 20 mL of sterile water to wash (2500 × g, 5 min) 8-12 times. Use 10 mL of anhydrous ethanol (dry) to wash 1-5 times, centrifuge to remove the supernatant; use 10 mL of xylene to wash 2-5 times, centrifuge to remove the supernatant; add an appropriate amount of xylene to make the volume 5 mL, transfer to a 15 mL glass sample bottle for storage; take out 1 ml (100 mg) and transfer to a 15 mL glass sample bottle, centrifuge to remove the supernatant, and put it in an oven at 110 ° C for 10-14 hours.

[0025] The advantages of the present invention are:

[0026] 1. The titration method was introduced into the microsphere quality inspection for the first time, which can quickly and accurately inspect the incoming microspheres and effectively improve the accuracy of the inspection.

[0027] 2. Ethylenediamine, hydrochloric acid, 2,4-toluene diisocyanate and other reagents were used for titration. By recording the amount of hydrochloric acid consumed, the hydroxyl groups connected to the microspheres can be quantitatively analyzed. By controlling the number of hydroxyl groups connected in each batch of microspheres, the number of probes connected to the microspheres during each test can be effectively controlled; and it is helpful for the quantitative calculation of subsequent reactions and saves the amount of other experimental reagents.

[0028] 3. The detection method of the present invention is simple and fast through the steps of heating, centrifugation, drying, titration, etc., and can quickly and effectively perform quality inspection on microspheres, thereby improving detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 .The experimental principle reaction formula of the present invention. DETAILED DESCRIPTION

[0030] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0031] Example 1: Solvent configuration

[0032] (1) Prepare a 1.22 mg / mL xylene solution of 2,4-toluene diisocyanate:

[0033] Take 50 μL of 2,4-toluene diisocyanate and make up to 50 mL with xylene.

[0034] (2) Prepare a xylene solution of 1.0295 mg / mL diethylamine:

[0035] Take 72.5 μL of diethylamine and dilute to 50 mL with xylene.

[0036] (3) Prepare 1g / L bromocresol green indicator:

[0037] Weigh 0.05 g of bromocresol green, dissolve it in ethanol, transfer it to a 50 mL volumetric flask, and make up to volume.

[0038] (4) Configuration 4.26*10 -3 mg / mL N,N-dimethylcyclohexylamine in xylene:

[0039] Take 25 μL of N,N-dimethylcyclohexylamine and add 975 μL of xylene to dilute to 1 mL to prepare the stock solution; take 10 μL of the stock solution and dilute to 50 mL with xylene.

[0040] Example 2: Titration of unacidified microspheres

[0041] (1) Weigh 100 mg of untreated silica microspheres into a 1.5 mL centrifuge tube, add 1 mL of a 1.22 mg / mL xylene solution of 2,4-toluene diisocyanate and 100 μL of 4.26*10-3 Prepare a xylene solution of N,N-dimethylcyclohexylamine at a concentration of 100 mg / mL and shake for 3-5 hours.

[0042] (2) Transfer the shaken mixed microsphere suspension to a 25 mL conical flask, add 1 mL of 1.0295 mg / mL ethylenediamine in xylene solution, 2 mL of xylene, 2 mL of isopropanol, and 3 drops of bromocresol green indicator, shake well, and titrate with 0.3 mmol / L hydrochloric acid, recording the amount of hydrochloric acid consumed.

[0043] Calculation of hydroxyl content: The amount of hydrochloric acid consumed in the back titration method is the amount of hydroxyl groups attached to the untreated silica microspheres, and the hydroxyl content on the surface of the untreated silica microspheres is calculated from this.

[0044] Example 3: Acidified microsphere titration

[0045] (1) Weigh 500 mg of silica microspheres and add them to a 4 mL glass bottle. Add 1.33 mL of concentrated sulfuric acid and 600 μL of hydrogen peroxide. After ultrasonic dispersion, place in a 90 °C water bath for heating and acidification for 1-4 h. Add 10 mL of sterile water to a 15 mL centrifuge tube, add the acidified microsphere suspension to the centrifuge tube, centrifuge to remove the supernatant, add 20 mL of sterile water to wash (2500 × g, 5 min) 8-12 times. Use 10 mL of anhydrous ethanol (dry) to wash 1-5 times, centrifuge to remove the supernatant; use 10 mL of xylene to wash 2-5 times, centrifuge to remove the supernatant; add an appropriate amount of xylene to make the volume 5 mL, transfer to a 15 mL glass sample bottle for storage; take out 1 ml (100 mg) and transfer to a 15 mL glass sample bottle, centrifuge to remove the supernatant, and place in an oven at 110 °C for 10-14 h to dry.

[0046] (2) Take 100 mg of dried acidified silica microspheres and place them in a 1.5 mL centrifuge tube. Add 1 mL of a 1.22 mg / mL xylene solution of 2,4-toluene diisocyanate and 100 μL of 4.26*10 -3 100 mg / mL solution of N,N-dimethylcyclohexylamine in xylene, shake for 4 hours.

[0047] (3) Transfer the shaken mixed microsphere suspension to a 25 mL conical flask, add 1 mL of 1.0295 mg / mL ethylenediamine in xylene solution, 2 mL of xylene, 2 mL of isopropanol, and 3 drops of bromocresol green indicator, shake well, and titrate with 0.3 mmol / L hydrochloric acid, recording the amount of hydrochloric acid consumed.

[0048] Calculation of hydroxyl content: The amount of hydrochloric acid consumed in the back titration method is the amount of hydroxyl groups attached to the untreated silica microspheres, and the hydroxyl content on the surface of the untreated silica microspheres is calculated from this.

[0049] Example 4: Test results and analysis

[0050] Table 1

[0051]

[0052] Experimental results analysis:

[0053] As shown in Table 1, the titration method can be used to effectively perform quantitative analysis and quality inspection on untreated silica microspheres. By performing quality inspection on the microspheres, the accuracy of the test results can be effectively guaranteed.

[0054] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for measuring the surface hydroxyl content of unmodified silica microspheres in a high-density gene chip, characterized in that: The method comprises the following steps: A. Weigh 100 mg of silica microspheres into a 1.5 mL centrifuge tube, add 1 mL of 2,4-toluene diisocyanate solution and 100 μL of N,N-dimethylcyclohexylamine solution, and shake for 3-5 hours; the 2,4-toluene diisocyanate solution is a xylene solution of 1.22 mg / mL 2,4-toluene diisocyanate; the N,N-dimethylcyclohexylamine solution is 4.26*10 -3 mg / mL N,N-dimethylcyclohexylamine in xylene; B. Transfer the shaken mixed microsphere suspension to a 25 mL conical flask, add 1 mL ethylenediamine solution, 2 mL xylene, 2 mL isopropanol and 3 drops of 1 g / L bromocresol green indicator, shake evenly, titrate with 0.3 mmol / L hydrochloric acid, and record the amount of hydrochloric acid consumed; the ethylenediamine solution is a xylene solution of 1.0295 mg / mL diethylamine; C. Calculation of hydroxyl content: The amount of hydrochloric acid consumed in the back titration method is the amount of hydroxyl groups connected to the silica microspheres, and the hydroxyl content on the surface of the silica microspheres is calculated from this; The silica microspheres are acidified silica microspheres; the acidification method is as follows: weigh 500 mg of silica microspheres and add them to a 4 mL glass bottle, add 1.33 mL of concentrated sulfuric acid and 600 μL of hydrogen peroxide, and after ultrasonic dispersion, place them in a 90° C. water bath for heating and acidification for 1-4 h; add 10 mL of sterile water to a 15 mL centrifuge tube, add the acidified microsphere suspension to the centrifuge tube, centrifuge at 2500×g for 5 min, remove the supernatant, add 20 mL of sterile water to wash 8-12 times; use 10 mL of anhydrous ethanol to wash 1-5 times, centrifuge to remove the supernatant; use 10 mL of xylene to wash 2-5 times, centrifuge to remove the supernatant; add xylene to make the volume 5 mL, transfer to a 15 mL glass sample bottle for storage; take out 1 ml and transfer to a 15 mL glass sample bottle, centrifuge to remove the supernatant, and put it in an oven at 110° C. for 10-14 h for drying.

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