A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation

Abstract

The present invention discloses a method for minimizing non-specific adsorption caused by carbodiimide bioconjugation, comprising the following steps: mixing bovine serum albumin and chlortetracycline hydrochloride solution, adding carbodiimide and stirring for reaction to obtain a conjugate product; placing the conjugate product in a phosphate buffer solution, dialyzing and concentrating the conjugate product, and diluting the conjugate product to 1.0-2.0 mg / mL; fixing a colloidal crystal film of silica gel with silica gel and a glass slide to form a reaction chamber, and fixing the reaction chamber on an inverted optical microscope; blocking the colloidal crystal film of silica gel with bovine serum albumin or polyethylene glycol; introducing the conjugate product into the reaction chamber, and confirming non-specific adsorption by reflection interference method; adding N-hydroxysulfosuccinimide during the conjugation process. The present invention can solve the strong non-specific adsorption that cannot be solved by traditional blocking reagents, control the non-specific adsorption on the surface of the conjugate by changing the reaction path, and ensure good sensitivity of biological recognition.

Description

Technical Field

[0001] The present invention relates to the field of bioconjugation, and specifically to a method for minimizing non-specific adsorption caused by carbodiimide bioconjugation. Background Art

[0002] Bioconjugation technology is of great significance for the design and development of new biomolecules and biomaterials, can explain complex biological processes, and has generated a large number of new applications in the fields of medicine, diagnostics, microelectronics, and materials science. Typical bioconjugate preparation can covalently link a small molecule to a large molecule, or immobilize an affinity ligand on a nanoparticle or other surface. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (carbodiimide) is a coupling agent widely used in bioconjugation. However, an important property of this coupling agent has not received much attention. Briefly speaking, carbodiimide can cause additional positive charges on the surface of the conjugate, resulting in non-specific adsorption of the conjugate during many applications. Traditionally, non-specific adsorption can be controlled by blocking reagents such as bovine serum albumin and polyethylene glycol, as well as surfactants. However, on the one hand, strong non-specific adsorption is difficult to solve through the above traditional methods, and on the other hand, the adsorption of a large amount of blocking reagents itself will lead to a decrease in the sensitivity of biological recognition. There is an urgent need for a method to control non-specific adsorption without reducing the sensitivity of biological recognition. Summary of the Invention

[0003] Object of the Invention: In order to overcome the deficiencies in the prior art, the object of the present invention is to provide a method that can control non-specific adsorption on the surface of the conjugate and minimize non-specific adsorption caused by carbodiimide bioconjugation.

[0004] Technical Solution: A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation according to the present invention includes the following steps:

[0005] Step 1, mix bovine serum albumin and chlortetracycline hydrochloride solution, add carbodiimide, and stir and react in the dark to obtain a conjugate product;

[0006] Step 2, place the conjugate product obtained in Step 1 in a phosphate buffer solution, perform dialysis, concentrate the conjugate product with polyethylene glycol, and after concentration, dilute the product concentration to 1.0 - 2.0 mg / mL, where the product concentration is calculated based on the concentration of bovine serum albumin;

[0007] Step 3, fix a colloidal crystal thin film of silica gel, silica gel, and a glass slide to form a reaction chamber, fix the reaction chamber on an inverted optical microscope, adjust the light spot to focus on the colloidal crystal thin film of silica gel, and after the light is reflected, pass through an optical fiber and finally reach a spectrometer, and the reflected interference spectrum of the colloidal crystal thin film of silica gel is formed after being processed by the spectrometer signal software;

[0008] Step 4: Inject bovine serum albumin or polyethylene glycol into the reaction chamber prepared in Step 3, keep it for 2 - 3 h to block the surface of the silica colloidal crystal film, and then inject phosphate buffer solution at the same flow rate to remove weakly bound or unbound bovine serum albumin or polyethylene glycol. Measure and record the real-time optical thickness change by reflectance interference measurement.

[0009] Step 5: Pass the product obtained in Step 2 into the reaction chamber treated in Step 4. The coupling product is non-specifically adsorbed on the blocked film surface due to the increased surface positive charge. After the reaction, inject phosphate buffer solution to remove weakly bound or unbound coupling products, control the refractive index of the solution, and use reflectance interference measurement to record the signal to confirm non-specific adsorption.

[0010] Step 6: Repeat Steps 1 - 5 once, and add N-hydroxysulfosuccinimide in Step 1 to change the reaction path. At this time, N-hydroxysulfosuccinimide will change the structure of the intermediate originally formed by carbodiimide, and this structure will not cause surface charge change, thus minimizing non-specific adsorption.

[0011] Furthermore, in Step 1, the concentration of the mixed bovine serum albumin is 4.0 - 6.0 mg / mL, the pH of the chlortetracycline hydrochloride solution is 7.0. The reaction time is 10 - 16 h, and the molar ratio of carbodiimide to bovine serum albumin is 10 - 10000.

[0012] Furthermore, in Step 2, the concentration of the phosphate buffer solution is 0.01 - 0.03 M, and the pH is 7.2 - 7.4. The dialysis time is 2 - 4 days, and a total of 12 - 16 replacements are made during this period. The molecular weight of polyethylene glycol is 20000 - 90000, and the blocking effect is better.

[0013] Furthermore, the injection rates of bovine serum albumin, polyethylene glycol, and phosphate buffer solution in Step 4 are the same as the injection rate of bovine serum albumin in Step 5. The injection rate is 0.3 - 0.5 mL / min.

[0014] Furthermore, the temperature of the reaction chamber in Steps 3 and 4 is 20 - 25 °C.

[0015] Furthermore, in Step 6, the mass of N-hydroxysulfosuccinimide is 0.016 - 160 mg, and the mass of carbodiimide is 0.72 - 720 mg.

[0016] Reaction principle: Using reflection interference spectroscopy, chlortetracycline hydrochloride is immobilized on bovine serum albumin through carbodiimide and introduced onto the sealed substrate, thereby dynamically exploring the relationship between the amount of carbodiimide and the degree of non-specific adsorption in real time. From the perspective of changing the reaction path, adding N-hydroxysulfosuccinimide changes the structure of the intermediate originally formed by carbodiimide, which will not cause changes in surface charge, thus minimizing non-specific adsorption.

[0017] Advantages: Compared with the prior art, the present invention has the following significant features:

[0018] 1. It can solve the strong non-specific adsorption that cannot be solved by traditional blocking reagents. By changing the reaction path to control the non-specific adsorption on the surface of the conjugate, the negative impacts brought by traditional non-specific adsorption can be avoided, ensuring better sensitivity of biological recognition.

[0019] 2. Through the silica colloidal crystal film and combining its reflection interference spectrum, substances can be bound to the film. The change in the refractive index of the system will lead to a change in the optical thickness, thereby causing the migration of interference peaks, and the signal is finally captured and displayed by the spectrometer, providing a new tool for the monitoring of non-specific adsorption.

[0020] 3. The periodic structure of the silica colloidal crystal film is obvious, with a high porosity and good preparation repeatability. Stable Fabry-Perot fringes can be obtained, and signal acquisition can be carried out from the back of the film, without being interfered by the properties of the fluid, and the signal in complex fluids can be monitored. Description of the drawings

[0021] Figure 1 is the preparation flow chart of the present invention;

[0022] Figure 2 is the reaction schematic diagram of the present invention;

[0023] Figure 3 is the measurement principle diagram of the reflection interference method of the present invention;

[0024] Figure 4 is the chemical equation schematic diagram of the side reaction of non-specific adsorption caused by carbodiimide in the present invention;

[0025] Figure 5 is the chemical equation schematic diagram of adding N-hydroxysulfosuccinimide to change the reaction path to solve non-specific adsorption in the present invention;

[0026] Figure 6 is the data of blocking and non-specific adsorption recorded in Example 3 of the present invention;

[0027] Figure 7 is the data of minimizing non-specific adsorption recorded in Example 4 of the present invention;

[0028] Figure 8 are the blocking and non-specific adsorption data of the conjugate containing Tween 20 as a control in Comparative Example 1 of the present invention;

[0029] Figure 9 are the data in Comparative Example 2 of the present invention demonstrating that non-specific adsorption is not caused by chlortetracycline hydrochloride. Detailed implementation mode

[0030] Example 1

[0031] As Figure 1 , a method for minimizing non-specific adsorption caused by carbodiimide bioconjugation, comprising the following steps:

[0032] Step 1: Dissolve chlortetracycline hydrochloride with 1M sodium hydroxide, adjust the pH back to 7.0, and then prepare 5.0 mL of 5.0 mg / mL chlortetracycline hydrochloride. Add 25.0 mg of bovine serum albumin to the solution to make the final concentration of bovine serum albumin reach 5.0 mg / mL. Add 0.72 mg of carbodiimide and react for 12 h in the dark to obtain a conjugate; the molar ratio of carbodiimide to bovine serum albumin is 10;

[0033] Step 2: Dialyze the conjugate obtained in Step 1 with a phosphate buffer solution of pH 7.2 and a concentration of 0.01M for 3 days, changing the solution a total of 12 times during this period. Concentrate the conjugate with polyethylene glycol - 20000 and dilute it to a bovine serum albumin concentration of 1.0 mg / mL. The product is a complex of binding chlortetracycline to bovine serum albumin. Dialysis will dialyze out the unbound small molecule chlortetracycline, while the product will remain in the dialysis bag. After concentration, it is difficult to determine the concentration of chlortetracycline on the product, but the concentration of bovine serum albumin is easy to determine. Therefore, the concentration of bovine serum albumin is used to represent the product concentration.

[0034] Step 3: Fix the colloidal crystal film of silica gel with silica gel and a glass slide to form a reaction chamber. Fix the reaction chamber on an inverted optical microscope, adjust the light spot to focus on the colloidal crystal film of silica gel, and perform reflection interference measurement with a self-developed optical system and software, as Figure 2 shown. After the light is reflected, it passes through an optical fiber and finally reaches a spectrometer. The reflection interference spectrum of the colloidal crystal film of silica gel is formed after being processed by the spectrometer signal software, as Figure 3 shown;

[0035] Step 4: Pump bovine serum albumin into the reaction chamber prepared in Step 3 at a speed of 0.4 mL / min, keep it for 2.5 h to block the surface of the colloidal crystal film of silica gel. After that, inject the phosphate buffer solution at the same flow rate to remove the weakly bound or unbound bovine serum albumin, and record the real-time optical thickness change through reflection interference measurement. The experimental temperature is 21 °C;

[0036] Step 5: Introduce the product obtained in Step 2 into the reaction chamber processed in Step 4 at the same flow rate. Due to the increased positive charge on the surface, the coupling product is non-specifically adsorbed on the surface of the sealed film. After the reaction, inject phosphate buffer at the same flow rate to remove weakly bound or unbound coupling products. Control the refractive index of the solution and record the signal using reflectance interference measurement to confirm non-specific adsorption. The experimental temperature is 21°C.

[0037] Step 6: Maintain the mass of carbodiimide at 0.72 mg in Step 1 and add 0.16 mg of N-hydroxysulfosuccinimide. The obtained coupling product also undergoes Steps 2 to 5.

[0038] Example 2

[0039] A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation, comprising the following steps:

[0040] Step 1: Dissolve chlortetracycline hydrochloride with 1 M sodium hydroxide, adjust the pH back to 7.0, and prepare 5.0 mL of 5.0 mg / mL chlortetracycline hydrochloride solution. Add 25.0 mg of bovine serum albumin to the solution to make the final concentration of bovine serum albumin reach 5.0 mg / mL. Add 7.2 mg of carbodiimide and react for 12 h in the dark to obtain a coupling product. There are inevitable side reactions in the reaction, such as Figure 4 shown; the molar ratio of carbodiimide to bovine serum albumin is 100;

[0041] Step 2: Dialyze the coupling product obtained in Step 1 with phosphate buffer at pH 7.2 and a concentration of 0.01 M for 3 d, changing the buffer a total of 12 times during this period. Concentrate the coupling product with polyethylene glycol-20000 and dilute it to a bovine serum albumin concentration of 1.0 mg / mL.

[0042] Step 3: Fix the colloidal silica crystal film with silica gel and a glass slide to form a reaction chamber. Fix the reaction chamber on an inverted optical microscope, adjust the light spot to focus on the colloidal silica crystal film, and perform reflectance interference measurement using a self-developed optical system and software. The reflected light passes through an optical fiber and finally reaches a spectrometer, and the signal on the spectrometer software is processed to form the reflectance interference spectrum of the colloidal silica crystal film.

[0043] Step 4: Pump bovine serum albumin into the reaction chamber prepared in Step 3 at a rate of 0.4 mL / min and maintain for 2.5 h to block the surface of the colloidal silica crystal film. After completion, inject phosphate buffer at the same flow rate to remove weakly bound or unbound bovine serum albumin. Measure the real-time optical thickness change through reflectance interference measurement and record it. The experimental temperature is 20°C.

[0044] Step 5: Feed the product obtained in Step 2 into the reaction chamber processed in Step 4 at the same flow rate. The coupling product is non-specifically adsorbed on the surface of the sealed film due to the increased positive charge on its surface. After the reaction, inject phosphate buffer at the same flow rate to remove weakly bound or unbound coupling products. Control the refractive index of the solution and record the signal using reflectance interference measurement to confirm non-specific adsorption. The experimental temperature is 20 °C.

[0045] Step 6: Maintain the mass of carbodiimide at 7.2 mg during Step 1 and add 1.6 mg of N-hydroxysulfosuccinimide. As Figure 5 shown, the coupling product obtained is also subjected to Steps 2 to 5.

[0046] Example 3

[0047] A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation, comprising the following steps:

[0048] Step 1: Dissolve chlortetracycline hydrochloride with 1 M sodium hydroxide, adjust the pH back to 7.0, and prepare 5.0 mL of 5.0 mg / mL chlortetracycline hydrochloride. Add 25.0 mg of bovine serum albumin to the solution to make the final concentration of bovine serum albumin reach 5.0 mg / mL. Add 720 mg of carbodiimide and react for 12 h in the dark to obtain a coupling product; the molar ratio of carbodiimide to bovine serum albumin is 10,000.

[0049] Step 2: Dialyze the coupling product obtained in Step 1 with a phosphate buffer at pH 7.2 and a concentration of 0.01 M for 3 days, changing the buffer a total of 12 times during this period. Concentrate the coupling product with polyethylene glycol-20000 and dilute it to a bovine serum albumin concentration of 1.0 mg / mL.

[0050] Step 3: Fix the colloidal silica crystal film with silica gel and a glass slide to form a reaction chamber. Fix the reaction chamber on an inverted optical microscope, adjust the light spot to focus on the colloidal silica crystal film, and perform reflectance interference measurement using a self-developed optical system and software. The reflected light passes through an optical fiber and finally reaches a spectrometer, and the reflected interference spectrum of the colloidal silica crystal film is formed after processing by the spectrometer signal software.

[0051] Step 4: Pump polyethylene glycol-20000 into the reaction chamber prepared in Step 3 at a speed of 0.4 mL / min and maintain for 2.5 h to block the surface of the colloidal silica crystal film. After completion, inject phosphate buffer at the same flow rate to remove weakly bound or unbound polyethylene glycol-20000, and record the real-time optical thickness change through reflectance interference measurement. The experimental temperature is 25 °C.

[0052] Step 5: Feed the product obtained in Step 2 into the reaction chamber treated in Step 4 at the same flow rate. The coupling product is non-specifically adsorbed on the surface of the sealed film due to the increased positive charge on its surface. After the reaction, inject phosphate buffer at the same flow rate to remove the weakly bound or unbound coupling products. Control the refractive index of the solution and record the signal using reflection interference measurement to confirm non-specific adsorption, as Figure 6 shown, the experimental temperature is 23 °C;

[0053] Step 6: Maintain the concentration of carbodiimide at 720 mg during Step 1 and add 16 mg of N-hydroxysulfosuccinimide. The obtained coupling product also goes through Steps 2 to 5.

[0054] Example 4

[0055] A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation, comprising the following steps:

[0056] Step 1: Dissolve chlortetracycline hydrochloride with 1 M sodium hydroxide, adjust the pH back to 7.0, and prepare 5.0 mL of 5.0 mg / mL chlortetracycline hydrochloride solution. Add 25.0 mg of bovine serum albumin to the solution so that the final concentration of bovine serum albumin reaches 5.0 mg / mL. Add 72 mg of carbodiimide and react in the dark for 12 h to obtain a coupling product; the molar ratio of carbodiimide to bovine serum albumin is 1000;

[0057] Step 2: Dialyze the coupling product obtained in Step 1 with a phosphate buffer at pH 7.2 and a concentration of 0.01 M for 3 days, changing the buffer a total of 12 times during this period. Concentrate the coupling product with polyethylene glycol-20000 and dilute it to a bovine serum albumin concentration of 1.0 mg / mL.

[0058] Step 3: Fix the colloidal silica crystal film with silica gel and a glass slide to form a reaction chamber. Fix the reaction chamber on an inverted optical microscope, adjust the light spot to focus on the colloidal silica crystal film, and perform reflection interference measurement using a self-developed optical system and software. The reflected light passes through an optical fiber and finally reaches a spectrometer, and the reflection interference spectrum of the colloidal silica crystal film is formed after processing by the spectrometer signal software;

[0059] Step 4: Pump bovine serum albumin into the reaction chamber prepared in Step 3 at a speed of 0.4 mL / min and keep it for 2.5 h to block the surface of the colloidal silica crystal film. After that, inject phosphate buffer at the same flow rate to remove the weakly bound or unbound bovine serum albumin. Measure and record the real-time optical thickness change through reflection interference measurement, and the experimental temperature is 23 °C;

[0060] Step 5: Introduce the product obtained in Step 2 into the reaction chamber processed in Step 4 at the same flow rate. The coupling product is non-specifically adsorbed on the surface of the sealed film due to the increased positive charge on its surface. After the reaction, inject phosphate buffer at the same flow rate to remove the weakly bound or unbound coupling products. Control the refractive index of the solution and record the signal using reflection interference measurement to confirm non-specific adsorption. The experimental temperature is 23 °C, and the signal increases by approximately 33 nm, indicating strong non-specific adsorption.

[0061] Step 6: Maintain the concentration of carbodiimide at 72 mg during Step 1 and add 160 mg, 16 mg, 1.6 mg, 0.16 mg, and 0.016 mg of N-hydroxysulfosuccinimide respectively. The obtained coupling products also go through Steps 2 to 5. As Figure 7 shown, the non-specific adsorption is significantly reduced.

[0062] Comparative Example 1

[0063] A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation, comprising the following steps:

[0064] Step 1: Dissolve chlortetracycline hydrochloride in 1 M sodium hydroxide, adjust the pH back to 7.0, and prepare 5.0 mL of 5.0 mg / mL chlortetracycline hydrochloride solution. Add 25.0 mg of bovine serum albumin to the solution to make the final concentration of bovine serum albumin reach 5.0 mg / mL. Add 360 mg of carbodiimide and react for 12 h in the dark to obtain the coupling product.

[0065] Step 2: Dialyze the coupling product against 0.01 M phosphate buffer at pH 7.2 for 3 days, changing the buffer a total of 12 times during this period. Concentrate the coupling product using polyethylene glycol-20000 and dilute it to a bovine serum albumin concentration of 1.0 mg / mL.

[0066] Step 3: Fix the colloidal silica crystal film to the silica gel and the glass slide to form a reaction chamber. Fix the reaction chamber on an inverted optical microscope, adjust the light spot to focus on the colloidal silica crystal film, and perform reflection interference measurement using a self-developed optical system and software. The reflected light passes through an optical fiber and finally reaches a spectrometer, and the signal on the spectrometer software is processed to form the reflection interference spectrum of the colloidal silica crystal film.

[0067] Step 4: First, pump bovine serum albumin into the reaction chamber prepared in Step 3 at a speed of 0.4 mL / min for 2.5 h to block the film surface. After that, inject phosphate buffer at the same flow rate to remove the weakly bound or unbound bovine serum albumin, and record the signal using reflection interference measurement. The experimental temperature is 23 °C.

[0068] Step 5: Add 0.25% Tween-20 to the coupling product obtained in Step 2 and introduce it into the reaction chamber treated in Step 4 at the same rate as in Step 4. Due to the increased positive charge on the surface, the coupling product is non-specifically adsorbed on the surface of the blocked film. After the reaction, inject phosphate buffer at the same flow rate to remove weakly bound or unbound coupling products and achieve the purpose of controlling the refractive index of the solution. Also record the signal using reflectance interference measurement, as Figure 8 shown. The signal increased by about 12 nm. The non-specific adsorption weakened slightly but was still strong. The experimental temperature was 23 °C.

[0069] Comparative Example 2

[0070] A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation, comprising the following steps:

[0071] Step 1: Pump bovine serum albumin into the prepared silica film reaction chamber at a rate of 0.4 mL / min for 2.5 h to block the film surface. After completion, inject phosphate buffer at the same flow rate to remove weakly bound or unbound bovine serum albumin. Record the signal using reflectance interference measurement. The experimental temperature was 24 °C;

[0072] Step 2: Introduce 1 mg / mL chlortetracycline hydrochloride into the reaction chamber at the same rate as in Step 1, and then rinse with phosphate buffer. It can be seen that the optical thickness is restored, as Figure 9 shown, indicating that chlortetracycline hydrochloride itself does not cause non-specific adsorption.

[0073] Example 5

[0074] A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation, comprising the following steps:

[0075] Step 1: Dissolve chlortetracycline hydrochloride with 1 M sodium hydroxide, adjust the pH back to 7.0, and prepare 5.0 mL of 5.0 mg / mL chlortetracycline hydrochloride. Add 20.0 mg of bovine serum albumin to the solution so that the final concentration of bovine serum albumin reaches 4.0 mg / mL. Add 360.0 mg of carbodiimide and react in the dark for 10 h to obtain a coupling product; the molar ratio of carbodiimide to bovine serum albumin is 6300;

[0076] Step 2: Dialyze the coupling product obtained in Step 1 with phosphate buffer at pH 7.4 and a concentration of 0.03 M for 2 d, changing a total of 16 times during this period. Concentrate the coupling product with polyethylene glycol-90000 and dilute it to a bovine serum albumin concentration of 2.0 mg / mL.

[0077] Step 3: Fix the silica colloidal crystal film with silica gel and a glass slide to form a reaction chamber. Fix the reaction chamber on an inverted optical microscope, adjust the light spot to focus on the silica colloidal crystal film, perform reflection interference measurement using a self-developed optical system and software. After the light is reflected, it passes through an optical fiber and finally reaches a spectrometer. The signal of the spectrometer is processed by software to form the reflection interference spectrum of the silica colloidal crystal film.

[0078] Step 4: Pump polyethylene glycol-90000 into the reaction chamber prepared in Step 3 at a rate of 0.3 mL / min, and keep it for 2 h to block the surface of the silica colloidal crystal film. After that, inject phosphate buffer solution at the same flow rate to remove weakly bound or unbound polyethylene glycol-90000, and record the real-time optical thickness change through reflection interference measurement. The experimental temperature is 25 °C.

[0079] Step 5: Pass the product obtained in Step 2 into the reaction chamber processed in Step 4 at the same flow rate. The coupling product is non-specifically adsorbed on the blocked film surface due to the increased positive charge on the surface. After the reaction, inject phosphate buffer solution at the same flow rate to remove weakly bound or unbound coupling products, control the refractive index of the solution, record the signal using reflection interference measurement, and confirm non-specific adsorption. The experimental temperature is 25 °C.

[0080] Step 6: Maintain the concentration of carbodiimide at 360.0 mg during the process of Step 1 and add 80 mg of N-hydroxysulfosuccinimide. The obtained coupling product also goes through Steps 2 to 5.

[0081] Example 6

[0082] A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation, comprising the following steps:

[0083] Step 1: Dissolve chlortetracycline hydrochloride with 1 M sodium hydroxide, adjust the pH back to 7.0, and prepare 5.0 mL of 6.0 mg / mL chlortetracycline hydrochloride solution. Add 30.0 mg of bovine serum albumin to the solution so that the final concentration of bovine serum albumin reaches 6.0 mg / mL. Add 720.0 mg of carbodiimide and react for 16 h in the dark to obtain a coupling product; the molar ratio of carbodiimide to bovine serum albumin is 10000.

[0084] Step 2: Dialyze the coupling product obtained in Step 1 with a phosphate buffer solution of pH 7.3 and a concentration of 0.02 M for 4 d, changing the solution a total of 14 times during this period. Concentrate the coupling product with polyethylene glycol-60000 and dilute it to a bovine serum albumin concentration of 1.5 mg / mL.

[0085] Step 3: Fix the silica colloidal crystal film with silica gel and a glass slide to form a reaction chamber. Fix the reaction chamber on an inverted optical microscope, adjust the light spot to focus on the silica colloidal crystal film, and perform reflection interference measurement using a self-developed optical system and software. After the light is reflected, it passes through an optical fiber and finally reaches a spectrometer. The signal of the spectrometer is processed by software to form the reflection interference spectrum of the silica colloidal crystal film;

[0086] Step 4: Pump polyethylene glycol-60000 into the reaction chamber prepared in Step 3 at a rate of 0.5 mL / min for 3 h to block the surface of the silica colloidal crystal film. After that, inject phosphate buffer solution at the same flow rate to remove weakly bound or unbound polyethylene glycol-60000. Record the real-time optical thickness change through reflection interference measurement. The experimental temperature is 24 °C;

[0087] Step 5: Introduce the product obtained in Step 2 into the reaction chamber treated in Step 4 at the same flow rate. The coupling product is non-specifically adsorbed on the blocked film surface due to the increased surface positive charge. After the reaction, inject phosphate buffer solution at the same flow rate to remove weakly bound or unbound coupling products. Control the refractive index of the solution and record the signal using reflection interference measurement to confirm non-specific adsorption. The experimental temperature is 24 °C;

[0088] Step 6: Maintain the concentration of carbodiimide at 720 mg during the process of Step 1 and add 160 mg of N-hydroxysulfosuccinimide. The obtained coupling product also goes through Steps 2 to 5.

Claims

1. A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation, characterized in that, It includes the following steps: Step 1: Mix bovine serum albumin and chlortetracycline hydrochloride solution, add carbodiimide, and stir and react in the dark to obtain a coupling product; Step 2: Place the coupling product obtained in Step 1 in a phosphate buffer solution, perform dialysis, concentrate the coupling product with polyethylene glycol, and after concentration, dilute the product concentration to 1.0 - 2.0 mg / mL, where the product concentration is calculated based on the bovine serum albumin concentration; Step 3: Fix the colloidal crystal film of silica gel with silica gel and a glass slide to form a reaction chamber, fix the reaction chamber on an inverted optical microscope, adjust the light spot to focus on the colloidal crystal film of silica gel, and after the light is reflected, pass through an optical fiber and finally reach a spectrometer, and the reflected interference spectrum of the colloidal crystal film of silica gel is formed after being processed by the spectrometer signal software; Step 4: Inject bovine serum albumin or polyethylene glycol into the reaction chamber prepared in Step 3, keep it for 2 - 3 h to block the surface of the colloidal crystal film of silica gel, and then inject the phosphate buffer solution at the same flow rate, and measure and record the real-time optical thickness change by reflection interference; Step 5: Pass the product obtained in Step 2 into the reaction chamber processed in Step 4, the coupling product is non-specifically adsorbed on the surface of the blocked film, inject the phosphate buffer solution, control the refractive index of the solution, and use reflection interference to measure and record the signal to confirm non-specific adsorption; Step 6: Repeat Steps 1 - 5 once, and add N-hydroxysulfosuccinimide in Step 1 to change the reaction path.

2. A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation according to claim 1, characterized in that: In Step 1, the concentration of the mixed bovine serum albumin is 4.0 - 6.0 mg / mL, and the pH of the chlortetracycline hydrochloride solution is 7.

0.

3. A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation according to claim 1, characterized in that: In Step 1, the reaction time is 10 - 16 h, and the molar ratio of carbodiimide to bovine serum albumin is 10 - 10000.

4. A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation according to claim 1, characterized in that: In Step 2, the concentration of the phosphate buffer solution is 0.01 - 0.03 M, and the pH is 7.2 - 7.

4.

5. A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation according to claim 1, characterized in that: In Step 2, the dialysis time is 2 - 4 days, and the total number of replacements during this period is 12 - 16 times.

6. A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation according to claim 1, characterized in that: In Step 2, the molecular weight of polyethylene glycol is 20000 - 90000.

7. A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation according to claim 1, characterized in that: The injection rates of bovine serum albumin, polyethylene glycol, and phosphate buffer solution in Step 4 are the same as the injection rate of bovine serum albumin in Step 5.

8. A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation according to claim 7, characterized in that: The injection rate is 0.3 - 0.5 mL / min.

9. A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation according to claim 1, characterized in that: The temperature of the reaction chamber in Steps 3 and 4 is 20 - 25 °C.

10. A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation according to claim 1, characterized in that: In Step 6, the mass of N-hydroxysulfosuccinimide is 0.016 - 160 mg, and the mass of carbodiimide is 0.72 - 720 mg.

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