CN116678956A - A Method to Minimize Nonspecific Adsorption Induced by Carbodiimide Bioconjugation - Google Patents

Abstract

The invention discloses a method for minimizing nonspecific adsorption caused by carbodiimide bioconjugate, which comprises the following steps: mixing bovine serum albumin and chlortetracycline hydrochloride solution, adding carbodiimide, stirring and reacting to obtain a coupling product; placing the coupling product in a phosphate buffer solution, dialyzing and concentrating the coupling product, and diluting the coupling product to 1.0-2.0 mg/mL; fixing a silica gel crystal film, silica gel and a glass slide to form a reaction chamber, and fixing the reaction chamber on an inverted optical microscope; blocking the silica colloidal crystal film with bovine serum albumin or polyethylene glycol; introducing the coupled product into a reaction chamber, and confirming nonspecific adsorption by using a reflection interferometry; n-hydroxysulfosuccinimide was added during the coupling. The invention can solve the problem of strong nonspecific adsorption which cannot be solved by the traditional blocking reagent, and ensures better sensitivity of biological recognition by changing the reaction path to control the nonspecific adsorption on the surface of the conjugate.

Description

A Method to Minimize Nonspecific Adsorption Induced by Carbodiimide Bioconjugation

technical field

The invention relates to the field of biocouplings, in particular to a method for minimizing non-specific adsorption caused by carbodiimide biocouplings.

Background technique

Bioconjugation technology is of great significance to the design and development of new biomolecules and biomaterials. It can explain complex biological processes and generate a large number of new applications in the fields of medicine, diagnostics, microelectronics and materials science. Typical bioconjugates are prepared by covalently linking a small molecule to a large molecule, or by immobilizing affinity ligands to nanoparticles or other surfaces. 1-Ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (carbodiimide) is a widely used coupling agent for bioconjugation, however one of such coupling agents Important properties have not been appreciated, simply put, carbodiimides cause additional positive charges on the surface of the conjugate, which leads to non-specific adsorption of the conjugate during many applications. Traditionally, although non-specific adsorption can be controlled by blocking reagents such as bovine serum albumin, polyethylene glycol, and surfactants, on the one hand, strong non-specific adsorption is difficult to solve through the above-mentioned traditional solutions, and on the other hand, the adsorption of a large number of blocking reagents itself It will lead to a decrease in the sensitivity of biological recognition, and there is an urgent need for a method to control non-specific adsorption without reducing the sensitivity of biological recognition.

Contents of the invention

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

Technical solution: A method for minimizing non-specific adsorption caused by carbodiimide biocoupling according to the present invention comprises the following steps:

Step 1, mixing bovine serum albumin and aureomycin hydrochloride solution, adding carbodiimide, stirring and reacting in a dark state, to obtain a coupling product;

Step 2: Place the coupled product obtained in Step 1 in phosphate buffer, dialyze, concentrate the coupled product with polyethylene glycol, and dilute the product concentration to 1.0-2.0 mg/mL after concentration, where the product concentration is Bovine serum albumin concentration meter;

Step 3: Fix the silica colloidal crystal film with silica gel and glass slides 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 the light passes through the optical fiber after reflection and finally reaches Spectrometer, reflection interference spectrum of silica colloidal crystal film formed after processing on 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 hours, seal the surface of the silica colloidal crystal film, and then inject phosphate buffer at the same flow rate to remove weakly bound or unbound Bovine serum albumin or polyethylene glycol, measure and record real-time optical thickness changes by reflection interferometry;

Step 5: Pass the product obtained in Step 2 into the reaction chamber after the treatment in Step 4. The coupling product is non-specifically adsorbed on the surface of the sealed film due to the increase in positive surface charge. After the reaction is completed, phosphate buffer is injected to remove the weak binding or unbound coupling products, control the refractive index of the solution, and use reflection interferometry to record signals to confirm non-specific adsorption;

Step 6, repeat steps 1 to 5 once, and add N-hydroxysulfosuccinimide in step 1 to change the reaction path. At this time, N-hydroxysulfosuccinimide will change the original carbodiimide. This structure does not cause a change in surface charge, thereby minimizing non-specific adsorption.

Further, in step one, the concentration of the mixed bovine serum albumin is 4.0-6.0 mg/mL, and the pH of the aureomycin hydrochloride solution is 7.0. The reaction time is 10-16 hours, and the molar ratio of carbodiimide to bovine serum albumin is 10-10000.

Further, in step 2, the concentration of the phosphate buffer is 0.01-0.03M, and the pH is 7.2-7.4. The dialysis time is 2 to 4 days, with a total of 12 to 16 changes during the period. The molecular weight of polyethylene glycol is 20000-90000, and the sealing effect is better.

Further, the speed of injecting bovine serum albumin, polyethylene glycol, and phosphate buffer in step 4 is the same as that of injecting bovine serum albumin in step 5. The injection rate is 0.3-0.5mL/min.

Further, the temperature of the reaction chamber in Step 3 and Step 4 is 20-25°C.

Further, in step six, the mass of N-hydroxysulfosuccinimide is 0.016-160 mg, and the mass of carbodiimide is 0.72-720 mg.

Reaction principle: Using reflection interference spectroscopy, aureomycin hydrochloride was immobilized on bovine serum albumin by carbodiimide, and passed into the blocked substrate, so as to dynamically explore the relationship between the amount of carbodiimide and the non-specificity caused by it in real time. relationship with the degree of adsorption. From the perspective of changing the reaction pathway, the addition of N-hydroxysulfosuccinimide changes the structure of the intermediates originally generated from carbodiimide, which does not cause changes in surface charges, thereby minimizing non-specific adsorption.

Beneficial effects: compared with the prior art, the present invention has the following remarkable features:

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, it can avoid the negative impact of traditional non-specific adsorption and ensure better sensitivity of biological recognition;

2. Through the silica colloidal crystal thin film, combined with its reflection interference spectrum, the substance can be bound to the thin film. The change of the refractive index of the system will lead to the change of the optical thickness, which will lead to the migration of the interference peak. The signal is finally captured and displayed by the spectrometer, which is non-specific The monitoring of heterosorption provides new tools;

3. Silica colloidal crystal film has obvious periodic structure, high porosity, good preparation repeatability, and can obtain stable Fabry-Perot fringes. Signal collection can be carried out from the back of the film without interference from fluid properties, and can be monitored Signals in complex fluids.

Description of drawings

Fig. 1 is a preparation flow chart of the present invention;

Fig. 2 is the reaction schematic diagram of the present invention;

Fig. 3 is the measurement schematic diagram of reflection interferometry of the present invention;

Fig. 4 is the schematic diagram of the chemical equation of non-specific adsorption side reaction caused by carbodiimide of the present invention;

Fig. 5 is a schematic diagram of the chemical equation for solving non-specific adsorption by adding N-hydroxysulfosuccinimide in the present invention to change the reaction path;

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

Fig. 7 is the data of minimizing non-specific adsorption recorded in Example 4 of the present invention;

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

Fig. 9 is the data proving that the non-specific adsorption is not caused by aureomycin hydrochloride in Comparative Example 2 of the present invention.

Detailed ways

Example 1

As shown in Figure 1, a method to minimize non-specific adsorption caused by carbodiimide bioconjugation includes the following steps:

Step 1, dissolve aureomycin hydrochloride with 1M sodium hydroxide, adjust the pH back to 7.0 and prepare 5.0mL 5.0mg/mL aureomycin hydrochloride, add 25.0mg bovine serum albumin to the solution, so that the final concentration of bovine serum albumin When the concentration reaches 5.0 mg/mL, add 0.72 mg carbodiimide and react for 12 hours in the dark to obtain a coupled product; the molar ratio of carbodiimide to bovine serum albumin is 10;

Step 2: Dialyze the coupling product obtained in Step 1 with pH 7.2 and 0.01M phosphate buffer for 3 days, during which a total of 12 changes were made, and the coupling product was concentrated with polyethylene glycol-20000 and diluted to the concentration of bovine serum albumin 1.0 mg/mL. The product is a complex that binds aureomycin to bovine serum albumin. Dialysis will dialyze out the unbound small molecule aureomycin, and the product will stay in the dialysis bag and be concentrated. The concentration of aureomycin on the product It is difficult to determine, but the concentration of bovine serum albumin is easy to determine. Therefore, the concentration of bovine serum albumin is used to represent the concentration of the product.

Step 3: Fix the silicon dioxide colloidal crystal film, silica gel and 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 use the self-developed optical system and software Perform reflection interferometry, as shown in Figure 2. After the light is reflected, it passes through the optical fiber and finally reaches the spectrometer, and the signal software of the spectrometer is processed to form the reflection interference spectrum of the silica colloidal crystal film, as shown in Figure 3;

Step 4: Pump bovine serum albumin into the reaction chamber prepared in Step 3 at a rate of 0.4 mL/min, keep it for 2.5 hours, seal the surface of the silica colloidal crystal film, and inject phosphate buffer at the same flow rate after completion to remove weakly bound or For unbound bovine serum albumin, real-time optical thickness changes were measured and recorded by reflection interferometry, and the experimental temperature was 21°C;

Step 5: Pass the resultant from 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 increase in positive surface charge. After the reaction, inject phosphoric acid at the same flow rate. Buffer, remove weakly bound or unbound coupling products, control the refractive index of the solution, use reflection interferometry to record signals, and confirm non-specific adsorption. The experimental temperature is 21°C;

Step 6, maintain the mass of carbodiimide in step 1 at 0.72 mg and add 0.16 mg of N-hydroxysulfosuccinimide, and the obtained coupling product also goes through steps 2 to 5.

Example 2

A method of minimizing nonspecific adsorption by carbodiimide bioconjugation comprising the steps of:

Step 1, dissolve aureomycin hydrochloride with 1M sodium hydroxide, adjust the pH back to 7.0 and prepare 5.0mL 5.0mg/mL aureomycin hydrochloride, add 25.0mg bovine serum albumin to the solution, so that the final concentration of bovine serum albumin When the concentration reached 5.0 mg/mL, 7.2 mg of carbodiimide was added to react for 12 hours in the dark, and the coupling product was obtained. There were unavoidable side reactions in the reaction, as shown in Figure 4; The ratio is 100;

Step 2: Dialyze the coupling product obtained in Step 1 with pH 7.2 and 0.01M phosphate buffer for 3 days, during which a total of 12 changes were made, and the coupling product was concentrated with polyethylene glycol-20000 and diluted to the concentration of bovine serum albumin 1.0 mg/mL.

Step 3: Fix the silicon dioxide colloidal crystal film, silica gel and 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 use the self-developed optical system and software Carry out reflection interferometry, after the light is reflected, it passes through the optical fiber and finally reaches the spectrometer, and the signal software of the spectrometer is processed to form the reflection interference spectrum of the silica colloidal crystal film;

Step 4: Pump bovine serum albumin into the reaction chamber prepared in Step 3 at a rate of 0.4 mL/min, keep it for 2.5 hours, seal the surface of the silica colloidal crystal film, and inject phosphate buffer at the same flow rate after completion to remove weakly bound or For unbound bovine serum albumin, real-time optical thickness changes were measured and recorded by reflection interferometry, and the experimental temperature was 20°C;

Step 5: Pass the resultant from 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 increase in positive surface charge. After the reaction, inject phosphoric acid at the same flow rate. Buffer, remove weakly bound or unbound coupling products, control the refractive index of the solution, use reflection interferometry to record signals, and confirm non-specific adsorption. The experimental temperature is 20°C;

Step 6: Maintain the mass of carbodiimide at 7.2 mg in step 1 and add 1.6 mg of N-hydroxysulfosuccinimide. As shown in Figure 5, the obtained coupling product also undergoes steps 2 to 5.

Example 3

A method of minimizing nonspecific adsorption by carbodiimide bioconjugation comprising the steps of:

Step 1, dissolve aureomycin hydrochloride with 1M sodium hydroxide, adjust the pH back to 7.0 and prepare 5.0mL 5.0mg/mL aureomycin hydrochloride, add 25.0mg bovine serum albumin to the solution, so that the final concentration of bovine serum albumin When the concentration reaches 5.0 mg/mL, add 720 mg of carbodiimide and react for 12 hours in the dark to obtain a coupled product; the molar ratio of carbodiimide to bovine serum albumin is 10,000;

Step 2: Dialyze the coupling product obtained in Step 1 with pH 7.2 and 0.01M phosphate buffer for 3 days, during which a total of 12 changes were made, and the coupling product was concentrated with polyethylene glycol-20000 and diluted to the concentration of bovine serum albumin 1.0 mg/mL.

Step 3: Fix the silicon dioxide colloidal crystal film, silica gel and 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 use the self-developed optical system and software Carry out reflection interferometry, after the light is reflected, it passes through the optical fiber and finally reaches the spectrometer, and the signal software of the spectrometer is processed to form the reflection interference spectrum of the silica colloidal crystal film;

Step 4: Pump polyethylene glycol-20000 into the reaction chamber prepared in Step 3 at a rate of 0.4 mL/min, keep it for 2.5 hours, seal the surface of the silica colloidal crystal film, and inject phosphate buffer at the same flow rate after the end to remove Weakly bound or unbound polyethylene glycol-20000, the real-time optical thickness change is measured and recorded by reflection interferometry, and the experimental temperature is 25°C;

Step 5: Pass the resultant from 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 increase in positive surface charge. After the reaction, inject phosphoric acid at the same flow rate. Buffer, remove weakly bound or unbound coupling products, control the refractive index of the solution, use reflection interferometry to record signals, and confirm non-specific adsorption. As shown in Figure 6, the experimental temperature is 23°C;

Step 6, maintain the carbodiimide concentration of 720 mg in the process of step 1 and add 16 mg of N-hydroxysulfosuccinimide, and the obtained coupling product also undergoes steps 2 to 5.

Example 4

A method of minimizing nonspecific adsorption by carbodiimide bioconjugation comprising the steps of:

Step 1, dissolve aureomycin hydrochloride with 1M sodium hydroxide, adjust the pH back to 7.0 and prepare 5.0mL 5.0mg/mL aureomycin hydrochloride, add 25.0mg bovine serum albumin to the solution, so that the final concentration of bovine serum albumin When the concentration reaches 5.0 mg/mL, add 72 mg of carbodiimide and react for 12 hours in the dark to obtain a coupling product; the molar ratio of carbodiimide to bovine serum albumin is 1000;

Step 2: Dialyze the coupling product obtained in Step 1 with pH 7.2 and 0.01M phosphate buffer for 3 days, during which a total of 12 changes were made, and the coupling product was concentrated with polyethylene glycol-20000 and diluted to the concentration of bovine serum albumin 1.0 mg/mL.

Step 3: Fix the silicon dioxide colloidal crystal film, silica gel and 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 use the self-developed optical system and software Carry out reflection interferometry, after the light is reflected, it passes through the optical fiber and finally reaches the spectrometer, and the signal software of the spectrometer is processed to form the reflection interference spectrum of the silica colloidal crystal film;

Step 4: Pump bovine serum albumin into the reaction chamber prepared in Step 3 at a rate of 0.4 mL/min, keep it for 2.5 hours, seal the surface of the silica colloidal crystal film, and inject phosphate buffer at the same flow rate after completion to remove weakly bound or For unbound bovine serum albumin, real-time optical thickness changes were measured and recorded by reflection interferometry, and the experimental temperature was 23°C;

Step 5: Pass the resultant from 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 increase in positive surface charge. After the reaction, inject phosphoric acid at the same flow rate. Buffer, remove weakly bound or unbound coupling products, control the refractive index of the solution, use reflection interferometry to record signals, and confirm non-specific adsorption. The experimental temperature is 23°C, the signal rises by about 33nm, and the non-specific adsorption is strong;

Step 6, maintain the carbodiimide concentration of 72 mg in the process of step 1 and add 160 mg, 16 mg, 1.6 mg, 0.16 mg, and 0.016 mg of N-hydroxysulfosuccinimide respectively, and the obtained coupling product also undergoes step 2 ~Step 5, as shown in Figure 7, the non-specific adsorption was significantly reduced.

Comparative example 1

A method of minimizing nonspecific adsorption by carbodiimide bioconjugation comprising the steps of:

Step 1, dissolve aureomycin hydrochloride with 1M sodium hydroxide, adjust the pH back to 7.0 and prepare 5.0mL 5.0mg/mL aureomycin hydrochloride, add 25.0mg bovine serum albumin to the solution, so that the final concentration of bovine serum albumin When the concentration reaches 5.0 mg/mL, add 360 mg carbodiimide and react for 12 hours in the dark to obtain the coupling product;

Step 2: Dialyze the coupling product with pH 7.2 and 0.01M phosphate buffer for 3 days, during which a total of 12 changes were made, and the coupling product was concentrated with polyethylene glycol-20000 and diluted to a bovine serum albumin concentration of 1.0 mg/mL ;

Step 3: Fix the silicon dioxide colloidal crystal film, silica gel and 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 use the self-developed optical system and software Carry out reflection interferometry, after the light is reflected, it passes through the optical fiber and finally reaches the spectrometer, and the signal software of the spectrometer is processed to form the reflection interference spectrum of the silica colloidal crystal film;

Step 4: first pump bovine serum albumin into the reaction chamber prepared in step 3 at a rate of 0.4 mL/min for 2.5 hours to seal the surface of the film, and then inject phosphate buffer at the same flow rate to remove weakly bound or unbound bovine serum Protein, using reflection interferometry to record signals, the experimental temperature is 23°C;

Step 5, add 0.25% Tween-20 to the coupling product obtained in Step 2 and pass it into the reaction chamber treated in Step 4 at the same speed as in Step 4. The coupling product is non-specifically adsorbed on the surface of the blocked film due to the increase of the positive charge on the surface. After the reaction, the phosphate buffer is injected at the same flow rate to remove the weakly bound or unbound coupling product and achieve the purpose of controlling the refractive index of the solution. The signal was recorded by reflection interferometry, as shown in Figure 8, the signal increased by about 12nm, the non-specific adsorption was slightly weakened but still strong, and the experimental temperature was 23°C.

Comparative example 2

A method of minimizing nonspecific adsorption by carbodiimide bioconjugation comprising the steps of:

Step 1: Pump bovine serum albumin into the prepared silica film reaction chamber at a rate of 0.4 mL/min for 2.5 hours to seal the film surface, and then inject phosphate buffer at the same flow rate to remove weakly bound or unbound bovine serum Protein, using reflection interferometry to record signals, the experimental temperature is 24°C;

Step 2: Pass 1mg/mL aureomycin hydrochloride into the reaction chamber at the same speed as in step 1, and then rinse with phosphate buffer, and the optical thickness can be seen to recover, as shown in Figure 9, which shows that aureomycin hydrochloride itself Does not cause non-specific adsorption.

Example 5

A method of minimizing nonspecific adsorption by carbodiimide bioconjugation comprising the steps of:

Step 1, dissolve aureomycin hydrochloride with 1M sodium hydroxide, adjust the pH back to 7.0 and prepare 5.0mL 5.0mg/mL aureomycin hydrochloride, add 20.0mg bovine serum albumin to the solution, so that the final concentration of bovine serum albumin When the concentration reaches 4.0 mg/mL, add 360.0 mg carbodiimide and react for 10 hours in the dark to obtain a coupling product; the molar ratio of carbodiimide to bovine serum albumin is 6300;

Step 2: Dialyze the coupling product obtained in step 1 with a phosphate buffer solution with a pH of 7.4 and a concentration of 0.03M for 2 days, during which a total of 16 changes were made, and the coupling product was concentrated with polyethylene glycol-90000 and diluted to the concentration of bovine serum albumin 2.0 mg/mL.

Step 3: Fix the silicon dioxide colloidal crystal film, silica gel and 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 use the self-developed optical system and software Carry out reflection interferometry, after the light is reflected, it passes through the optical fiber and finally reaches the spectrometer, and the signal software of the spectrometer is processed to form the reflection interference spectrum of the silica colloidal crystal film;

Step 4: Pump polyethylene glycol-90000 into the reaction chamber prepared in Step 3 at a rate of 0.3 mL/min, keep it for 2 hours, seal the surface of the silica colloidal crystal film, and inject phosphate buffer at the same flow rate after the end to remove weak Combined or unbound polyethylene glycol-90000, the real-time optical thickness change is measured and recorded by reflection interferometry, and the experimental temperature is 25°C;

Step 5: Pass the resultant from 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 increase in positive surface charge. After the reaction, inject phosphoric acid at the same flow rate. Buffer, remove weakly bound or unbound coupling products, control the refractive index of the solution, use reflection interferometry to record signals, and confirm non-specific adsorption. The experimental temperature is 25°C;

Step 6, maintain the carbodiimide concentration of 360.0 mg in the process of step 1 and add 80 mg of N-hydroxysulfosuccinimide, and the obtained coupling product also undergoes steps 2 to 5.

Example 6

A method of minimizing nonspecific adsorption by carbodiimide bioconjugation comprising the steps of:

Step 1, dissolve aureomycin hydrochloride with 1M sodium hydroxide, adjust the pH back to 7.0 and prepare 5.0mL 6.0mg/mL aureomycin hydrochloride, add 30.0mg bovine serum albumin to the solution, so that the final concentration of bovine serum albumin When the concentration reaches 6.0 mg/mL, add 720.0 mg carbodiimide and react for 16 hours in the dark to obtain the coupling product; the molar ratio of carbodiimide to bovine serum albumin is 10000;

Step 2: Dialyze the coupled product obtained in Step 1 with pH 7.3 and a concentration of 0.02M phosphate buffer for 4 days, during which a total of 14 changes were made, and the coupled product was concentrated with polyethylene glycol-60000 and diluted to the concentration of bovine serum albumin 1.5 mg/mL.

Step 3: Fix the silicon dioxide colloidal crystal film, silica gel and 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 use the self-developed optical system and software Carry out reflection interferometry, after the light is reflected, it passes through the optical fiber and finally reaches the spectrometer, and the signal software of the spectrometer is processed to form the reflection interference spectrum of the silica colloidal crystal film;

Step 4: Pump polyethylene glycol-60000 into the reaction chamber prepared in Step 3 at a rate of 0.5 mL/min, keep it for 3 hours, seal the surface of the silica colloidal crystal film, and inject phosphate buffer at the same flow rate after the end to remove weak Combined or unbound polyethylene glycol-60000, the real-time optical thickness change was measured and recorded by reflection interferometry, and the experimental temperature was 24°C;

Step 5: Pass the resultant from 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 increase in positive surface charge. After the reaction, inject phosphoric acid at the same flow rate. Buffer, remove weakly bound or unbound coupling products, control the refractive index of the solution, use reflection interferometry to record signals, and confirm non-specific adsorption. The experimental temperature is 24°C;

Step 6, maintain the carbodiimide concentration of 720 mg in the process of step 1 and add 160 mg of N-hydroxysulfosuccinimide, and the obtained coupling product also undergoes steps 2 to 5.

Claims (10)

1. A method for minimizing the non-specific adsorption caused by carbodiimide biocoupling, characterized in that, comprising the following steps: Step 1, mixing bovine serum albumin and aureomycin hydrochloride solution, adding carbodiimide, stirring and reacting in a dark state, to obtain a coupling product; Step 2: Place the coupled product obtained in Step 1 in phosphate buffer, dialyze, concentrate the coupled product with polyethylene glycol, and dilute the product concentration to 1.0-2.0 mg/mL after concentration, where the product concentration is Bovine serum albumin concentration meter; Step 3: Fix the silica colloidal crystal film with silica gel and glass slides 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 the light passes through the optical fiber after reflection and finally reaches Spectrometer, reflection interference spectrum of silica colloidal crystal film formed after processing on 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 hours, seal the surface of the silica colloidal crystal film, then inject phosphate buffer at the same flow rate, and measure real-time optical thickness by reflection interferometry change and record; Step 5: Pass the product obtained in Step 2 into the reaction chamber after the treatment in Step 4, the coupling product is non-specifically adsorbed on the surface of the sealed film, inject phosphate buffer solution, control the refractive index of the solution, and record the signal by reflection interferometry, Confirm non-specific adsorption; Step six, repeat steps one to five once, and add N-hydroxysulfosuccinimide in step one to change the reaction path. 2. A method for minimizing non-specific adsorption caused by carbodiimide biocoupling according to claim 1, characterized in that: in said step 1, the concentration of bovine serum albumin after mixing is 4.0-6.0 mg/mL, the pH of aureomycin hydrochloride solution is 7.0. 3. A method for minimizing non-specific adsorption caused by carbodiimide biocoupling according to claim 1, characterized in that: in the step 1, the reaction time is 10-16h, and the carbodiimide and The bovine serum protein molar ratio is 10-10000. 4. A method for minimizing non-specific adsorption caused by carbodiimide biocoupling according to claim 1, characterized in that: in the second step, the concentration of the phosphate buffer is 0.01-0.03M, pH 7.2 to 7.4. 5. A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation according to claim 1, characterized in that: in the second step, the dialysis time is 2 to 4 days, during which a total of 12 ~16 times. 6. A method for minimizing non-specific adsorption caused by carbodiimide bioconjugation according to claim 1, characterized in that: in the second step, the molecular weight of polyethylene glycol is 20,000-90,000. 7. a kind of method for minimizing the non-specific adsorption that carbodiimide biocoupling causes according to claim 1 is characterized in that: in described step 4, inject bovine serum albumin, polyethylene glycol, phosphate buffer The speed is the same as the speed of injecting bovine serum albumin in step five. 8. A method for minimizing non-specific adsorption caused by carbodiimide biocoupling 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 biocoupling according to claim 1, characterized in that: the temperature of the reaction chamber in Step 3 and Step 4 is 20-25°C. 10. A method of minimizing the non-specific adsorption caused by carbodiimide biocoupling according to claim 1, characterized in that: in the step 6, the quality of N-hydroxysulfosuccinimide is 0.016~160mg, carbodiimide mass is 0.72~720mg.