阅读说明：本技术 一种利用生物膜层干涉技术测定氧化石墨烯对小球藻胞外聚合物亲和力的方法 (Method for determining affinity of graphene oxide to chlorella extracellular polymeric substances by utilizing biofilm interference technology ) 是由 周启星 吴康迎 欧阳少虎 于 2021-09-01 设计创作，主要内容包括：本发明公开了一种利用生物膜层干涉技术测定氧化石墨烯对小球藻胞外聚合物亲和力的方法。首先通过选取APS传感器,将GO可以固化到APS传感器上,构建APS-GO传感器。其次,APS-GO传感器借助BLI分子相互作用仪可与不同浓度梯度的EPS进行亲和实验测试,目标EPS被固化在APS表面的GO特异性捕获结合。接着,将APS-GO传感器捕获EPS。最后,基于结合和解离实验的数据,分析出GO对EPS的吸附常数K-(on)、解离常数K-(dis)和亲和力常数K-(D)。本发明利用生物膜层干涉技术可在30分钟内实现GO对小球藻EPS亲和力常数的测定,具有无需借助人工标签,可直接检测,且耗量少,结果准确等优点。(The invention discloses a method for determining the affinity of graphene oxide to chlorella extracellular polymeric substances by utilizing a biofilm interference technology. The APS-GO sensor is constructed by first selecting an APS sensor and solidifying GO onto the APS sensor. Secondly, the APS-GO sensor can be subjected to affinity experimental tests with EPS with different concentration gradients by means of a BLI molecular interaction instrument, and the target EPS is specifically captured and bound by GO solidified on the surface of APS. Next, the APS-GO sensor is captured of the EPS. Finally, analyzing the adsorption constant K of GO to EPS based on the data of combination and dissociation experiments on Dissociation constant K dis And affinity constant K D . The method can realize the determination of the GO on the EPS affinity constant of the chlorella in 30 minutes by utilizing the biomembrane interference technology, and has the advantages of no need of manual labeling, direct detection, low consumption, accurate result and the like.)

1. A method for measuring the affinity of graphene oxide to chlorella extracellular polymeric substances by using a biofilm interference technology is characterized by comprising the following steps:

(1) preparation of sample plate: selecting a buffer solution and preparing GO and EPS aqueous solutions, wherein the buffer solution selects ultrapure water, the GO and chlorella EPS use the ultrapure water as a solvent, and the pH value is adjusted to be equal to the pH value of the selected buffer solution by ice bath ultrasonic treatment to prepare the aqueous solution; adding the buffer solution, the GO solution and the chlorella EPS solution into a totally black 96-hole plate according to a certain arrangement sequence to be detected;

(2) constructing an APS-GO sensor: binding GO to an Aminopropylsilane sensor using non-covalent binding and hydrophobic interactions, immobilizing GO to an APS sensor;

(3) and affinity testing: the affinity test comprises five steps: a. balancing a base line; solidifying GO to the surface of the APS sensor, namely constructing the APS-GO sensor; c. baseline balancing again; determination of binding constant K by APS-GO sensor and EPS binding experiment_on(ii) a Determination of dissociation constant K by APS-GO sensor and EPS dissociation experiment_dis；

(4) Analyzing data by using data analysis Software 9.0 Software carried by a BLI molecular interaction instrument platform, and calculating to obtain an EPS (expandable polystyrene) affinity constant K of GO to chlorella_D。

2. The method for measuring the affinity of graphene oxide for chlorella extracellular polymeric substances by using the biofilm interference technology as claimed in claim 1, wherein in the step (1), the buffer solution is ultrapure water, the EPS detection range is 10000-90000 nM, GO and two EPS types take the ultrapure water as a solvent, and the specific parameters are as follows: carrying out ice bath ultrasonic treatment on GO for 30min, wherein the concentration is 100 mg/L; carrying out ice bath ultrasound on soluble extracellular polymer and combined extracellular polymer for 30min, setting a plurality of concentration gradients in a detection range, and sequentially adjusting the pH values of GO and EPS aqueous solutions to be close to that of a buffer solution, wherein the model of a total black 96 pore plate is Grenier 655209.

3. The method for measuring the affinity of graphene oxide for chlorella extracellular polymeric substances by using the biofilm layer interference technology as claimed in claim 1, wherein in the step (2), the pre-wetting equilibration time in the buffer solution before the use of the APS sensor is 10 min.

4. The method for determining the affinity of graphene oxide for chlorella extracellular polymeric substances by using a biofilm layer interference technology as claimed in claim 1, wherein in the step (3), the affinity test parameters are as follows: a. baseline equilibration time was 60 s; the curing time of GO is 5-10 min; c. the baseline equilibration time was again 60 s; d. the combination experiment time is 5-10 min; e. the dissociation experiment time is 5-30 min, and the temperature is set to be 25-30 ℃.

Technical Field

The invention relates to a method for measuring the affinity of graphene oxide to chlorella extracellular polymeric substances, in particular to a method for measuring the affinity of graphene oxide to chlorella extracellular polymeric substances by utilizing a biofilm interference technology.

Background

Graphene Oxide (GO) is a two-dimensional nanomaterial that is exfoliated from graphite oxide with a single atomic layer thickness. GO is widely applied to the fields of chemistry, physics, biology, manufacturing, environmental protection and the like due to the excellent performance of GO. With the wide application and rapid increase of the yield of GO in various fields, the risk of GO being exposed to the environment is higher and higher, and the GO has excellent dispersibility in an aqueous solution, so that the understanding of the environmental behavior and the biological effect of GO in a water body is very necessary, chlorella is taken as a model organism for aquatic toxicological research, and the research on the interaction of GO and chlorella Extracellular Polymeric Substances (EPS) is of great significance for exploring the environmental behavior and the biological effect of GO in the water body.

The bio-film interference (BLI) technique is a non-labeling technique based on the principle of optical interference, and can detect the interference signal change caused by the surface thickness change in real time by means of an optical fiber biosensor, and the change detected in real time can be used for calculating the speed of binding and dissociation, and the like. The technology has the advantages of a non-labeled biological analysis method, simple operation, nondestructive detection, low sample consumption, real-time provision of direct information and interaction conditions of analytes and the like. Due to these advantages, BLI technology has been widely used for kinetic analysis and rapid detection of biomolecules. Meanwhile, the BLI technique is gradually applied to kinetic analysis of nanomaterials. Therefore, the BLI technology has wide development prospect as a method for determining the affinity of GO. In this patent, BLI technology will be used to explore the binding affinity between GO and chlorella EPS.

Disclosure of Invention

The invention aims to solve the problems and the prior art, and provides a method for determining the EPS affinity of GO to chlorella by utilizing BLI technology, which is realized by a molecular interaction instrument filled with BLI. The affinity of GO to EPS can be analyzed quickly, accurately and quantitatively.

The purpose of the invention is realized by the following technical scheme: a method for measuring the affinity of graphene oxide to chlorella extracellular polymeric substances by using a biofilm interference technology comprises the following steps:

(1) preparation of sample plate: selecting a buffer solution and preparing GO and EPS aqueous solutions, wherein the buffer solution selects ultrapure water, the GO and chlorella EPS use the ultrapure water as a solvent, and the pH value is adjusted to be equal to the pH value of the selected buffer solution by ice bath ultrasonic treatment to prepare the aqueous solution; and adding the buffer solution, the GO solution and the chlorella EPS solution into a total black 96-well plate according to a certain arrangement sequence to be detected.

(2) Constructing an APS-GO sensor: the GO is immobilized to an APS sensor by binding to the Aminoropropylaniline (APS) sensor very well using non-covalent binding and hydrophobic interactions.

(3) And affinity testing: the affinity test comprises five steps: a. baseline balance (Baseline); solidifying (Loading) GO to the surface of the APS sensor, namely constructing the APS-GO sensor; c. baseline balance again (Baseline); determination of binding constant K by APS-GO sensor and EPS binding experiment (association)_on(association rate); determination of dissociation constant K by APS-GO sensor and EPS dissociation (dissociation) experiment_dis(dissociationrate)。

(4) Analyzing Data by using Data Analysis Software 9.0 Software carried by a BLI molecular interaction instrument platform, and calculating to obtain the EPS affinity constant (K) of GO to chlorella_D)。

In the step (1), through a pre-experiment, the detection range of the buffer solution type which is ultrapure water and EPS is 10000-90000 nM, the GO and the two EPS types which use the ultrapure water as a solvent, and the specific parameters are as follows: carrying out ice bath ultrasonic treatment on GO for 30min, wherein the concentration is 100 mg/L; soluble extracellular polymeric substrates (S-EPS) and bound extracellular polymeric substrates (B-EPS) were subjected to ultrasonic treatment in ice bath for 30min, and several concentration gradients were set within the detection range. And sequentially adjusting the pH values of the GO and EPS aqueous solutions to be close to the pH value of the buffer solution. The model of the 96-well plate is Grenier 655209.

In the step (2), the APS sensor needs to be pre-wetted in the buffer solution for an equilibrium time of about 10min before use.

In the step (3), the affinity test experiment parameters are as follows: a. the baseline balance time is about 60 s; the curing time of GO is 5-10 min; c. the baseline balance time is about 60s again; d. the combination experiment time is 5-10 min; e. the dissociation experiment time is 5-30 min. The temperature is set to 25 to 30 ℃.

The technical progress of the invention is represented as follows: the method can rapidly measure the affinity constants of the graphene oxide and the two extracellular polymers at high flux by utilizing a biomembrane interference technology, can directly detect and analyze the biomolecule sample without an artificial label, avoids the influence of a chemical label on a research result, and has the advantages of low consumption and accurate result. Provides a new idea for determining the affinity between the nanometer material and the biological macromolecules.

Drawings

FIG. 1 is a schematic diagram of the present invention GO attached to an APS sensor by non-covalent bonding and hydrophobic interaction and tested for affinity with EPS in a fully black microplate;

FIG. 2 is a schematic diagram showing the arrangement sequence of the affinity test experiment sample plates in the example of the present invention;

FIG. 3 is a graph of BLI raw data for graphene oxide versus two extracellular polymeric substances according to an embodiment of the present invention, FIG. 3a is a graph of BLI raw data for GO versus S-EPS, and FIG. 3B is a graph of BLI raw data for GO versus B-EPS;

fig. 4 is a graph of binding-dissociation kinetics of two extracellular polymers on a graphene oxide surface in an embodiment of the present invention, fig. 4a is a graph of BLI binding-dissociation kinetics of S-EPS on a GO surface, and fig. 4B is a graph of BLI binding-dissociation kinetics of B-EPS on a GO surface;

table 1 shows experimental procedures and test schedules for BLI affinity testing in examples of the present invention;

table 2 shows the binding-dissociation kinetic parameters of different fractions of EPS on the GO surface in the examples of the invention.

Detailed Description

The invention is further described below with reference to the following figures and examples.

FIG. 1 is a schematic diagram of the present invention GO attached to an APS sensor by non-covalent bonding and hydrophobic interaction and tested for affinity with EPS in a fully black microplate; the embodiment of the invention selects chlorella to extract two levels of extracellular polymeric substances through different centrifugal forces and temperatures: soluble extracellular polymeric substrates (S-EPS), bound extracellular polymeric substrates (B-EPS).

Examples

(1) Weight average molecular weights (M) of both EPS types_W) The determination of (1): the weight average molecular weights (M) of the S-EPS and B-EPS were determined by Gel Permeation Chromatography (GPC)_W) 9.904kDa and 8.733kDa respectively.

(2) Preparation of sample plate: according to the result of the preliminary experiment, ultrapure water is selected as the buffer solution, GO and two EPS are dissolved in ultrapure water, ice-bath ultrasonic treatment is carried out for 30min, the pH value is adjusted to be approximately equal to the pH value of the ultrapure water, and an aqueous solution is prepared; adding the buffer solution, the GO solution and the EPS solution into a 96-well plate according to the sequence of the figure 2 to be detected according to a certain arrangement sequence, namely: the method comprises the following steps: adding a buffer solution into the column 1; step two: add GO solution in column 2; step three: adding a buffer solution into the column 3; step four: adding EPS buffer solution into the 4 th row; step five: buffer solution was added in column 5. For the GO solution, the concentration was fixed at 100 mg/L; for the S-EPS solution, the molar concentration gradient was set at 10100nM, 20190nM, 60580nM, 80780 nM; for the B-EPS solution, the concentration gradient was set at 11400nM, 34200nM, 45600nM, 68400 nM.

(3) And affinity experiment testing: the affinity test experiment comprises five steps of a, Baseline balance (Baseline)60 s; go curing (Loading) to APS sensor surface 300 s; c. baseline balance again (Baseline)60 s; d. determination of the binding constant K by binding experiments (association)_on(association rate)420 s; e. dissociation (dissociation) experiment determination of dissociation constant K_dis(dissociation rate)420s。

TABLE 1

And sequentially setting on an OCTET software operation interface: firstly, arranging a sample plate in a sample plate 'plate definition' according to the position of actual sample adding; next, the step setting is made at "assist definition", and "Shake speed" is set to 1000 rpm. Adjusting according to the actual setting parameters of the affinity test experiment; setting the position of the sensor in the sensor assignment; and finally setting an experiment data file storage directory, namely experiment temperature, wherein the experiment temperature is set to be 25 ℃. After the setting is finished, clicking an icon button 'GO' on the platform to start an experiment.

(4) After the step (3) is operated, an operation interface displays a real-time binding curve, after the experiment is finished, Data Analysis Software 9.0 Software carried by a BLI molecular interaction instrument platform is used for analyzing Data, and affinity constants (K) of graphene oxide to two kinds of extracellular polymeric substances of chlorella are calculated_D). The results are shown in FIG. 3, FIG. 4 and Table 2, where the ordinate is the binding signal. The affinity of GO to S-EPS is measured to be K by using BLI technology_D(5.88E-07M) GO has a K affinity for B-EPS_D＝9.05E-07M。

TABLE 2

Note: k_on(M^-1s^-1) Binding Rate, K_dis(1/s), dissociation rate. Affinity constant K_DThe value (M) is equal to K_disAnd K_onThe ratio of (a) to (b).