CN101231260B - Method for preparing biosensor using ionic liquid as green medium - Google Patents

Abstract

The invention discloses a method for preparing a biosensor using ionic liquid as a green medium. The main steps are: (1) purifying the ionic liquid, dissolving the crude ionic liquid in 200ml of acetone, and filtering to remove inorganic salts. (2) Embedding of enzymes. In 100ml of phosphate buffer solution, add 30mg of enzyme, stir well to dissolve it completely. Add 10ml of the ionic liquid purified by the method (1) to the above solution, then vigorously stir for 2 hours (2500 rpm), leave to separate layers, discard the aqueous phase, and collect the lower ionic liquid. (3) Biosensor preparation. Inject 0.1ml of the ionic liquid embedding solution prepared according to (2) into a polypropylene tube, seal the lower end of the tube with a modified cellulose membrane, draw in a platinum wire from the upper end to a distance of 0.5 cm from the bottom, and use a conductive Glue seals to make biosensors. The preparation method of the invention is simple, the electrochemical window width of the obtained biosensor is greater than 4V, the peak current of the substrate is only several nA, the electrochemical response is fast, and the stability is good.

Description

Method for preparing biosensor using ionic liquid as green medium

technical field

The invention relates to a method for preparing a sensor, in particular to a method for preparing a biosensor using an ionic liquid as a green medium.

Background technique

Biosensors are immobilized biological components (enzymes, proteins, DNA, antibodies, antigens, biofilms, etc.) or organisms themselves (cells, microorganisms, tissues, etc.) as sensitive materials, combined with appropriate chemical transducers to generate a A device for rapid detection of physical, chemical, and biomass. The attractive application prospects of biosensors make it rapidly become a research hotspot in modern analytical chemistry, and play an increasingly important role in rapid detection in the fields of environment, biology, medicine and food safety.

The core work of making biosensors is enzyme immobilization. Traditional immobilization methods include adsorption, embedding, cross-linking and covalent bonding. Their biggest disadvantage is that they lead to a significant decrease in enzyme activity. In order to overcome this deficiency, in recent years people have tried to use some biophilic materials as carriers such as poly-L-tyrosine (Xie Yi, Yuan Ruo, Chai Yaqin, Gao Fengxian, Tang Mingyu, Analytical Laboratory, 2007, 26(9): 1) , peroxide polypyrrole film (Jiang Xiaohua, Liu Weiqiang, Chen Jianjun, Chemical Journal of Chinese Universities, 2007, 28(3): 450) and polyethylene (Jiang Y, Wang AY, Kan JQ, Sensors and Actuators B, 2007, 124: 529 )wait. Although these biophilic materials have played a certain role in improving electrode stability and conductivity, they are difficult to provide an optimal microenvironment for enzyme catalysis.

In recent years, a new type of green solvent-room temperature ionic liquid has attracted great interest and has been widely used in analytical chemistry (AndersonJL, Armstrong DW, Wei GT, Analytical Chemistry, 2007, 79(11): 4247; Rizvi SAA, Shamsi SA, Analytical Chemistry, 2006, 78(19):7061). A large number of facts show that ionic liquids have good compatibility with enzymes, substrates and other biophilic materials, and can significantly improve the activity and stability of enzymes. These characteristics have laid the foundation for ionic liquids to replace traditional adhesives or polymer materials as biosensor media. Solid foundation (Lee JK, Kim MJ, Journal of Organic Chemistry, 2002, 67: 6845; Maminska R, Dybko A, Wroblewski W, Sensors and Actuators B, 2006, 115: 552; Lesniewski A, Niedziolka J, Palys B, Rizzi C, Gaillon L, Opallo M, Electrochemistry Communication, 2007, 9: 2580). Recently, Liu Yang, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, etc. disclosed a method for embedding enzyme biosensors in ionic liquid sol-gel composite membranes (Chinese patents, application numbers 200410011340.0 and 200610017201.8). However, the hydrogen atom at the 2-position of the imidazole ring in traditional 1-alkyl-3-methylimidazole ionic liquids is relatively active and easily oxidized, resulting in a narrow electrochemical window, which limits their wide application in biosensors and related fields. application.

Contents of the invention

An object of the present invention is to provide a method for preparing a biosensor using ionic liquid as a green medium. The substrate of the obtained biosensor has small peak current, fast response and good stability. The enzyme embedded in the electrode has no loss and has a long service life.

The technical scheme provided by the invention is as follows:

(1) Purification of ionic liquids, 50-100g crude ionic liquids are dissolved in 200-1000mL acetone, filtered to remove inorganic salts; decolorized with 50-250g ultra-fine activated carbon and chromatographic grade alumina respectively, and filtered to obtain a colorless solution; Acetone, water and organic raw materials were removed by distillation under reduced pressure to prepare a colorless, transparent oily liquid;

(2) Encapsulation of enzyme, in 100-200mL phosphate buffer solution (0.1-0.5mol/L, pH 7-7.5), add 30-50mg of enzyme, stir well to dissolve it completely; add 10-15mL to the above solution The ionic liquid purified by the method (1) was vigorously stirred for 2 hours (2500 rpm), then left to stand for stratification, discarding the aqueous phase, and collecting the lower ionic liquid;

1～1.2mm)至离聚丙烯管底部0.2-0.5cm处，上端用导电胶封位制成生物传感器。(3) Biosensor preparation, inject 0.1-0.2mL ionic liquid embedding solution prepared according to (2) method into polypropylene tube ( 2 ~ 5mm), the lower end of the tube is sealed with a modified cellulose membrane (pore size 0.1 ~ 0.15 μm), and a platinum wire (

1-1.2mm) to 0.2-0.5cm away from the bottom of the polypropylene tube, and the upper end is sealed with conductive glue to make a biosensor.

When embedding, the enzyme is first dissolved in a phosphate buffer solution with a pH of 7.0-7.5, and then uniformly embedded in an ionic liquid through an extraction process.

The enzyme is one of catalase, horseradish peroxidase, glucose oxidase, dehydrogenase or polyphenol oxidase.

The ionic liquid used is an ionic liquid with a 1-alkyl-2,3-methylimidazole structure. The alkyl groups in the cations include various alkyl groups with 2 to 12 carbon atoms, and the anions include PF ₆ ^- and N(CF ₃ ) ₂ ⁻ .

There is no obvious absorption peak when the purified ionic liquid is scanned between 400-800nm, indicating that the colored compound in the ionic liquid has been removed; After heating for 1 to 2 hours, 1200L GC/MS-MS gas chromatography was used to analyze the components floating above the ionic liquid, and no obvious low boiling point compounds were detected.

Since the 2-position hydrogen atom of the imidazole ring in the ionic liquid of the 1-alkyl-2,3-methylimidazole structure is replaced by an inert methyl group, the electrochemical stability of the ionic liquid is improved, and the prepared electrode has a wide range Electrochemical window, thereby eliminating the interference caused by the oxidation-reduction of the electrode material itself during the detection process, and expanding the application range of the electrode. However, electrochemical research has strict requirements on the purity of materials used, and ionic liquids synthesized by existing methods cannot meet the needs. The main impurity components in ionic liquids come from the impurity of raw materials and the oxidation of colored compounds during the reaction process. The former can be solved by using high-purity raw materials and re-distilling before use. The latter is difficult to remove by conventional decolorization methods due to the high viscosity of ionic liquids, which has become a difficult point in the purification of ionic liquids (Earle MJ, Gordon CM, Plechkova NV, Seddon KR , Welton T, Analytical Chemistry, 2007, 79 (11): 4247), but adding a suitable diluent to the ionic liquid greatly reduces its viscosity, and the above problems are easily solved.

Compared with the prior art, the present invention has the following advantages: 1. The electrochemical window is wide, and the ionic liquid of the adopted 1-alkyl-2,3-methylimidazole structure has a 2-position hydrogen atom on the cationic imidazole ring Substituted by a methyl group, the ionization or oxidation of the hydrogen atom is fundamentally eliminated; the anion part adopts electrochemically inert PF ₆ ^- and N(CF ₃ ) ₂ ^- , this type of ionic liquid has very ideal stability, electrochemical The windows are all greater than 4V. The above properties not only avoid the interference caused by the oxidation-reduction reaction of the medium during the detection process, but also greatly expand the application range of the biosensor. 2. The matrix current is small. Since the ionic liquid itself is conductive, it can be used as a conductive medium. The ionic liquid used in the present invention has good stability, and its charging current is only in the range of several nA (see Figure 1), which is much smaller than the carbon paste electrode (at mA level) commonly used now. The matrix current is small, which is beneficial to reduce the matrix interference and detection limit in the detection process. 3. The service life is long, and the ionic liquid used has good biophilicity, which greatly improves the activity and stability of the enzyme. The electrochemical properties of the biosensor made by the present invention are maintained for one year, and the data are basically unchanged.

Description of drawings

Fig. 1 is a cyclic voltammogram of a blank electrode of a 1-pentyl-2,3-dimethylimidazolium hexafluorophosphate ionic liquid membrane.

Detailed ways

(1) Purification of ionic liquids

50 g of crude 1-pentyl-2,3-methylimidazole ionic liquid was dissolved in 200 mL of acetone, and the inorganic salt was removed by filtration. Decolorize with 50 g of ultrafine activated carbon and chromatographic grade alumina for 4 hours, and filter to obtain a colorless solution. The filtrate was distilled off under reduced pressure to remove acetone, water and organic materials to prepare a colorless, transparent oily liquid.

(2) Embedding of catalase

In 100mL of phosphate buffer solution (0.1mol/L, pH7), add 30mg of catalase, stir well to dissolve the horn completely. Add 10 mL of purified ionic liquid to the above solution, and then vigorously stir for 2 hours (2500 rpm), then, let stand to separate layers, discard the aqueous phase, and collect the ionic liquid in the lower layer.

(3) Preparation of hydrogen peroxide biosensor

1mm)至离底部0.5cm处，然后，上端用导电胶封位制成生物传感器。Inject 0.1 mL of ionic liquid embedding solution into a polypropylene tube ( 2 mm), the lower end of the tube was sealed with a modified cellulose membrane (pore size 0.1 μm), and a platinum wire (

1mm) to 0.5cm from the bottom, and then, the upper end is sealed with conductive glue to make a biosensor.

(4) Electrochemical detection method

Using a three-electrode system (hydrogen peroxide biosensor as working electrode, Pt wire electrode as auxiliary electrode and Ag/AgCl standard electrode as reference electrode), 0.1mol/L Na ₂ HPO ₄ ·12H ₂ O-KH ₂ PO _4. Buffer solution (pH7) was used as the test base solution, and high-purity nitrogen gas was introduced to remove dissolved oxygen, and the cyclic voltammogram (CV) of the system was measured on an electrochemical workstation. The peak current value of CV reduction decreases linearly with the increase of hydrogen peroxide concentration, which is used for the determination of hydrogen peroxide concentration.

(5) Analysis characteristics

Using 1-pentyl-2,3-dimethylimidazolium hexafluorophosphate/enzyme electrode as the working electrode, the CV analysis was carried out according to the experimental method. From the CV diagram, we can clearly see that the reduction peak current increases with the increase of the concentration of hydrogen peroxide in the bottom solution, and when the concentration of hydrogen peroxide is in the range of 3.17-12.4 μmol/L, the relationship is linear, and the linear equation is y =-1.6776x-1.5577, R ² =0.9979. Among them, y is the electrode reduction peak current value (μA), x is the concentration of hydrogen peroxide in the bottom solution (mol/L), and the detection limit is 1.1×10 ^-6 mol/L. The enzyme electrode measures the response current of hydrogen peroxide to be stable within 5s. The electrochemical performance of the enzyme electrode did not change significantly after being stored at 4°C for 30 days. In addition, the enzyme electrode has better selectivity. When measuring 1×10 ^-5 mol/L hydrogen peroxide in water, Ca ²⁺ and Mg ²⁺ more than 1000 times, and ascorbic acid, glucose, citric acid, ethanol, acetic acid and dopamine at 5 times the concentration do not produce interference, which is It may be related to the selective extraction of ionic liquids.

(6) Determination of trace hydrogen peroxide in environmental water samples

The hydrogen peroxide biosensor has been applied to the determination of hydrogen peroxide in environmental water samples, and the results are listed in Table 1. The recoveries of hydrogen peroxide were in the range of 95.3-100.8%, which showed that the method had good accuracy.

Table 1: Determination of hydrogen peroxide in environmental water samples

Sample Added(10 ^-5 mol/L ^-1 ) /Found(10 ^-5 mol/L ^-1 ) Recovery(%) river water groundwater lake water sewage 0.002.280.005.720.007.840.0010.79 0.002.300.005.680.008.230.8211.19 100.899.395.396.2

Claims (3)

1. A method for preparing a biosensor with an ionic liquid as a green medium, the main steps of which are: (1) Purification of ionic liquids, 50-100g crude ionic liquids are dissolved in 200-1000mL acetone, filtered to remove inorganic salts; decolorized with 50-250g ultra-fine activated carbon and chromatographic grade alumina respectively, and filtered to obtain a colorless solution; Acetone, water and organic raw materials were removed by distillation under reduced pressure to prepare a colorless, transparent oily liquid; (2) Encapsulation of enzymes: Add 30-50 mg of enzymes to 100-200 mL of phosphate buffer solution, stir thoroughly to dissolve them completely; add 10-15 mL of ionic liquid purified by the method (1) to the above solution, and then vigorously Stir for 2 hours, the rotation speed during stirring is 2500 rpm; then stand for stratification, discard the water phase, and collect the ionic liquid in the lower layer; the concentration of the phosphate buffer solution is 0.1-0.5mol/L, pH 7-7.5; (3) For the preparation of biosensors, inject 0.1-0.2mL of the ionic liquid embedding solution prepared according to (2) with a diameter of

In the polypropylene tube, the lower end of the tube is sealed with a modified cellulose membrane with a pore size of 0.1-0.15 μm, and a tube with a diameter of

The platinum wire is placed 0.2-0.5cm away from the bottom of the polypropylene tube, and the upper end is sealed with conductive glue to make a biosensor; The ionic liquid used is an ionic liquid with a 1-alkyl-2,3-methylimidazole structure. The alkyl groups in the cations include various alkyl groups with 2 to 12 carbon atoms, and the anions include PF ₆ ^- and N(CF ₃ ) ₂ ⁻ . 2. the method for preparing biosensor with ionic liquid as green medium as claimed in claim 1, is characterized in that, described enzyme is catalase, horseradish peroxidase, glucose oxidase, dehydrogenase or polyphenol one of the oxidases. 3. as claimed in claim 1, prepare the method for biosensor with ionic liquid as green medium, it is characterized in that, there is no obvious absorption peak when carrying out spectral scanning to the ionic liquid after the purification between 400～800nm; Then ionic liquid Heated at 250-300°C and under vacuum conditions of less than 0.01MPa for 1-2 hours, and analyzed the components floating above the ionic liquid by 1200L GC/MS-MS gas chromatography, and no obvious low boiling point compounds were detected.

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