CN100427941C - A chiral sensor based on bovine serum albumin and its preparation method - Google Patents

Abstract

The invention relates to the fields of nanotechnology, biotechnology and electrochemical research. Specifically, the invention provides a bovine serum albumin-based chiral sensor and a preparation method thereof. Different chiral isomers have obvious differences in biological activities, pharmacological effects, and metabolic processes in vivo. Therefore, the development of simple, accurate, and fast chiral recognition methods has become the main direction of chiral analysis in recent years. The invention provides a chiral sensor based on bovine serum albumin, which utilizes bovine serum albumin (BSA) as a chiral selector to identify isomer samples labeled with metal nanoparticles, and detects by electrochemical detection means for chiral recognition. The chiral sensor of the invention can be efficiently applied to the detection of chiral amino acids and chiral drugs. Experiments show that the chiral sensor can detect amino acid isomers at a concentration of 0.1 picomolar.

Description

A chiral sensor based on bovine serum albumin and its preparation method

technical field

The invention relates to the fields of nanotechnology, biotechnology and electrochemical research. Specifically, the invention provides a chiral sensor based on bovine serum albumin (BSA) and a preparation method thereof.

Background technique

Chiral compounds are closely related to the life process. Different chiral isomers have obvious differences in biological activities, pharmacological effects, and metabolic processes in organisms. main direction of analysis. In recent years, the research of chiral sensor has obtained certain development, wherein the chiral selector major part is to borrow the chiral stationary phase (A.Tsourkas, O.Hofstetter, H.Hofstetter, R.Weissleder, L. Josephson, Angew. Chem. Int. Edit. 2004, 43, 2395). Due to the complex and transformable conformation of proteins, chiral isomers can be separated well on chromatography, and domestic patents (patent application number: 99113091.X, publication number: CN 1280986A) have reported the use of proteins as chromatographic hand Sexual stationary phase. For example, BSA is usually used to separate chiral amino acids and chiral drugs on chromatography, and the separation effect is very good, and experiments and structural analysis have proved that the chiral selectivity of BSA is mainly derived from its interaction with chiral compounds (host-guest). Between stereoselectivity, hydrogen bonding and hydrophilic hydrophobicity (M.Kato, K.Sakai-Kato, N.Matsumoto, T.Toyo'oka, Anal.Chem.2002,74,1915; C.V.Kumar, A. Buranaprapuk, H.C.Sze, Chem.Comm.2001, 3, 297), however, so far, BSA has not been used in chiral sensors for chiral recognition. Therefore, starting from the combination of the good chiral selectivity of BSA and the advantages of simple, rapid and accurate determination of analytes, the development of chiral sensors will have important theoretical and practical research significance.

On the other hand, nanomaterials are considered to be a hotspot in the field of material research across the century. The quantum size effect, small size effect and surface effect of nanomaterials make them have many unique properties, so they are widely used in catalysis, biomedicine and new materials. The synthesis (P.Selvakannan, S.Mandal, S.Phadtare, A.Gole, R.Pasricha, S.D.Adyanthaya, M.Sastry, J.Colloid.Interf.Sci.2004, 269 , 97), opening up broad prospects for the application of nanomaterials in the biological field, such as biomarkers and drug delivery. For chiral sensors with proteins as chiral selectors, metal nanoparticles will be used to label chiral amino acid isomers, thereby detecting chiral recognition results by electrochemically detecting metal nanoparticles.

Contents of the invention

The purpose of the present invention is to provide a chiral sensor based on BSA, which makes chiral recognition easy to operate and high in detection sensitivity.

Another object of the present invention is to provide a method for preparing the above-mentioned sensor.

The invention provides a bovine serum albumin-based chiral sensor. In the sensor, the surface of an electrode is covered with an alumina sol-gel film containing bovine serum albumin, and the thickness of the film ranges from 20 to 40 nm.

Experimental results demonstrate that the sensor not only enables efficient chiral recognition, but also sensitively detects amino acid isomers and drug isomers.

In the sensor of the present invention, the electrodes may be screen-printed electrodes or glassy carbon electrodes.

The present invention also provides a preparation method for the above-mentioned sensor, that is, mixing bovine serum albumin with a concentration of 0.05-0.2 mg mL ^-1 with alumina sol-gel, and the volume ratio of bovine serum albumin and alumina sol-gel is 0.5:1-1.2:1; cover the mixed solution on the electrode, stand and cure at 0-4°C for 60-80 hours, and the thickness of the obtained film is in the range of 20-40nm; finally, connect the electrode prepared above to the electrochemical device , to make a sensor.

During the preparation process of the sensor of the present invention, the mixed solution is covered on the electrode by means of dipping, coating or dropping. The total volume of the mixed solution used to cover one electrode can be 2-20 μL.

During the preparation process of the sensor of the present invention, when the concentration of bovine serum albumin used is 0.09-0.15 mg mL ^-1 , the result is more optimal.

During the preparation process of the sensor of the present invention, the electrodes used may be screen-printed electrodes or glassy carbon electrodes.

During the preparation process of the sensor of the present invention, the sensor glassy carbon electrode used is rod-shaped, and its diameter may be 2-4 mm.

The sensor is mainly prepared by immobilizing BSA with alumina sol-gel at low temperature, the preparation method is simple, and the protein activity is well maintained. For chiral recognition, it is only necessary to incubate the sensor with the amino acid isomer solution labeled with metal nanoparticles for 8-12 minutes, and then obtain the chiral recognition result through electrochemical detection. The operation is simple, time-saving, and has a high Detection sensitivity.

When the sensor of the present invention is applied, two of the sensors obtained above may be used, and placed in the phosphate buffer solution containing the metal nanoparticle-labeled chiral isomer to be tested for incubation for 8-12 minutes. The pH range of the phosphate buffer solution is 6-9; After hatching, rinse the electrode with distilled water and perform electrochemical determination.

In the application process of the sensor of the present invention, the metal nanoparticles used may be gold particles, silver colloidal particles or copper sulfide nanoparticles.

In the application process of the sensor of the present invention, the isomers of the samples to be tested are tryptophan isomers, phenylalanine isomers or tyrosine isomers, and may also be chiral drugs.

The above-mentioned metal nanoparticles can be gold colloid, which is used to electrochemically detect the silver oxidation signal after staining with gold standard silver; it can also be silver colloid and Ag@Au or Cu@Au alloy nanoparticles, etc., to directly detect metal nanoparticles. The objects of chiral recognition can be tryptophan isomers, phenylalanine, tyrosine and some chiral drugs.

In the application process of the sensor of the present invention, a three-electrode system is used for electrochemical determination, the platinum wire is the counter electrode, the saturated calomel electrode is the reference electrode, and the sensor electrode or the carbon fiber electrode after chirality recognition is the working electrode. In mol L ^-1 acetic acid buffer solution (pH 4.8-5.5) or 0.08-0.12mol L ^-1 nitric acid base solution, record the differential pulse voltammetry curve.

The invention provides a chiral sensor based on bovine serum albumin. The chiral sensor is simple to prepare, can stably immobilize the protein, maintain the activity of the protein, and has relatively high detection sensitivity. The chiral sensor of the invention can be efficiently applied to the detection and separation of chiral amino acids and chiral drugs, and experiments show that the chiral sensor can detect amino acid isomers with a concentration of 0.1 picomolar.

Description of drawings

Figure 1 is a schematic diagram of the chiral recognition process.

Fig. 2 is a result diagram of chiral tryptophan identification. Curves a and b represent the chiral recognition results of the sensor for L- and D-tryptophan, respectively. The isomer selectivity coefficient α is defined as the ratio of silver oxidation peak currents obtained by the sensor for identifying L- and D-amino acids. Then the isomer selectivity coefficient of the sensor to tryptophan is 2.3. Experimental results show that the detection limit of the sensor for tryptophan isomers reaches 0.1 picomolar concentration.

Fig. 3 is a diagram showing the variation of peak current and isomer selectivity coefficient of the sensor with the concentration of tryptophan isomers.

Detailed ways

Example 1

The present invention will be further described below by using the sensor to recognize gold colloid-labeled tryptophan isomers.

(1) Preparation of gold colloid

Heat 500mL of 1mM _HAuCl4 solution to boiling, then add 50mL of 38.8mM trisodium citrate solution, continue to heat and boil for 10min, then stop heating and stirring for 15min to obtain a gold colloid with a diameter of about 13-15nm. After filtering through a 0.22μm filter membrane, store in a brown bottle at 0-4°C.

(2) Preparation of gold colloid-labeled tryptophan isomers

Add 1mL of 0.01mM tryptophan isomer solution to 9mL of the freshly prepared gold colloid solution above, and assemble at room temperature 20-30°C for 48 hours.

(3) Sensor preparation

Add 5 μL ( ¹ :1 v/v) of 0.1 mg mL-1 mixed solution of BSA and alumina sol-gel dropwise on a glassy carbon electrode (Φ=4), and let it stand and cure for 72 hours at 0-4°C to form The alumina sol-gel film thickness is 25±3nm.

(4) chiral recognition

Add 10 μL of the gold colloid-labeled tryptophan isomer solution prepared in step (2) to 10 mL of phosphate buffer solution (pH: 7), and then incubate the two sensors in it for 10 minutes, then rinse them thoroughly with distilled water .

(5) Gold standard silver dyeing

Place the two sensors after chiral recognition in silver staining solution (a mixed solution of 150 μL 0.22% silver nitrate solution and 150 μL 1% hydroquinone citrate buffer solution (pH=3.8)) to avoid light After reacting for 8 minutes, fully rinse with distilled water, then place in 2.5% Na ₂ S ₂ O ₃ aqueous solution for 3 minutes, and finally rinse fully with distilled water.

(6) Electrochemical determination

A three-electrode system was adopted, with platinum wire as the counter electrode, a saturated calomel electrode as the reference electrode, a silver-stained glassy carbon electrode as the working electrode, and 0.1mol/L acetic acid-sodium acetate buffer solution (pH 5.2) as the bottom solution. Record the differential pulse voltammetry (DPV) curve. Scanning range: 0.1V-0.8V (vs SCE). The reference electrode uses a double salt bridge to isolate it from the bottom solution, avoiding the continuous release of Cl ^- and silver to form AgCl precipitation to interfere with the determination.

The chiral sensor performs chiral recognition of tryptophan according to the above-mentioned embodiment. The chiral recognition process is shown in FIG. 1 , and the obtained chiral recognition results are shown in FIGS. 2 and 3 . In Fig. 2, curves a and b represent the chiral recognition results of the sensor for L- and D-tryptophan, respectively. The isomer selectivity coefficient α is defined as the ratio of silver oxidation peak currents obtained by the sensor for identifying L- and D-amino acids. Then the isomer selectivity coefficient of the sensor to tryptophan is 2.3. The experimental results show that the detection limit of the sensor for tryptophan isomers reaches 0.1 picomolar concentration. Fig. 3 shows the changes of the detected peak current and isomer selectivity coefficient of the sensor with the change of the concentration of tryptophan isomers.

Example 2

The present invention will be further described below by using the sensor to identify the isomer of phenylalanine labeled with gold colloid.

(1) The preparation of gold colloid and the preparation of the phenylalanine labeled by gold colloid are the same as in Example 1

(2) Sensor preparation

Add 10 μL ⁽ 1:1 v/v) of 0.1 mg mL-1 mixed solution of BSA and alumina sol-gel dropwise on a glassy carbon electrode (Φ=2mm), and let it stand and cure for 72 hours at 0-4°C to form The alumina sol-gel film thickness is 31±2nm.

(3) chiral recognition

Add 10 μL of the gold colloid-labeled phenylalanine isomer solution prepared in step (1) to 10 mL of phosphate buffer solution (pH: 7.4), and then incubate the two sensors in it for 11 minutes, then rinse with distilled water. rinse.

(4) Gold standard silver dyeing

Place the two sensors after chiral recognition in silver staining solution (a mixed solution of 150 μL 0.22% silver nitrate solution and 150 μL 1% hydroquinone citrate buffer solution (pH=3.8)) to avoid light After reacting for 8.5 minutes, fully rinse with distilled water, then place in 2.5% Na ₂ S ₂ O ₃ aqueous solution for 3 minutes, and finally rinse fully with distilled water.

(5) Electrochemical determination method is the same as embodiment 1

The chiral sensor performs chiral recognition on phenylalanine isomers according to the above embodiment, and the obtained isomer selectivity coefficient is 1.3.

Example 3

In the following, the present invention will be further described through the recognition of gold colloid-labeled tyrosine isomers by the sensor.

(1) Preparation of gold colloid The preparation of gold colloid and the preparation of the tyrosine labeled by gold colloid are the same as in Example 1

(2) Sensor preparation

Add 15 μL ⁽ 1:1 v/v) of 0.1 mg mL-1 mixed solution of BSA and alumina sol-gel dropwise on a glassy carbon electrode (Φ=4), and let it stand and solidify at 0-4°C for 72 hours to form The alumina sol-gel film thickness is 36±2nm.

(3) chiral recognition

Add 10 μL of the gold gel-labeled tyrosine isomer solution prepared in step (1) to 10 mL of phosphate buffer solution (pH: 8), and then incubate the two sensors in it for 12 minutes, then rinse them thoroughly with distilled water .

(4) Gold standard silver dyeing

Place the two sensors after chiral recognition in silver staining solution (a mixed solution of 150 μL 0.22% silver nitrate solution and 150 μL 1% hydroquinone citrate buffer solution (pH=3.8)) to avoid light After reacting for 9 minutes, fully rinse with distilled water, then place in 2.5% Na ₂ S ₂ O ₃ aqueous solution for 3 minutes, and finally fully rinse with distilled water.

(5) Electrochemical determination method is the same as embodiment 1

The chiral sensor performs chiral recognition of tyrosine according to the above embodiment, and the obtained isomer selectivity coefficient is 1.36.

Example 4

The present invention will be further described below by using the sensor to identify silver colloid-labeled tryptophan isomers.

(1) Preparation of silver colloid

Slowly add 10mL 3×10 ^-3 mol L ^-1 NaBH ₄ dropwise to 10mL 10 ^-3 mol L ^-1 AgNO ₃ solution, and stir in an ice bath. After the dropwise addition, leave the ice bath and keep stirring Until the temperature of the solution is gradually raised to room temperature, the obtained clear transparent light yellow colloid is then purified by dialysis and purification in a semipermeable membrane for 24 hours, and the obtained brownish yellow silver colloid, that is, the nano silver colloid to be prepared, has a particle size distribution of Within the range of 20-25nm.

(2) Preparation of silver colloid-labeled tryptophan isomers

Add 1mL of 0.01mM tryptophan isomer solution to 9mL of the freshly prepared silver colloid solution, and assemble at room temperature 20-30°C for 48 hours.

(3) Sensor preparation

Add 0.1 mg mL ^-1 of BSA and alumina sol-gel mixed solution 5 μL (1:1 v/v) dropwise on the glassy carbon electrode (Φ=4), and let it stand and solidify at 0-4°C for 72 hours.

(4) chiral recognition

Add 10 μL of the silver colloid-labeled tryptophan isomer solution prepared in step (2) to 10 mL of 0.01 M phosphate buffer solution (pH: 7), and then incubate the two sensors in it for 8 minutes, then rinse with distilled water Rinse well.

(5) Oxidation and dissolution of silver

Immerse the two sensors that have completed the chiral recognition reaction into 150 μL of 50% HNO ₃ solution respectively, shake to dissolve the silver oxidation, take out the sensor after 5 min, and add 0.8mL 10mM HNO ₃ -KNO ₃ solution as a supporting electrolyte in this solution Perform electrochemical detection

(6) Electrochemical determination

A three-electrode system was adopted, platinum wire was used as counter electrode, saturated calomel electrode was used as reference electrode, and 5 μm carbon fiber microelectrode was used as working electrode. In the above solution, Ag ⁺ was determined by anodic stripping voltammetry. After the electrode was pre-enriched at -0.5V for 150s, it was dissolved in the reverse direction within the range of 0.0-+0.6V, and the dissolution DPV signal was recorded. The reference electrode uses a double salt bridge to isolate it from the bottom solution, avoiding the continuous release of Cl ^- and silver to form AgCl precipitation to interfere with the determination.

Example 5

The present invention will be further described below by using the sensor to identify the isomer of phenylalanine labeled with silver colloid.

(1) The preparation of silver colloid and the preparation of the phenylalanine isomers marked by silver colloid are the same as in Example 1

(2) Sensor preparation

Add 10 μL ( ¹ :1 v/v) of 0.1 mg mL-1 mixed solution of BSA and alumina sol-gel dropwise on the glassy carbon electrode (Φ=3), and let it stand at 0-4°C for 72 hours to cure.

(3) chiral recognition

Add 15 μL of the silver gel-labeled phenylalanine isomer solution prepared in step (1) to 10 mL of 0.02 M phosphate buffer solution (pH: 8), and then incubate the two sensors in it for 10 minutes, then use Rinse thoroughly with distilled water.

(4) Oxidation and dissolution of silver

Immerse the two sensors that have completed the chiral recognition reaction in 200 μL of 50% HNO3 solution, shake to dissolve the silver oxidation, take out the glassy carbon electrode after 5 minutes, and add 1mL of 10mM HNO ₃ -KNO ₃ solution as a supporting electrolyte in this solution Perform electrochemical detection

(5) Electrochemical determination method is the same as embodiment 1

Claims (7)

1. the chiral sensor based on bovine serum albumin(BSA) is characterized in that, electrode surface covers the alumina sol-gel mould that contains bovine serum albumin(BSA) in this sensor, and the thickness range of film is 20～40nm; And the concentration of the bovine serum albumin(BSA) of alumina sol-gel mixing is 0.05-0.2mg mL ^-1, the volume ratio of bovine serum albumin(BSA) and alumina sol-gel is 0.5: 1-1.2: 1.

2. sensor according to claim 1 is characterized in that electrode is screen printing electrode or glass-carbon electrode in this sensor.

3. the preparation method of sensor according to claim 1 is characterized in that, is 0.05-0.2mg mL with concentration ^-1Bovine serum albumin(BSA) and alumina sol-gel mix, the volume ratio of bovine serum albumin(BSA) and alumina sol-gel is 0.5: 1-1.2: 1; Mixed solution is covered on the electrode, and 0-4 ℃ leaves standstill curing 60-80 hour, and the film thickness scope that obtains is 20～40nm; At last the above-mentioned electrode that makes is connected with electrochemical appliance, makes sensor.

4. the preparation method of sensor as claimed in claim 3 is characterized in that, mixed solution covers on the electrode by dipping, coating or dropping method.

5. the preparation method of sensor as claimed in claim 3 is characterized in that, the concentration of bovine serum albumin(BSA) is 0.09-0.15mg mL ^-1

6. as the preparation method of sensor as described in the claim 3, it is characterized in that electrode is screen printing electrode or glass-carbon electrode in this sensor.

7. as the preparation method of sensor as described in the claim 3, it is characterized in that glass-carbon electrode is shaft-like in this sensor, diameter is the 2-4 millimeter.

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