CN109975382A - A kind of tyrosinase biosensor of phosphorus doping MXene modified electrode and preparation method and application - Google Patents
A kind of tyrosinase biosensor of phosphorus doping MXene modified electrode and preparation method and application Download PDFInfo
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Abstract
A kind of tyrosinase biosensor of phosphorus doping MXene modified electrode and preparation method and application, belongs to electrochemica biological sensor technical field.The present invention includes glass-carbon electrode；The glass-carbon electrode is surface modification tyrosinase/phosphorus doping MXene/ chitosan glass-carbon electrode.Preparation method are as follows: configuration tyrosinase/phosphorus doping MXene/ chitosan mixed solution is added drop-wise to glassy carbon electrode surface, obtains tyrosinase biosensor.It is applied to catechol, i.e., in the detection of catechol.The features such as biosensor preparation method of the invention has simple, low in cost, production quickly, and enzymatic activity is high, precision and efficiency of detecting is high, detection limit is low, may be implemented the efficient detection to catechol.
Description
Technical field
The present invention relates to tyrosinase biosensor of a kind of phosphorus doping MXene modified electrode and preparation method thereof and answer
With, and in particular to a kind of tyrosinase biosensor of the trace detection for catechol belongs to electrochemica biological sensor
Technical field.
Background technique
Catechol, scientific name catechol are widely present in tealeaves, fruit and vegetables, are the weights of enzymatic browning reaction
One of substrate is wanted, also there is wide application in pharmacy and Chemical Manufacture.In recent years, researcher is for pyrocatechol in tealeaves
The anti-oxidant, antiviral of substance, such as protocatechuic acid, ellagic acid etc., anticancer effect expand extensive research.Currently, catechu
The measurement of phenol is mainly gas-chromatography, Capillary Electrophoresis or high performance liquid chromatography etc..Such method high sensitivity, stability
It is good, but the time-consuming and laborious and equipment of detection is expensive, complicated for operation, detection limit is still higher.Therefore, simplicity, high sensitivity, inspection are developed
The trace detection method of the low catechol of rising limit is of great significance for the availability of research catechol in vivo.
Tyrosinase (or polyphenol oxidase) is a kind of copper-containing metal enzyme, is widely present in animal, plant and microbial body
It is interior, it is the dominant catalyst that melanin is formed.It has double-function catalyzing effect, can be catalyzed the single phenol that ortho position is free of substituent group
The hydroxylating of class or polyphenols, and the dehydrogenation reaction of catechol can be catalyzed.In analysis field, it is based on tyrosine
The catalytic mechanism of enzyme, tyrosinase are often used to prepare electrochemica biological sensor and detect to extensive phenolic compound.
Therefore, in terms of environment pollution detection, food composition detection, health care, tyrosinase biosensor has wide
Wealthy application prospect and commercial applications potentiality.Although currently based on tyrosinase electrochemica biological sensor research work not
The problems such as few, but it cannot handle complicated actual constituent detection, detection performance is not high and unstable, still needs to solve.
MXene material is a kind of novel two-dimensional layer material, is carved in a solution of hydrofluoric acid by lamellar compound MAX material
Erosion processing is made.Wherein, M is transition metal element, and A III, IV major element, X is C or N element.Because having and graphene phase
As two-dimensional structure and name MXene.MXene material has high-specific surface area, high conductivity and chemical stability, has been reported
For fields such as biosensor and pharmaceutical carriers, it to be used for the electronics conduction that modified biological sensor electrode not only improves enzyme
Property, its detection limit and sensitivity are improved, the stability of enzyme can be also improved, there are huge applications potentiality.The existing miscellaneous original of document report
Son doping (N, S etc.) MXene can be further improved its electric conductivity and specific surface area.
Summary of the invention
The purpose of the present invention is overcoming above-mentioned shortcoming, a kind of tyrosinase of phosphorus doping MXene modified electrode is provided
Biosensor and preparation method and application, the tyrosinase biosensor is good with selectivity, catalytic activity is high, anti-
The advantages that speed is fast, the range of linearity is wide, Monitoring lower-cut is low, storage stability is good is answered, trace analysis can be carried out to catechol.It is made
Preparation Method is simple, it is at low cost, can on-line checking, be suitable for application in food, biological monitoring.
Technical solution of the present invention, a kind of tyrosinase biosensor based on phosphorus doping MXene modified electrode are described
Biosensor includes a glass-carbon electrode, and the glass-carbon electrode is surface modification tyrosinase/phosphorus doping MXene/ chitosan
Glass-carbon electrode.
The present invention also provides a kind of systems of above-mentioned tyrosinase biosensor based on phosphorus doping MXene modified electrode
Preparation Method, comprising the following steps:
(1) it configures tyrosinase solution: the use of concentration being the phosphate buffer solution that 1 ~ 100mmol/L, pH are 3.5 ~ 9.5, configuration
Concentration is the tyrosinase solution of 1 ~ 10 mg/mL；
(2) it configures phosphorus doping MXene solution: the use of water being solvent, configuration concentration is the MXene solution of 1 ~ 10mg/mL, ultrasound
0.5h or more；
(3) configure chitosan solution: using water be solvent, configuration concentration be 1 ~ 10mg/mL chitosan solution, ultrasonic 0.5h with
On；
(4) it prepares tyrosinase/phosphorus doping MXene/ chitosan mixed solution: step (1), step (2) and step (3) is made
Standby acquired solution mixes, and hybrid mode is stirring or ultrasound, and incorporation time is 10min or more, tyrosine in final mixed solution
Enzyme concentration is 1 ~ 10mg/mL, and phosphorus doping MXene concentration is 0.1 ~ 10mg/mL, and chitosan concentration is 1 ~ 10mg/mL.
(5) it prepares surface modification tyrosinase/phosphorus doping MXene/ chitosan glass-carbon electrode: taking that 2 ~ 20 μ L's is above-mentioned
(4) tyrosinase/phosphorus doping MXene/ chitosan mixed solution in is dripped in polished glassy carbon electrode surface, nitrogen
It is dry, obtain tyrosinase biosensor.
Further, the atom doped content of P elements is the 0.1% of total atom number in phosphorus doping MXene described in step (2)
~10%。
Further, phosphorus doping MXene preparation step described in the step (2) is as follows: by MXene material and it is phosphorous before
Body mixing is driven, grinding is placed in protection gas and is heat-treated, phosphorus doping MXene material is made.
In the above preparation method, it is preferable that the temperature of the heat treatment is 300 ~ 1000 DEG C, and the time is 1 ~ 12h.
In the above preparation method, it is preferable that the time of the grinding is 0.1 ~ 2h.
In the above preparation method, the protection gas bag includes N2Or inert gas, such as Ar；Protect gas flow velocity be 20 ~
200mL/min。
In the above preparation method, it is preferable that the phosphorous presoma includes one in triphenylphosphine, phosphoric acid or phosphate
Kind.
In the above preparation method, it is preferable that the mass ratio of the MXene material and the phosphorous presoma is (0.02
~ 1): 1；It is highly preferred that the mass ratio of the MXene material and the presoma is (0.1 ~ 1): 1.
In the above preparation method, it is preferable that further include removing remaining phosphorous presoma after obtained phosphorus doping MXene material
The step of.Specially remaining presoma is removed with deionized water or ethanol washing.
In the above preparation method, it is preferable that the MXene material includes Ti3C2、Ti2C and Ti3One of CN or several
The combination of kind.MXene material is by MAX phase material by being made with the etching reaction of HF solution, the two dimension with class graphene
Layer structure material.
The present invention also provides the applications of the tyrosinase biosensor of the phosphorus doping MXene modified electrode, by it
Applied to catechol, the i.e. detection of catechol；
Specific step is as follows: using the tyrosinase biosensor as working electrode, Ag/AgCl is reference electrode, platinum
Electrode is that electrode is established three-electrode system, the three-electrode system connect with electrochemical workstation, by the inspection of working electrode
It surveys end to be placed in solution to be measured, reduction current when being electrochemically reacted in solution to be measured is detected by electrochemical workstation
Size can qualitatively or quantitatively measure then according to the equation of linear regression of the concentration of catechol and reduction current variation
Phenol or catechol in solution to be measured；
The equation of linear regression of the catechol concentration and reduction current variation are as follows:
P=1.9743×10-7+0.4584C；Wherein, P is current variation value when catechol detects, unit A；C is to be measured
The concentration value of catechol in solution, unit mol/L；The linear detection range of the catechol is 1.0 × 10-7~3.6×
10-5Mol/L, Monitoring lower-cut 5nmol/L.
In above-mentioned application, it is 4 ~ 9 that the three-electrode system, which detects the electrolyte preferable ph used when solution to be measured,
Phosphate buffer solution.
Beneficial effects of the present invention: the tyrosinase biosensor of the invention based on phosphorus doping MXene modified electrode
It is low in cost, production is simple, stability is good, be suitble to large-scale production.Use phosphorus doping MXene as the carrier of tyrosinase, no
It is only capable of improving electric conductivity, improves the transfer velocity of electronics between biosensor and solution to be measured, stable sound can be quickly obtained
Electric current is answered, the load capacity of enzyme is also added, keeps the activity of enzyme, making tyrosinase biosensor, the service life is longer, junket ammonia
Phytase activity is higher, substantially increases the stability of biosensor, the reliability of repeatability and sensor structure, improves existing
There is the detection level of biosensor.
Detailed description of the invention
Fig. 1 is the stereoscan photograph of the phosphorus doping MXene in embodiment 1.
Fig. 2 is the corresponding current signal response curve of the different solubility catechols of biosensor dropwise addition in embodiment 2.
Fig. 3 is the relation curve of the catechol concentration that Fig. 2 curve calculates in embodiment 2 and current signal.
Specific embodiment
A kind of tyrosinase biosensor of the phosphorus doping MXene modified electrode of embodiment 1
A kind of tyrosinase biosensor of the invention based on phosphorus doping MXene modified electrode, the biosensor packet
A glass-carbon electrode is included, the glass-carbon electrode is surface modification tyrosinase/phosphorus doping MXene/ chitosan glass-carbon electrode.Specifically
Preparation step is as follows:
(1) preparation of phosphorus doping graphene: 1g Ti is taken3C2Material and 1g triphenylphosphine, which are put into mortar, grinds 30min, then
It is put into tube furnace, is passed through Ar gas, controlling its flow velocity is 100mL/min, then heats to 800 DEG C of heat treatment 2h, subsequent tube furnace
It is down to room temperature, takes out sample, is cleaned for several times and is dried with deionized water, obtain phosphorus doping Ti3C2Material.The present embodiment is made
Phosphorus doping Ti3C2Material is characterized, as shown in the scanning electron microscope (SEM) photograph of Fig. 1, phosphorus doping Ti made from the present embodiment3C2Material
For layer structure, the atom doped content of P elements is 1.32%, specific surface area 49m2/g。
(2) preparation of tyrosinase biosensor: being the phosphate buffer solution that 10mmol/L, pH are 6.0 using concentration,
Configuration concentration is the tyrosinase solution of 10mg/mL, ultrasonic 10min；Configuration concentration is the water of the phosphorus doping MXene of 10mg/mL
Solution, ultrasonic 30min；Configuration concentration is 1 ~ 10mg/mL chitosan aqueous solution, ultrasonic 30min；Above-mentioned three kinds of solution is mixed,
It is made into that tyrosinase concentration is 2.5mg/mL, phosphorus doping MXene concentration is 0.5mg/mL and chitosan concentration is 1.5mg/mL's
Mixed solution；Above-mentioned tyrosinase/phosphorus doping MXene/ chitosan mixed solution of 10 μ L is taken, is dripped in successively with 1 μ
M, the glassy carbon electrode surface of 0.3 μm and 0.05 μm of aluminum oxide powder polishing, the dry 4h of nitrogen, it is raw to obtain tyrosinase
Object sensor, it is spare under the conditions of being placed in 4 DEG C.
The application of the tyrosinase biosensor of phosphorus doping MXene modified electrode described in embodiment 2
Detection process is as follows: the tyrosinase biosensor based on phosphorus doping MXene modified electrode of above-described embodiment 1 is made
For working electrode, Ag/AgCl electrode is used as reference electrode, platinum electrode to electrode, three-electrode system is established, by three electrode systems
System is connect with electrochemical workstation, is detected to the catechol concentration in solution to be measured.
The testing principle of tyrosinase biosensor of the invention are as follows: tyrosinase catalysis aoxidizes catechol into adjacent benzene
Diquinone, adjacent benzene diquinone obtains electronics on the electrode, and is reduced into catechol, is quantified by detecting the size of reduction current
Indicate the concentration of catechol in prepare liquid.
The main reaction formula of detection process of the present invention is as follows:
C6H6O2(catechol)+tyrosinase (oxidized form) → C6H4O2(adjacent benzene diquinone)+2H++2e-→ C6H6O2(adjacent benzene two
Phenol)+tyrosinase (reduced form)+O2→ tyrosinase (oxidized form)+H2O
Catechol concentration is measured using chronoamperometry (i-t), the phosphate that 50mmol/L, pH=6.0 are added in measuring cell is slow
It rushes solution (PBS), a magnetic stir bar is placed in the inside, and the electric current for detecting various concentration substrate (catechol concentration) is corresponding
Value, it is as a result as shown in Figures 2 and 3 respectively.
As seen from Figure 3, the equation of linear regression of catechol concentration and reduction current variation are as follows:
P=1.9743×10-7+ 0.4584C (1)；
In formula (1), P is current variation value when catechol detects, unit A；C is the concentration of catechol in solution to be measured
Value, unit mol/L；The linear detection range of the catechol is 1.0 × 10-7 ~3.6×10-5Mol/L, Monitoring lower-cut
For 5nmol/L.
The above is only a preferred embodiment of the present invention, protection scope of the present invention is not limited merely to above-mentioned implementation
Example.All technical solutions belonged under thinking of the present invention all belong to the scope of protection of the present invention.It is noted that for the art
Those of ordinary skill for, improvements and modifications without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of tyrosinase biosensor of phosphorus doping MXene modified electrode, it is characterised in that: including glass-carbon electrode；Institute
Stating glass-carbon electrode is surface modification tyrosinase/phosphorus doping MXene/ chitosan glass-carbon electrode.
2. the preparation method of the tyrosinase biosensor of phosphorus doping MXene modified electrode according to claim 1, special
Sign is that steps are as follows:
(1) it configures tyrosinase solution: the use of concentration being the phosphate buffer solution that 1 ~ 100mmol/L, pH are 3.5 ~ 9.5, configuration
Concentration is the tyrosinase solution of 1 ~ 10mg/mL；
(2) it configures phosphorus doping MXene solution: the use of water being solvent, configuration concentration is the MXene solution of 1 ~ 10mg/mL, super to it
0.5 h or more of sound；
(3) it configures chitosan solution: the use of water being solvent, configuration concentration is the chitosan solution of 1 ~ 10mg/mL, to its ultrasound
0.5 h or more；
(4) it prepares tyrosinase/phosphorus doping MXene/ chitosan mixed solution: step (1), step (2) and step (3) is made
Standby acquired solution mixes, and hybrid mode is stirring or ultrasound, and incorporation time is 10 min or more, tyrosine in final mixed solution
Enzyme concentration is 1 ~ 10mg/mL, and phosphorus doping MXene concentration is 0.1 ~ 10mg/mL, and chitosan concentration is 1 ~ 10mg/mL；
(5) surface modification tyrosinase/phosphorus doping MXene/ chitosan glass-carbon electrode is prepared: in the step of taking 2 ~ 20 μ L (4)
Tyrosinase/phosphorus doping MXene/ chitosan mixed solution, for drop in polished glassy carbon electrode surface, nitrogen is dry,
Obtain tyrosinase biosensor.
3. the tyrosinase biosensor of phosphorus doping MXene modified electrode according to claim 2, it is characterised in that: step
Suddenly in (2), the atom doped content of P elements is the 0.1% ~ 10% of total atom number in the phosphorus doping MXene.
4. the tyrosinase biosensor of phosphorus doping MXene modified electrode according to claim 2, it is characterised in that: step
Suddenly the preparation of phosphorus doping MXene described in (2) is as follows: MXene material being mixed with phosphorous presoma, is ground, is placed in protection gas
It is heat-treated, phosphorus doping MXene material is made.
5. the tyrosinase biosensor of phosphorus doping MXene modified electrode according to claim 4, it is characterised in that described
Phosphorus doping MXene prepares specific as follows: MXene material and phosphorous presoma 0.02 ~ 1:1 in mass ratio being sufficiently mixed, then
0.1 ~ 2h is ground, is heat-treated 1 ~ 12h at 300 ~ 1000 DEG C in inert gas shielding device, protecting the flow velocity of gas is 20 ~ 200mL/
Phosphorus doping MXene material is made in min.
6. the tyrosinase biosensor of phosphorus doping MXene modified electrode according to claim 5, it is characterised in that: institute
MXene material is stated to mix with phosphorous presoma 0.1 ~ 1:1 in mass ratio.
7. the tyrosinase biosensor of phosphorus doping MXene modified electrode according to claim 5, it is characterised in that: institute
The preparation for stating phosphorus doping MXene further includes the steps that removing remaining phosphorous presoma；Specially use deionized water or ethanol washing
Remove remaining phosphorous presoma.
8. the tyrosinase biosensor of phosphorus doping MXene modified electrode according to claim 5, it is characterised in that: institute
Stating phosphorous presoma is specially one of triphenylphosphine, phosphoric acid or phosphate；
The MXene material includes Ti3C2、Ti2C and Ti3The combination of one or more of CN；Further, the MXene material
Material is by MAX phase material by being made with the etching reaction of HF solution, the material of the two-dimensional layered structure with class graphene.
9. the application of the tyrosinase biosensor of phosphorus doping MXene modified electrode described in claim 1, it is characterised in that:
It is applied to catechol, the i.e. detection of catechol；Specific step is as follows: using the tyrosinase biosensor as
Working electrode, Ag/AgCl are reference electrode, and platinum electrode is to establish three-electrode system to electrode, by the three-electrode system and electricity
Chem workstation connection, the test side of working electrode is placed in solution to be measured, is detected by electrochemical workstation to be measured molten
Then reduction current size when being electrochemically reacted in liquid changes according to the concentration of catechol with reduction current linear
Regression equation measures the catechol in solution to be measured qualitatively or quantitatively.
10. the application of the tyrosinase biosensor of phosphorus doping MXene modified electrode according to claim 9, feature
It is the equation of linear regression of the catechol concentration and reduction current variation specifically: P=1.9743 × 10-7+0.4584C；
Wherein, P is current variation value when catechol detects, unit A；C is the concentration value of catechol in solution to be measured, unit
For mol/L；
The linear detection range of the catechol is 1.0 × 10-7~3.6×10-5Mol/L, Monitoring lower-cut 5nmol/L；
The three-electrode system detects the phosphate buffer solution that the electrolyte preferable ph used when solution to be measured is 4 ~ 9.
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