CN109797186A - A kind of bacterial laccase sensor and its application based on Direct Electrochemistry - Google Patents
A kind of bacterial laccase sensor and its application based on Direct Electrochemistry Download PDFInfo
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- CN109797186A CN109797186A CN201910009422.8A CN201910009422A CN109797186A CN 109797186 A CN109797186 A CN 109797186A CN 201910009422 A CN201910009422 A CN 201910009422A CN 109797186 A CN109797186 A CN 109797186A
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Abstract
A kind of preparation method of the bacterial laccase sensor based on Direct Electrochemistry, which is characterized in that comprise the following steps: S1: bacterial laccase CotA expression obtains；S2: the building of Direct Electrochemistry type bacterial laccase sensing electrode.The present invention detects bilirubin in the presence of meta-alkalescence condition and a certain concentration sodium salt, not only compensates for traditional sensors to the detection higher defect of environmental requirement, also provides new tool for Future Development bilirubin detecting line sensor.
Description
Technical field
The present invention designs bio-sensing field, more specifically a kind of bacterial laccase sensor based on Direct Electrochemistry and its
Using.
Background technique
Bilirubin (bilirubin) is the primary pigments in a kind of human bile, as the main of internal ferriporphyrin compound
Metabolite, have toxicity, brain and nervous system can be caused the important indicator of irreversible damage and liver function it
One, it is the important evidence for clinically determining jaundice.
General bilirubin detection using heavy nitrogen, vanadic acid oxidation method, enzymatic measurement etc., these methods handle sample it is cumbersome,
Detection accuracy is inadequate, test result is affected by many factors larger causes result less reliable etc..Therefore, in bilirubin detection side
Face possesses response quickly, the electrochemical sensor for the features such as detection accuracy is high is by numerous studies, development and application.However, using
Although the sensing electrode of bilirubin oxidase is sensitive to bilirubin response, specificity is high, and detection environmental requirement is higher, is limited by
The neutral or limitation such as alkalinity and high-concentration chlorine ion.
Therefore, this field need to develop a kind of bacterial laccase sensor based on Direct Electrochemistry, can be realized red to gallbladder
Element measures higher accuracy, while overcoming detection environmental requirement higher, is limited by neutral or alkaline and high-concentration chlorine ion
Limitation.
Summary of the invention
In order to solve the above-mentioned technical problem, the first aspect of the present invention provides a kind of bacterium paint based on Direct Electrochemistry
The preparation method of enzyme sensor, comprises the following steps:
S1: bacterial laccase CotA expression obtains；
S2: the building of Direct Electrochemistry type bacterial laccase sensing electrode.
As a kind of perferred technical scheme, the expression of the bacterial laccase CotA obtains, and comprises the following steps:
A1: carrying out full genome extraction to bacterial laccase, obtains mesh using PCR amplification of the design primer to target gene CotA
Gene order；
A2: construction of expression vector is connect with the carrier that sets out to A1 target gene obtained using a cloning process；
A3: the genetic engineering bacterium containing CotA gene, high efficient expression bacterial laccase gene are constructed with host strain；
A4: recombination bacterial laccase CotA is formed after purification using nickel ion column, concentration obtains the pure enzyme of CotA albumen.
As a kind of perferred technical scheme, the building of the Direct Electrochemistry type bacterial laccase sensing electrode, comprising such as
Lower step:
B1: gold electrode is pre-processed, and carries out electro-chemical test.After reaching electrochemistry experiment requirement, by gold electrode into
Row cleaning；
B2: the obtained gold electrode of B1 is placed in cysteine solution and is surface modified, keeps gold electrode surfaces immobilized
One layer of fine and close cysteine modified layer；
B3: the obtained gold electrode of B2 is subjected to surface and is fixed.
As a kind of perferred technical scheme, the bacterial laccase is selected from Bacillus subtilis 168.
As a kind of perferred technical scheme, the carrier that sets out is pET-28a (+), and the expression vector is pET-
cotA。
As a kind of perferred technical scheme, the host strain is E.coli BL21 (DE3), and the genetic engineering bacterium is
E.coli BL21(DE3-pET-cotA)。
As a kind of perferred technical scheme, the construction method of the E.coli BL21 (DE3-pET-cotA), comprising such as
Lower step:
C1: by pET-28a (+) recombinant plasmid transformed of the gene of CotA containing bacterial laccase to expressive host bacterium E.coliBL21
(DE3) recombinant microorganism E.coli BL21 (DE3-pET-cotA) is obtained；
C2: being transferred to the plate containing ABTS, kanamycins and IPTG for C1 recombinant microorganism obtained, and culture is chosen
Positive transformant is taken, is saved.
As a kind of perferred technical scheme, the condition of the culture is to cultivate 14-18h at 35-39 DEG C.
As a kind of perferred technical scheme, the fixed method in the surface are as follows: be placed in the obtained gold electrode of B2
In EDC/NHS reagent, 4-8h is stood at 2-6 DEG C.
The second aspect of the present invention provides a kind of bacterial laccase sensor for bilirubin detection, with above-mentioned preparation side
Method obtains.
As a kind of perferred technical scheme, the detection environment pH is 7.0-9.0.
The beneficial effects of the present invention are:
Bilirubin is detected in the presence of meta-alkalescence condition and a certain concentration sodium salt, not only compensates for traditional sensors to detection
The higher defect of environmental requirement also provides new tool for Future Development bilirubin detecting line sensor.
Detailed description of the invention
Fig. 1 is detection performance phenogram of the sensing electrode to bilirubin of building；1 represents pH=6.0；2 represent pH=
7.0；3 represent pH=8.0；4 represent pH=8.5；5 represent pH=9.0.
Fig. 2 is the response performance phenogram of sensing electrode bilirubin under condition of different pH of building.
Fig. 3 be building bacterial laccase sensing electrode from originated from fungus bilirubin oxidase electrode in the presence of different sodium salts
The response performance of bilirubin is compared；1 indicates comparative example 1, i.e. originated from fungus bilirubin oxidase electrode；2 represent embodiment 1,
That is bacterial laccase sensing electrode.
Specific embodiment
For the purpose of following detailed description, it should be understood that the present invention can be used various substitutions variation and step it is suitable
Sequence, unless specifically stated on the contrary.In addition, being indicated in the case where in addition in any operational instances or otherwise pointing out
Such as all numbers of the amount of ingredient used in description and claims should be understood in all cases by term
" about " it modifies.Therefore, unless indicated to the contrary, the numerical parameter otherwise illustrated in the following description and appended dependent claims is root
The approximation changed according to the expected performance of the invention to be obtained.It is at least not intended to for the applicable of doctrine of equivalents being limited in
In the scope of the claims, each numerical parameter should at least be given up according to the number of the effective digital of report and by the way that application is common
Enter technology to explain.
Although illustrating that broad range of numberical range and parameter of the invention are approximations, listed in specific example
Numerical value is reported as accurately as possible.However, any numerical value inherently includes the standard deviation by finding in its each self-test measurement
The certain errors necessarily led to.
In addition, it should be understood that any numberical range as described herein, which is intended to include, is included into all subranges therein.Example
Such as, the range of " 1 to 10 " is intended to include all sub- models between (and including) described minimum value 1 and the maximum value 10
It encloses, that is, there is the minimum value equal to or more than 1 and the maximum value equal to or less than 10.In specification, BOD represents bilirubin oxidation
Enzyme.
To solve the above-mentioned problems, the first aspect of the present invention provides a kind of bacterial laccase biography based on Direct Electrochemistry
The preparation method of sensor, comprises the following steps:
S1: bacterial laccase CotA expression obtains；
S2: the building of Direct Electrochemistry type bacterial laccase sensing electrode.
Bacterial laccase CotA refers to the tool active exine PROTEIN C otA gene of bacterial laccase.
In a particular embodiment, the expression of the bacterial laccase CotA obtains, and comprises the following steps:
A1: carrying out full genome extraction to bacterial laccase, obtains mesh using PCR amplification of the design primer to target gene CotA
Gene order；
A2: construction of expression vector is connect with the carrier that sets out to A1 target gene obtained using a cloning process；
A3: the genetic engineering bacterium containing CotA gene, high efficient expression bacterial laccase gene are constructed with host strain；
A4: recombination bacterial laccase CotA is formed after purification using nickel ion column, concentration obtains the pure enzyme of CotA albumen.
In the A1, the design of primer is using conventional technical means in the art.Round pcr refer to specific gene into
The external synthesis of row, and for the various genes for the purpose of detecting DNA/RNA.
In the A4, concentration is carried out using the super filter tube that molecule interception is 50kDa and obtains the pure enzyme of CotA albumen.It is described
CotA albumen alcoholase is the protein concentrated solution that concentration is 20mg/ml.
In a particular embodiment, the specific reaction condition of PCR amplification of the target gene CotA is as follows:
The PCR amplification of bacterial laccase CotA gene:
Gene order (the corresponding GenBank of CotA in the Bacillus subtilis 168 announced according to NCBI
Accession No. is BSU_06300)；
Primer cotA-28f (the AGCAAATGG of amplification CotA is devised using primer-design software Primer Premier
) and cotA-28r (CGGAGCTCGAATTCGGATCCGCTTTATGGG GTCGCGGATCCATGACACTTGAAAAATTTGTGGA
GATCAGTTATATCCAT).Using 168 full-length genome of Bacillus subtilis as template, cotA gene, PCR reaction are expanded
Condition:
Using the correctness of agarose gel electrophoresis identification PCR product, carried out after the completion of identification with gel purification kit pure
Change recycling, is placed in -20 DEG C and stores for future use.
In a preferred embodiment, the building of the Direct Electrochemistry type bacterial laccase sensing electrode includes following step
It is rapid:
The specific reaction condition of PCR amplification of the target gene CotA is as follows:
The PCR amplification of bacterial laccase CotA gene:
Gene order (the corresponding GenBank of CotA in the Bacillus subtilis 168 announced according to NCBI
Accession No. is BSU_06300)；
Primer cotA-28f (the AGCAAATGG of amplification CotA is devised using primer-design software Primer Premier
) and cotA-28r (CGGAGCTCGAATTCGGATCCGCTTTATGGG GTCGCGGATCCATGACACTTGAAAAATTTGTGGA
GATCAGTTATATCCAT).Using 168 full-length genome of Bacillus subtilis as template, cotA gene, PCR reaction are expanded
Condition:
Using the correctness of agarose gel electrophoresis identification PCR product, carried out after the completion of identification with gel purification kit pure
Change recycling, is placed in -20 DEG C and stores for future use.
In a particular embodiment, the building of the Direct Electrochemistry type bacterial laccase sensing electrode includes following step
It is rapid:
B1: gold electrode is pre-processed, and carries out electro-chemical test.After reaching electrochemistry experiment requirement, by gold electrode into
Row cleaning；
B2: the obtained gold electrode of B1 is placed in cysteine solution and is surface modified, keeps gold electrode surfaces immobilized
One layer of fine and close cysteine modified layer；
B3: the obtained gold electrode of B2 is subjected to surface and is fixed.
The electro-chemical test mode is the common test method of analytical chemistry field, such as cyclic voltammetry, electrochemistry
Impedance spectrum, chronoa mperometric plot method.
The cleaning process is clean using distilled water flushing, then successively in secondary distilled water, dehydrated alcohol, acetone, two
It is cleaned in secondary distilled water.
In a particular embodiment, the bacterial laccase is selected from Bacillus subtilis 168.
In a particular embodiment, the carrier that sets out is pET-28a (+), and the expression vector is pET-cotA.
PET-28a (+) carrier buys purple hundred Aurion of background and wins Science and Technology Ltd..
In a particular embodiment, the host strain is E.coli BL21 (DE3), and the genetic engineering bacterium is
E.coli BL21(DE3-pET-cotA).The purchase of host strain E.coli BL21 (DE3) strain has from Shanghai pool leaf biotechnology
Limit company.
In a particular embodiment, the method for used fixation is conventional technical means in the art.It does not do herein
Carefully state.
In a particular embodiment, the construction method of the E.coli BL21 (DE3-pET-cotA) includes following step
It is rapid:
C1: by pET-28a (+) recombinant plasmid transformed of the gene of CotA containing bacterial laccase to expressive host bacterium E.coli
BL21 (DE3) obtains recombinant microorganism E.coli BL21 (DE3-pET-cotA)；
C2: being transferred to the plate containing ABTS, kanamycins and IPTG for C1 recombinant microorganism obtained, and culture is chosen
Positive transformant is taken, is saved.
In a particular embodiment, the condition of the culture is to cultivate 14-18h at 35-39 DEG C；In preferred embodiment party
In formula, the condition of the culture is to cultivate 15-17h at 36-38 DEG C；In further preferred embodiment, the culture
Condition is to cultivate 16h at 37 DEG C.
In a particular embodiment, the positive transformant is the positive transformant that there is blue-green haloing on surface.
In a particular embodiment, the fixed method in the surface are as follows: the obtained gold electrode of B2 is placed in EDC/NHS
In reagent, 4-8h is stood at 2-6 DEG C；In a preferred embodiment, the fixed method in the surface are as follows: B2 is obtained
Gold electrode is placed in EDC/NHS reagent, stands 5-7h at 3-5 DEG C；In further preferred embodiment, the surface is solid
Fixed method are as follows: the obtained gold electrode of B2 is placed in EDC/NHS reagent, stands 6h at 4 DEG C.
In a particular embodiment, No. CAS of the EDC is 25952-53-8, and No. CAS of the NHS is 6606-
82-6.In EDC/NHS reagent, the weight ratio of EDC and NHS are (1-5): (5-1)；In a preferred embodiment, the EDC with
The weight ratio of NHS is 2:1.
In a particular embodiment, the plate is to contain 200mg/l ABTS, 50mg/l kanamycins and 24mg/l
The plate of IPTG.ABTS is a kind of mediator substance, and entitled 2, the 2'- of Chinese joins the bis- -3- ethyl benzo thiazole phenanthroline -6- sulfonic acid of nitrogen -.Institute
The IPTG stated is isopropylthiogalactoside.
In a particular embodiment, the building of recombinant plasmid pET28 (+)-cotA
PET-28a (+) plasmid carries out digestion with restriction enzyme BamHI, identifies its overall length.Endonuclease reaction system (20 μ
L) as follows:
Reaction condition is 37 DEG C of digestion 4h, utilizes 1.0% after 10 × Loading Buffer terminate liquid is added in system
Agarose gel electrophoresis separation, the ultraviolet plastic recovery kit specification quickly cutting glue purification and being provided referring to Beijing Zhuan Meng company
Recycle target fragment.
It is CotA full length gene by PCR product obtained in (1), needs to use at the both ends of PCR product plus plasmid fragments
It is connect in one-step cloning with plasmid, building principle is Fig. 1.Condition of contact are as follows:
The building of recombinant bacterial strain E.coli BL21/pET28 (+)-cotA
The recombinant plasmid solution of building is added to the suspension that 200mL competent cell is housed, after mixing ice bath
30min.After taking-up at 42 DEG C the accurate heat shock timing 90s of water-bath, ice bath 2min.A certain amount of LB culture medium is added, 37 DEG C,
1~2h of hatching culture under the conditions of 150r/min.The bacterium solution hatched in right amount is taken to be coated on containing 200mg/l ABTS, 50mg/l card
On the LB solid plate of that mycin and 24mg/l IPTG, 37 DEG C of culture 12h select the positive clone molecule of blue variable colour circle, warp
Sequence verification gene order is errorless.
The inducing expression and purifying of the recombinant bacteria laccase CotA:
Plate culture: bacterium solution is applied on plating medium, is placed in constant incubator, 37 DEG C of constant temperature incubation 12h.
The aerobic activation culture of strain: it after the strain for being stored in -80 DEG C of ultra low temperature freezers is thawed, is connect with 1% inoculum concentration
To in the test tube containing 5mL LB liquid medium, at 37 DEG C, 12h is cultivated under the conditions of 200r/min.
Aerobic shaking flask culture: the bacterium solution after Tube propagation 12h is inoculated by 1% inoculum concentration containing 50mLLB liquid
In the triangular flask of the 500mL specification of culture medium, at 37 DEG C, cultivated under the conditions of 200r/min.
Aerobic Fiber differentiation: with aerobic shaking flask culture, when thallus OD600 reaches 0.6 or so, IPTG is added
(isopropyl- β-D-1-thiogalactopyranpside, isopropyl-β-D-1- thiogalactoside) is extremely final concentration of
0.1mmol/L adds the CuSO4 of 0.25mM concentration, micro- aerobic induction is for 24 hours in 20 DEG C, 170r/min.
Fermentation liquid is resuspended after being centrifuged using the buffer (PBS pH5.0) of 50mL pre-cooling, and 4 DEG C, 10000rpm, 30min
Centrifugation.With ultrasonic cell disintegration method (220V, ultrasonic 3s, interval 5s, 30-50 minutes) smudge cells suspension until muddy
It is turbid to become limpid.It is spare to collect supernatant.
ReferencePrime Plus protein purification system prepares sample and purifying process:
1) by 0.22 μm of membrane filtration supernatant of the sample after ultrasonication.
2) it is switched on, illustrates installation and debugging purifying system by operating with, installation GE HisTrap FF 5mL histidine protein is pure
Change column.After cleaning, using 2mL syringe from sample loop sample introduction, instrument is run, while replacing buffer as required.
3) purifying protein is collected using 10mL centrifuge tube, collects pure protein using the retention super filter tube concentration of 50kDa molecular weight,
By albumen buffer exchange at 0.1mM pH 5.0PBS, be stored in 4 DEG C it is spare.
Above-mentioned sample detects the expression and purifying situation of CotA by 12%SDS polyacrylate hydrogel electrophoresis (SDS-PAGE),
And tentatively judge its molecular weight.
In a preferred embodiment, the building of recombinant plasmid pET28 (+)-cotA
PET-28a (+) plasmid carries out digestion with restriction enzyme BamHI, identifies its overall length.Endonuclease reaction system (20 μ
L) as follows:
Reaction condition is 37 DEG C of digestion 4h, utilizes 1.0% after 10 × Loading Buffer terminate liquid is added in system
Agarose gel electrophoresis separation, the ultraviolet plastic recovery kit specification quickly cutting glue purification and being provided referring to Beijing Zhuan Meng company
Recycle target fragment.
It is CotA full length gene by PCR product obtained in (1), needs to use at the both ends of PCR product plus plasmid fragments
It is connect in one-step cloning with plasmid, building principle is Fig. 1.Condition of contact are as follows:
The building of recombinant bacterial strain E.coli BL21/pET28 (+)-cotA
The recombinant plasmid solution of building is added to the suspension that 200mL competent cell is housed, after mixing ice bath
30min.After taking-up at 42 DEG C the accurate heat shock timing 90s of water-bath, ice bath 2min.A certain amount of LB culture medium is added, 37 DEG C,
1~2h of hatching culture under the conditions of 150r/min.The bacterium solution hatched in right amount is taken to be coated on containing 200mg/l ABTS, 50mg/l card
On the LB solid plate of that mycin and 24mg/l IPTG, 37 DEG C of culture 12h select the positive clone molecule of blue variable colour circle, warp
Sequence verification gene order is errorless.
The inducing expression and purifying of the recombinant bacteria laccase CotA:
Plate culture: bacterium solution is applied on plating medium, is placed in constant incubator, 37 DEG C of constant temperature incubation 12h.
The aerobic activation culture of strain: it after the strain for being stored in -80 DEG C of ultra low temperature freezers is thawed, is connect with 1% inoculum concentration
To in the test tube containing 5mL LB liquid medium, at 37 DEG C, 12h is cultivated under the conditions of 200r/min.
Aerobic shaking flask culture: the bacterium solution after Tube propagation 12h is inoculated by 1% inoculum concentration containing 50mLLB liquid
In the triangular flask of the 500mL specification of culture medium, at 37 DEG C, cultivated under the conditions of 200r/min.
Aerobic Fiber differentiation: with aerobic shaking flask culture, when thallus OD600 reaches 0.6 or so, IPTG is added
(isopropyl- β-D-1-thiogalactopyranpside, isopropyl-β-D-1- thiogalactoside) is extremely final concentration of
0.1mmol/L adds the CuSO4 of 0.25mM concentration, micro- aerobic induction is for 24 hours in 20 DEG C, 170r/min.
Fermentation liquid is resuspended after being centrifuged using the buffer (PBS pH5.0) of 50mL pre-cooling, and 4 DEG C, 10000rpm, 30min
Centrifugation.With ultrasonic cell disintegration method (220V, ultrasonic 3s, interval 5s, 30-50 minutes) smudge cells suspension until muddy
It is turbid to become limpid.It is spare to collect supernatant.
ReferencePrime Plus protein purification system prepares sample and purifying process:
1) by 0.22 μm of membrane filtration supernatant of the sample after ultrasonication.
2) it is switched on, illustrates installation and debugging purifying system by operating with, installation GE HisTrap FF 5mL histidine protein is pure
Change column.After cleaning, using 2mL syringe from sample loop sample introduction, instrument is run, while replacing buffer as required.
3) purifying protein is collected using 10mL centrifuge tube, collects pure protein using the retention super filter tube concentration of 50kDa molecular weight,
By albumen buffer exchange at 0.1mM pH 5.0PBS, be stored in 4 DEG C it is spare.
Above-mentioned sample detects the expression and purifying situation of CotA by 12%SDS polyacrylate hydrogel electrophoresis (SDS-PAGE),
And tentatively judge its molecular weight.
The second aspect of the present invention provides a kind of bacterial laccase sensor for bilirubin detection, using the above method
It is prepared.
In a particular embodiment, the bilirubin includes bilirubin direct, indirect bilirubin and total bilirubin.
In a particular embodiment, the detection environment pH is 7.0-9.0.
Bacterial laccase sensor of the invention can have excellent stability under alkaline condition, to realize red to gallbladder
The accurate and stable test of element.
In addition, the sensor that the present invention constructs, which has reached, carries out concentration survey to the bilirubin of low concentration under alkaline condition
Determine effect.
Illustrated below with specific embodiment.
Embodiment
Embodiment 1
The first aspect of embodiment 1 provides a kind of preparation method of bacterial laccase sensor based on Direct Electrochemistry,
It comprises the following steps:
S1: bacterial laccase CotA expression obtains；
S2: the building of Direct Electrochemistry type bacterial laccase sensing electrode；
The expression of the bacterial laccase CotA obtains, and comprises the following steps:
A1: carrying out full genome extraction to bacterial laccase, obtains mesh using PCR amplification of the design primer to target gene CotA
Gene order；
A2: construction of expression vector is connect with the carrier that sets out to A1 target gene obtained using a cloning process；
A3: the genetic engineering bacterium containing CotA gene, high efficient expression bacterial laccase gene are constructed with host strain；
A4: recombination bacterial laccase CotA is formed after purification using nickel ion column, concentration obtains the pure enzyme of CotA albumen；
The bacterial laccase is selected from Bacillus subtilis 168；The carrier that sets out is pET-28a (+), the table
It is pET-cotA up to carrier；The host strain is E.coli BL21 (DE3), and the genetic engineering bacterium is E.coli BL21
(DE3-pET-cotA)；
The specific reaction condition of PCR amplification of the target gene CotA is as follows:
The PCR amplification of bacterial laccase CotA gene:
Gene order (the corresponding GenBank of CotA in the Bacillus subtilis 168 announced according to NCBI
Accession No. is BSU_06300)；
Primer cotA-28f (the AGCAAATGG of amplification CotA is devised using primer-design software Primer Premier
) and cotA-28r GTCGCGGATCCATGACACTTGAAAAATTTGTGGA
(CGGAGCTCGAATTCGGATCCGCTTTATGGGGATCAGTTATATCCAT)。
Using 168 full-length genome of Bacillus subtilis as template, CotA gene is expanded, PCR reaction condition:
Using the correctness of agarose gel electrophoresis identification PCR product, carried out after the completion of identification with gel purification kit pure
Change recycling, is placed in -20 DEG C and stores for future use.
The building of the Direct Electrochemistry type bacterial laccase sensing electrode, comprises the following steps:
B1: gold electrode is pre-processed, and carries out electro-chemical test.After reaching electrochemistry experiment requirement, by gold electrode into
Row cleaning；
B2: the obtained gold electrode of B1 is placed in cysteine solution and is surface modified, keeps gold electrode surfaces immobilized
One layer of fine and close cysteine modified layer；
B3: the obtained gold electrode of B2 is subjected to surface and is fixed；
The construction method of the E.coli BL21 (DE3-pET-cotA), comprises the following steps:
C1: by pET-28a (+) recombinant plasmid transformed of the gene of CotA containing bacterial laccase to expressive host bacterium E.coliBL21
(DE3) recombinant microorganism E.coli BL21 (DE3-pET-cotA) is obtained；
C2: being transferred to the plate containing ABTS, kanamycins and IPTG for C1 recombinant microorganism obtained, and culture is chosen
Positive transformant is taken, is saved；
The condition of the culture is to cultivate 16h at 37 DEG C；The positive transformant is the positive that there is blue-green haloing on surface
Transformant；The fixed method in the surface are as follows: the obtained gold electrode of B2 is placed in EDC/NHS reagent, is stood at 4 DEG C
6h；The weight ratio of the EDC and NHS is 2:1；
The building of recombinant plasmid pET28 (+)-cotA
PET-28a (+) plasmid carries out digestion with restriction enzyme BamHI, identifies its overall length.Endonuclease reaction system (20 μ
L) as follows:
Reaction condition is 37 DEG C of digestion 4h, utilizes 1.0% after 10 × Loading Buffer terminate liquid is added in system
Agarose gel electrophoresis separation, the ultraviolet plastic recovery kit specification quickly cutting glue purification and being provided referring to Beijing Zhuan Meng company
Recycle target fragment.
It is CotA full length gene by PCR product obtained in (1), needs to use at the both ends of PCR product plus plasmid fragments
It is connect in one-step cloning with plasmid, building principle is Fig. 1.Condition of contact are as follows:
The building of recombinant bacterial strain E.coli BL21/pET28 (+)-cotA
The recombinant plasmid solution of building is added to the suspension that 200mL competent cell is housed, after mixing ice bath
30min.After taking-up at 42 DEG C the accurate heat shock timing 90s of water-bath, ice bath 2min.A certain amount of LB culture medium is added, 37 DEG C,
1~2h of hatching culture under the conditions of 150r/min.The bacterium solution hatched in right amount is taken to be coated on containing 200mg/l ABTS, 50mg/l card
On the LB solid plate of that mycin and 24mg/l IPTG, 37 DEG C of culture 12h select the positive clone molecule of blue variable colour circle, warp
Sequence verification gene order is errorless.
The inducing expression and purifying of the recombinant bacteria laccase CotA:
Plate culture: bacterium solution is applied on plating medium, is placed in constant incubator, 37 DEG C of constant temperature incubation 12h.
The aerobic activation culture of strain: it after the strain for being stored in -80 DEG C of ultra low temperature freezers is thawed, is connect with 1% inoculum concentration
To in the test tube containing 5mL LB liquid medium, at 37 DEG C, 12h is cultivated under the conditions of 200r/min.
Aerobic shaking flask culture: the bacterium solution after Tube propagation 12h is inoculated by 1% inoculum concentration containing 50mLLB liquid
In the triangular flask of the 500mL specification of culture medium, at 37 DEG C, cultivated under the conditions of 200r/min.
Aerobic Fiber differentiation: with aerobic shaking flask culture, when thallus OD600 reaches 0.6 or so, IPTG is added
(isopropyl- β-D-1-thiogalactopyranpside, isopropyl-β-D-1- thiogalactoside) is extremely final concentration of
0.1mmol/L adds the CuSO4 of 0.25mM concentration, micro- aerobic induction is for 24 hours in 20 DEG C, 170r/min.
Fermentation liquid is resuspended after being centrifuged using the buffer (PBS pH5.0) of 50mL pre-cooling, and 4 DEG C, 10000rpm, 30min
Centrifugation.With ultrasonic cell disintegration method (220V, ultrasonic 3s, interval 5s, 30-50 minutes) smudge cells suspension until muddy
It is turbid to become limpid.It is spare to collect supernatant.
ReferencePrime Plus protein purification system prepares sample and purifying process:
1) by 0.22 μm of membrane filtration supernatant of the sample after ultrasonication.
2) it is switched on, illustrates installation and debugging purifying system by operating with, installation GE HisTrap FF 5mL histidine protein is pure
Change column.After cleaning, using 2mL syringe from sample loop sample introduction, instrument is run, while replacing buffer as required.
3) purifying protein is collected using 10mL centrifuge tube, collects pure protein using the retention super filter tube concentration of 50kDa molecular weight,
By albumen buffer exchange at 0.1mM pH 5.0PBS, be stored in 4 DEG C it is spare.
Above-mentioned sample detects the expression and purifying situation of CotA by 12%SDS polyacrylate hydrogel electrophoresis (SDS-PAGE),
And tentatively judge its molecular weight.
The second aspect of embodiment 1 provides a kind of bacterial laccase sensor for bilirubin detection, using above-mentioned system
Preparation Method obtains.
Embodiment 2
Embodiment 2 provides a kind of preparation method of bacterial laccase sensor based on Direct Electrochemistry, includes following step
It is rapid:
S1: bacterial laccase CotA expression obtains；
S2: the building of Direct Electrochemistry type bacterial laccase sensing electrode；
The expression of the bacterial laccase CotA obtains, and comprises the following steps:
A1: carrying out full genome extraction to bacterial laccase, obtains mesh using PCR amplification of the design primer to target gene CotA
Gene order；
A2: construction of expression vector is connect with the carrier that sets out to A1 target gene obtained using a cloning process；
A3: the genetic engineering bacterium containing CotA gene, high efficient expression bacterial laccase gene are constructed with host strain；
A4: recombination bacterial laccase CotA is formed after purification using nickel ion column, concentration obtains the pure enzyme of CotA albumen；
The specific reaction condition of PCR amplification of the target gene CotA is as follows:
The PCR amplification of bacterial laccase CotA gene:
Gene order (the corresponding GenBank of CotA in the Bacillus subtilis 168 announced according to NCBI
Accession No. is BSU_06300)；
Primer cotA-28f (the AGCAAATGG of amplification CotA is devised using primer-design software Primer Premier
) and cotA-28r (CGGAGCTCGAATTCGGATCCGCTTTATGGG GTCGCGGATCCATGACACTTGAAAAATTTGTGGA
GATCAGTTATATCCAT).Using 168 full-length genome of Bacillus subtilis as template, cotA gene, PCR reaction are expanded
Condition:
Using the correctness of agarose gel electrophoresis identification PCR product, carried out after the completion of identification with gel purification kit pure
Change recycling, is placed in -20 DEG C and stores for future use.
The building of the Direct Electrochemistry type bacterial laccase sensing electrode, comprises the following steps:
B1: gold electrode is pre-processed, and carries out electro-chemical test.After reaching electrochemistry experiment requirement, by gold electrode into
Row cleaning；
B2: the obtained gold electrode of B1 is placed in cysteine solution and is surface modified, keeps gold electrode surfaces immobilized
One layer of fine and close cysteine modified layer；
B3: the obtained gold electrode of B2 is subjected to surface and is fixed；
The bacterial laccase is selected from Bacillus subtilis 168；The carrier that sets out is pET-28a (+), the table
It is pET-cotA up to carrier；The host strain is E.coli BL21 (DE3), and the genetic engineering bacterium is E.coliBL21 (DE3-
pET-cotA)；
The construction method of the E.coli BL21 (DE3-pET-cotA), comprises the following steps:
C1: by pET-28a (+) recombinant plasmid transformed of the gene of CotA containing bacterial laccase to expressive host bacterium E.coliBL21
(DE3) recombinant microorganism E.coli BL21 (DE3-pET-cotA) is obtained；
C2: being transferred to the plate containing ABTS, kanamycins and IPTG for C1 recombinant microorganism obtained, and culture is chosen
Positive transformant is taken, is saved；
The condition of the culture is to cultivate 15h at 36 DEG C；The positive transformant is the positive that there is blue-green haloing on surface
Transformant；The fixed method in the surface are as follows: the obtained gold electrode of B2 is placed in EDC/NHS reagent, is stood at 3 DEG C
5h；The weight ratio of the EDC and NHS is 2:1.
The building of recombinant plasmid pET28 (+)-cotA
PET-28a (+) plasmid carries out digestion with restriction enzyme BamHI, identifies its overall length.Endonuclease reaction system (20 μ
L) as follows:
Reaction condition is 37 DEG C of digestion 4h, utilizes 1.0% after 10 × Loading Buffer terminate liquid is added in system
Agarose gel electrophoresis separation, the ultraviolet plastic recovery kit specification quickly cutting glue purification and being provided referring to Beijing Zhuan Meng company
Recycle target fragment.
It is CotA full length gene by PCR product obtained in (1), needs to use at the both ends of PCR product plus plasmid fragments
It is connect in one-step cloning with plasmid, building principle is Fig. 1.Condition of contact are as follows:
The building of recombinant bacterial strain E.coli BL21/pET28 (+)-cotA
The recombinant plasmid solution of building is added to the suspension that 200mL competent cell is housed, after mixing ice bath
30min.After taking-up at 42 DEG C the accurate heat shock timing 90s of water-bath, ice bath 2min.A certain amount of LB culture medium is added, 37 DEG C,
1~2h of hatching culture under the conditions of 150r/min.The bacterium solution hatched in right amount is taken to be coated on containing 200mg/l ABTS, 50mg/l card
On the LB solid plate of that mycin and 24mg/l IPTG, 37 DEG C of culture 12h select the positive clone molecule of blue variable colour circle, warp
Sequence verification gene order is errorless.
The inducing expression and purifying of the recombinant bacteria laccase CotA:
Plate culture: bacterium solution is applied on plating medium, is placed in constant incubator, 37 DEG C of constant temperature incubation 12h.
The aerobic activation culture of strain: it after the strain for being stored in -80 DEG C of ultra low temperature freezers is thawed, is connect with 1% inoculum concentration
To in the test tube containing 5mL LB liquid medium, at 37 DEG C, 12h is cultivated under the conditions of 200r/min.
Aerobic shaking flask culture: the bacterium solution after Tube propagation 12h is inoculated by 1% inoculum concentration containing 50mLLB liquid
In the triangular flask of the 500mL specification of culture medium, at 37 DEG C, cultivated under the conditions of 200r/min.
Aerobic Fiber differentiation: with aerobic shaking flask culture, when thallus OD600 reaches 0.6 or so, IPTG is added
(isopropyl- β-D-1-thiogalactopyranpside, isopropyl-β-D-1- thiogalactoside) is extremely final concentration of
0.1mmol/L adds the CuSO4 of 0.25mM concentration, micro- aerobic induction is for 24 hours in 20 DEG C, 170r/min.
Fermentation liquid is resuspended after being centrifuged using the buffer (PBS pH5.0) of 50mL pre-cooling, and 4 DEG C, 10000rpm, 30min
Centrifugation.With ultrasonic cell disintegration method (220V, ultrasonic 3s, interval 5s, 30-50 minutes) smudge cells suspension until muddy
It is turbid to become limpid.It is spare to collect supernatant.
ReferencePrime Plus protein purification system prepares sample and purifying process:
1) by 0.22 μm of membrane filtration supernatant of the sample after ultrasonication.
2) it is switched on, illustrates installation and debugging purifying system by operating with, installation GE HisTrap FF 5mL histidine protein is pure
Change column.After cleaning, using 2mL syringe from sample loop sample introduction, instrument is run, while replacing buffer as required.
3) purifying protein is collected using 10mL centrifuge tube, collects pure protein using the retention super filter tube concentration of 50kDa molecular weight,
By albumen buffer exchange at 0.1mM pH 5.0PBS, be stored in 4 DEG C it is spare.
Above-mentioned sample detects the expression and purifying situation of CotA by 12%SDS polyacrylate hydrogel electrophoresis (SDS-PAGE),
And tentatively judge its molecular weight.
The second aspect of embodiment 2 provides a kind of bacterial laccase sensor for bilirubin detection, using above-mentioned system
Preparation Method obtains.
Embodiment 3
Embodiment 3 provides a kind of preparation method of bacterial laccase sensor based on Direct Electrochemistry, includes following step
It is rapid:
S1: bacterial laccase CotA expression obtains；
S2: the building of Direct Electrochemistry type bacterial laccase sensing electrode；
The expression of the bacterial laccase CotA obtains, and comprises the following steps:
A1: carrying out full genome extraction to bacterial laccase, obtains mesh using PCR amplification of the design primer to target gene CotA
Gene order；
A2: construction of expression vector is connect with the carrier that sets out to A1 target gene obtained using a cloning process；
A3: the genetic engineering bacterium containing CotA gene, high efficient expression bacterial laccase gene are constructed with host strain；
A4: recombination bacterial laccase CotA is formed after purification using nickel ion column, concentration obtains the pure enzyme of CotA albumen；
The bacterial laccase is selected from Bacillus subtilis 168；The carrier that sets out is pET-28a (+), the table
It is pET-cotA up to carrier；The host strain is E.coli BL21 (DE3), and the genetic engineering bacterium is E.coliBL21 (DE3-
pET-cotA)；
The specific reaction condition of PCR amplification of the target gene CotA is as follows:
The PCR amplification of bacterial laccase CotA gene:
Gene order (the corresponding GenBank of CotA in the Bacillus subtilis 168 announced according to NCBI
Accession No. is BSU_06300)；
Primer cotA-28f (the AGCAAATGG of amplification CotA is devised using primer-design software Primer Premier
) and cotA-28r (CGGAGCTCGAATTCGGATCCGCTTTATGGG GTCGCGGATCCATGACACTTGAAAAATTTGTGGA
GATCAGTTATATCCAT).Using 168 full-length genome of Bacillus subtilis as template, cotA gene, PCR reaction are expanded
Condition:
Using the correctness of agarose gel electrophoresis identification PCR product, carried out after the completion of identification with gel purification kit pure
Change recycling, is placed in -20 DEG C and stores for future use.
The building of the Direct Electrochemistry type bacterial laccase sensing electrode, comprises the following steps:
B1: gold electrode is pre-processed, and carries out electro-chemical test.After reaching electrochemistry experiment requirement, by gold electrode into
Row cleaning；
B2: the obtained gold electrode of B1 is placed in cysteine solution and is surface modified, keeps gold electrode surfaces immobilized
One layer of fine and close cysteine modified layer；
B3: the obtained gold electrode of B2 is subjected to surface and is fixed；
The construction method of the E.coli BL21 (DE3-pET-cotA), comprises the following steps:
C1: by pET-28a (+) recombinant plasmid transformed of the gene of CotA containing bacterial laccase to expressive host bacterium E.coliBL21
(DE3) recombinant microorganism E.coli BL21 (DE3-pET-cotA) is obtained；
C2: being transferred to the plate containing ABTS, kanamycins and IPTG for C1 recombinant microorganism obtained, and culture is chosen
Positive transformant is taken, is saved；
The condition of the culture is to cultivate 16h at 37 DEG C；The positive transformant is the positive that there is blue-green haloing on surface
Transformant；The fixed method in the surface are as follows: the obtained gold electrode of B2 is placed in EDC/NHS reagent, is stood at 4 DEG C
6h；The weight ratio of the EDC and NHS is 2:1；
The building of recombinant plasmid pET28 (+)-cotA
PET-28a (+) plasmid carries out digestion with restriction enzyme BamHI, identifies its overall length.Endonuclease reaction system (20 μ
L) as follows:
Reaction condition is 37 DEG C of digestion 4h, utilizes 1.0% after 10 × Loading Buffer terminate liquid is added in system
Agarose gel electrophoresis separation, the ultraviolet plastic recovery kit specification quickly cutting glue purification and being provided referring to Beijing Zhuan Meng company
Recycle target fragment.
It is CotA full length gene by PCR product obtained in (1), needs to use at the both ends of PCR product plus plasmid fragments
It is connect in one-step cloning with plasmid, building principle is Fig. 1.Condition of contact are as follows:
The building of recombinant bacterial strain E.coli BL21/pET28 (+)-cotA
The recombinant plasmid solution of building is added to the suspension that 200mL competent cell is housed, after mixing ice bath
30min.After taking-up at 42 DEG C the accurate heat shock timing 90s of water-bath, ice bath 2min.A certain amount of LB culture medium is added, 37 DEG C,
1~2h of hatching culture under the conditions of 150r/min.The bacterium solution hatched in right amount is taken to be coated on containing 200mg/l ABTS, 50mg/l card
On the LB solid plate of that mycin and 24mg/l IPTG, 37 DEG C of culture 12h select the positive clone molecule of blue variable colour circle, warp
Sequence verification gene order is errorless.
The inducing expression and purifying of the recombinant bacteria laccase CotA:
Plate culture: bacterium solution is applied on plating medium, is placed in constant incubator, 37 DEG C of constant temperature incubation 12h.
The aerobic activation culture of strain: it after the strain for being stored in -80 DEG C of ultra low temperature freezers is thawed, is connect with 1% inoculum concentration
To in the test tube containing 5mL LB liquid medium, at 37 DEG C, 12h is cultivated under the conditions of 200r/min.
Aerobic shaking flask culture: the bacterium solution after Tube propagation 12h is inoculated by 1% inoculum concentration containing 50mLLB liquid
In the triangular flask of the 500mL specification of culture medium, at 37 DEG C, cultivated under the conditions of 200r/min.
Aerobic Fiber differentiation: with aerobic shaking flask culture, when thallus OD600 reaches 0.6 or so, IPTG is added
(isopropyl- β-D-1-thiogalactopyranpside, isopropyl-β-D-1- thiogalactoside) is extremely final concentration of
0.1mmol/L adds the CuSO4 of 0.25mM concentration, micro- aerobic induction is for 24 hours in 20 DEG C, 170r/min.
Fermentation liquid is resuspended after being centrifuged using the buffer (PBS pH5.0) of 50mL pre-cooling, and 4 DEG C, 10000rpm, 30min
Centrifugation.With ultrasonic cell disintegration method (220V, ultrasonic 3s, interval 5s, 30-50 minutes) smudge cells suspension until muddy
It is turbid to become limpid.It is spare to collect supernatant.
ReferencePrime Plus protein purification system prepares sample and purifying process:
1) by 0.22 μm of membrane filtration supernatant of the sample after ultrasonication.
2) it is switched on, illustrates installation and debugging purifying system by operating with, installation GE HisTrap FF 5mL histidine protein is pure
Change column.After cleaning, using 2mL syringe from sample loop sample introduction, instrument is run, while replacing buffer as required.
3) purifying protein is collected using 10mL centrifuge tube, collects pure protein using the retention super filter tube concentration of 50kDa molecular weight,
By albumen buffer exchange at 0.1mM pH 5.0PBS, be stored in 4 DEG C it is spare.
Above-mentioned sample detects the expression and purifying situation of CotA by 12%SDS polyacrylate hydrogel electrophoresis (SDS-PAGE),
And tentatively judge its molecular weight.
The second aspect of embodiment 3 provides a kind of bacterial laccase sensor for bilirubin detection, using above-mentioned system
Preparation Method obtains.
Comparative example 1
Comparative example 1 provides a kind of pale red test method, and used is conventional prior, fungi red pigment oxidizing ferment
Sensing electrode.
Performance test evaluation
1) bacterial laccase sensing electrode under alkaline condition evaluates and tests the detection performance of bilirubin
Firstly, studying CotA using the preparation-obtained bacterial laccase sensor based on Direct Electrochemistry of embodiment 1
Electrochemical response performance to bilirubin of the Direct Electrochemistry type electrode of modification under condition of different pH, uses cyclic voltammetry
The performance of electrode response a certain concentration bilirubin is characterized.As can be seen from Figure 1, pH is obvious to electrode response effect of signals,
Wherein, pH partial neutral be 6,7 when, electrode to bilirubin almost without response performance, this is because bilirubin can only be dissolved in alcohols
In equal organic reagents or meta-alkalescence solution, precipitating is formed under the conditions of neutral or slant acidity, the CotA zymoprotein on electrode cannot
Oxidation catalysis is carried out to it, causes to be formed almost without response current, to only embody reduction of the Direct Electrochemistry to oxygen
Journey.And when pH is 8, bilirubin, which dissolves, to form bilirubin solution, and electrode is that one reduction peak of 0.45V or so formation is in current potential
The electric current of about 3 μ A sizes, and the reduction peak current of oxygen is accordingly reduced.This is because the oxidation process of bilirubin consumes
Oxygen of the part in electrode surface.8.5 are risen to pH value of solution, electrode illustrates the good response energy to bilirubin
Power and further decline along with oxygen reduction peak current.Therefore, it is 8.5 electricity as electrode detection bilirubin that pH, which may be selected,
Pressure value.And when pH arrival 9.0, electrode declines the oxidability of bilirubin, and is significantly reduced to the reducing power of oxygen,
This may be due to compared to pH8.5, and CotA albumen performance in pH 9.0 is suppressed, either directly to the reduction of oxygen also
It is that oxidation to bilirubin is affected, and detects change of the current potential with pH also to off-centring.
2) the Direct Electrochemistry characterization of bacterial laccase sensing electrode
The preparation-obtained bacterial laccase sensor based on Direct Electrochemistry of method provided using embodiment 1 is red to gallbladder
Element is detected.As can be seen from Figure 2, after the bilirubin of 10 μm of ol being added in setting detection architecture of the detection voltage as 0.45V, electricity
Electrode current does not obvious response to.Until response current when concentration is 40 μm of ol, which is added, begins with display, but response current is smaller.Through
Measurement, the range of linearity of this electrode is that 80 μm of ol to 640 μm of ol sensitivity are 3.2 μ A/mmol, and detection is limited to 9.5 μm of ol/L.
3) bacterial laccase sensing electrode and fungi bilirubin oxidase sensing electrode are in the presence of sodium salt to the inspection of bilirubin
Performance is surveyed to compare:
By the fungi bilirubin oxidase sensor in the bacterial laccase sensor obtained of embodiment 1 and comparative example 1 into
Row bilirubin detection performance compares.
In nearly human body environment, chloride ion is generally existing and concentration is 100mmol or so, to still can in such environment
Detection function is enough played, the stability of the biological elements enzyme molecule itself on bioelectrode surface is even more important.In the studies above
CotA albumen and bilirubin oxidase are compared to the tolerance of chloride ion.On the electrode, m- current scanning pair when we use
There are environment stability inferiors to be characterized in various concentration chloride ion for electrode.From figure 3, it can be seen that with continuous addition sodium chloride
Into detection environment, CotA modified electrode can still keep detection to stablize under 200mmol/L concentration, or even in higher concentration
Electrode can still retain 40% or more detection performance in (1mol/L), this and the chloride ion tolerance studies phase before in relation to CotA
Symbol.And in contrast, fungi bilirubin oxidase modified electrode is impacted more serious, reaches 100mmol in chlorine ion concentration
When electrode performance be just disturbed, cannot bring into normal play detection level.
Claims (10)
1. a kind of preparation method of the bacterial laccase sensor based on Direct Electrochemistry, which is characterized in that comprise the following steps:
S1: bacterial laccase CotA expression obtains；
S2: the building of Direct Electrochemistry type bacterial laccase sensing electrode.
2. the preparation method of bacterial laccase sensor described in claim 1, which is characterized in that the table of the bacterial laccase CotA
Up to acquisition, comprise the following steps:
A1: carrying out full genome extraction to bacterial laccase, obtains purpose base using PCR amplification of the design primer to target gene CotA
Because of sequence；
A2: construction of expression vector is connect with the carrier that sets out to A1 target gene obtained using a cloning process；
A3: the genetic engineering bacterium containing CotA gene, high efficient expression bacterial laccase gene are constructed with host strain；
A4: recombination bacterial laccase CotA is formed after purification using nickel ion column, concentration obtains the pure enzyme of CotA albumen.
3. the preparation method of bacterial laccase sensor described in claim 1, which is characterized in that the Direct Electrochemistry type bacterium
The building of laccase sensing electrode, comprises the following steps:
B1: gold electrode is pre-processed, and is cleaned；
B2: the obtained gold electrode of B1 being placed in cysteine solution and is surface modified, and makes immobilized one layer of gold electrode surfaces
Fine and close cysteine modified layer；
B3: the obtained gold electrode of B2 is subjected to surface and is fixed.
4. the preparation method of bacterial laccase sensor as claimed in claim 2, which is characterized in that the bacterial laccase is selected from
Bacillus subtilis 168。
5. the preparation method of bacterial laccase sensor as claimed in claim 2, which is characterized in that the carrier that sets out is pET-
28a (+), the expression vector are pET-cotA.
6. the preparation method of bacterial laccase sensor as claimed in claim 2, which is characterized in that the host strain is E.coli
BL21 (DE3), the genetic engineering bacterium are E.coli BL21 (DE3-pET-cotA).
7. the preparation method of bacterial laccase sensor as claimed in claim 6, which is characterized in that the E.coli BL21 (DE3-
PET-cotA construction method), comprises the following steps:
C1: by pET-28a (+) recombinant plasmid transformed of the gene of CotA containing bacterial laccase to expressive host bacterium E.coli BL21
(DE3) recombinant microorganism E.coli BL21 (DE3-pET-cotA) is obtained；
C2: being transferred to the plate containing ABTS, kanamycins and IPTG for C1 recombinant microorganism obtained, culture, picking sun
Property transformant, save.
8. the preparation method of bacterial laccase sensor as claimed in claim 3, which is characterized in that the fixed method in the surface
Are as follows: the obtained gold electrode of B2 is placed in EDC/NHS reagent, stands 4-8h at 2-6 DEG C.
9. a kind of bacterial laccase sensor for bilirubin detection, which is characterized in that described in any item with claim 1-8
Preparation method obtains.
10. bacterial laccase sensor as claimed in claim 9, which is characterized in that detection environment pH is 7.0-9.0.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111455018A (en) * | 2020-03-03 | 2020-07-28 | 天津大学 | Direct bilirubin detection kit containing bacillus subtilis laccase |
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| CN105388203A (en) * | 2015-10-18 | 2016-03-09 | 桂林理工大学 | Method for detecting carmine concentration |
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