CN106896147A - A kind of electrochemical sensor of Rapid Determination of Plasma indoles - Google Patents
A kind of electrochemical sensor of Rapid Determination of Plasma indoles Download PDFInfo
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Abstract
The invention discloses a kind of electrochemical sensor for detecting blood plasma indoles.The present invention is by screen printing electrode surface modification multi-walled carbon nano-tubes chitosan complexes, constructing a kind of electrochemical sensor for blood plasma indoles quantitative determination.The electrochemical sensor provided in the present invention combine nano material accelerate electron transmission and Electrochemical Detection response quickly, it is easy to operate the characteristics of, according to the linear relationship between gained electrochemical signals and testing concentration, the quantitative determination of indoles concentration in blood plasma is realized.The transducer sensitivity is high, response quickly, and it is convenient to prepare, and with low cost, single use can be mass.
Description
Technical field
The invention belongs to field of electrochemical detection, and in particular to a kind of electrochemical sensor of detection blood plasma indoles.
Background technology
Indoles is product of the tryptophan through intestinal flora metabolism, is widely present in enteron aisle.It is a kind of to be currently known indoles
Signaling molecule, mainly has both sides to act in vivo.On the one hand, participate in maintaining the stable state of gut flora, control various bacteriums
Function, such as promote biofilm formation, keep the stability of plasmid, the division of regulation bacterium and grow, induced synthesis antibiotic is resistance to
By property etc.；On the other hand, participate in human body physiological function regulation, be in particular in it is following some：(1) body bacterium infection is avoided；
(2) regulation causes inflammation/anti-inflammation gene expression, strengthens gut barrier function；(3) brain-gut axis function controlling is participated in, nervous system is influenceed
Development and function are formed；(4) hormone secretion is adjusted, prevents metabolic disease.Blood plasma indoles detection method is set up, is contributed to more preferably
Ground understands physiological function of the indoles in body, and for disease mechanisms research, clinical early diagnosis, drug therapy are provided, one kind is new to be thought
Road.
At present, detect that the method for indoles concentration has in blood：
(1) chromatography.It has that separative efficiency is high, detection sensitivity is high, amount of samples is few, selectivity is good, range of application
The features such as wide, but limited instrument and equipment factor and operating personnel require etc. that limitation is difficult to be widely used in common lab.
(2) chromatograph-mass spectrometer coupling method.It combines the power of chromatogram quantification and mass spectrometry, but expensive equipment, behaviour
Make complicated and need special messenger to operate, maintaining trouble, it is impossible to which popularization is used.
(3) indole test is improved.It is acted on Paradimethylaminobenzaldehyde by indoles and taken on a red color, then by colorimetric method
It is quantitative.Selectivity, high efficiency due to the reaction, it is to avoid the interference of sample Coexisting component, but the method sensitivity is relatively low.Due to blood
Indoles content is low in slurry, therefore its inapplicable blood sample.
Electrochemical analysis method is the electrochemical properties and its Changing Pattern according to material in the solution, is set up with electricity
On the basis of quantitative relation between the electrical quantities such as position, conductance, electric current and electricity and measured object quality, component is carried out it is qualitative and
Quantitative instrument analytical method.At present, the electrochemistry both at home and abroad also not on electrochemical analysis method detection blood plasma indoles is passed
The correlative study report of the preparation method and applications of sensor.
The content of the invention
This invention address that providing a kind of electrochemical sensor for detecting blood plasma indoles, the electrochemical sensing that the present invention makes
Device combine nano material accelerate electron transmission and Electrochemical Detection it is quick, sensitive, easy the features such as, by electrochemical credit
Analysis method realizes the fast and accurately quantitative determination of blood plasma indoles.
The Cleaning Principle of electrochemical sensor of the invention is：Can be in silk-screen printing using the double bond on indoles imidazole ring
Electrode surface is oxidized, while losing electronics produces electric signal, electron transmission is accelerated by multi-walled carbon nano-tubes；Using electrochemistry
Work station detects to the oxidation peak current produced by indoles, the concentration of its oxidation peak current and indoles in test plasma sample
It is linear within the specific limits, so as to realize the direct quantitative analysis to blood plasma indoles.
It is a kind of detect blood plasma indoles electrochemical sensor, including screen printing electrode and working electrode surface compound
Modification.
Wherein, the screen printing electrode includes the PET substrates of a printed electrode, PET bases exterior insulation printed on chip
The interface of one end on layer and substrate, it is characterised in that be also printed with three electrodes on described substrate, respectively carbon working electrode,
Ag/AgCl reference electrodes and to electrode, three electrodes form a circular work regions, and each electrode is by the company under mutually insulated film
Wire is connect to be connected with interface.
Wherein, the surface of the working electrode of the screen printing electrode is covered with modified coatings, using the strong absorption of shitosan
Property, the excellent multi-walled carbon nano-tubes of electric conductivity is securely attached to electrode surface：It is molten that 0.5wt.% shitosans are prepared first
Liquid：0.5g Chitosan powders are dissolved in 100mL 1.0% (v/v) acetic acid solution, and 4 DEG C save backup；Then prepare 1mgmL-1It is many
The chitosan dispersion of wall carbon nano tube：4.0mg functionalized multi-wall carbonnanotubes are added described in 4.0mL in chitosan solution,
Ultrasonic 90min；Then the working electrode surface for printing electrode for being cleaned in pure water is added dropwise the shell of multi-walled carbon nano-tubes described in 5 μ L
Glycan dispersion liquid, spontaneously dries；Finally obtain the screen printing electrode that surface is covered with modified coatings.
A kind of electrochemical sensor of described detection blood plasma indoles, comprises the following specific steps that：
1) 300 μ L plasma samples are taken respectively and 600 μ L ether are placed in test tube, and 18min is shaked with 250rpm in shaking table,
Subsequent 13300g, 4 DEG C of centrifugation 5min, then take supernatant and are dried up in 30 DEG C of nitrogen；
2) by step 1) sample that obtains, is redissolved with 150 μ L ultra-pure waters；
3) take isometric step 2) solution and phosphate buffer be vortexed mix, obtain reaction mixture；
4) the silk-screen printing modified electrode is connected with electrochemical workstation, by step 3) the μ L of reaction mixture 50 that obtain
It is added dropwise in the working region of the electrode, the oxidation peak current to indoles is directly determined, so as to in test plasma sample
Indoles carry out quantitative analysis, its parameter is set to：Low potential 0V, high potential+1.0V, current potential increment 0.005V, amplitude 0.1V,
Pulse width 0.05s, pulse period 0.2s.
More preferably, the phosphate buffering liquid concentration is 0.1mgmL-1, pH=7.0.
The electrochemical sensor of detection blood plasma indoles of the present invention has the beneficial effect that：
1) electrochemical sensor of the present invention, electrochemical workstation is coordinated to blood plasma by using screen printing electrode
Indoles is quantitative determined, and detecting instrument is simple to operate, cheap.
2) that is used in electrochemical sensor of the present invention prints electrode, low manufacture cost, makes and modification
Simply, quickly.
3) electrochemical sensor of the present invention, is single use, has both been avoided dull to conventional solid electrode dull
Sanding and polishing, while also avoid electrode reuse cause test cross pollution.
4) when electrochemical sensor of the present invention is used to detect blood plasma indoles, it is only necessary to simple pre- place is carried out to sample
Reason, agents useful for same species is few, consumption is small, significantly reduces testing cost.
5) electrochemical sensor of the present invention, accelerates electron transmission and Electrochemical Detection response fast with reference to nano material
Fast, easy to operate the features such as, with good precision, sensitivity, the degree of accuracy higher can be used to carry out blood plasma indoles soon
Fast accurate quantitative analysis.
Brief description of the drawings
Fig. 1 is screen printing electrode structure chart of the present invention and modification schematic diagram.
1- working electrodes in figure, 2- auxiliary electrodes, 3- reference electrodes, 4- circular works region, 5-PET substrates；Dotted line frame
In for working electrode modification and Cleaning Principle.
Fig. 2 is the Electrochemical Detection response signal contrast of indoles before and after electrode modification.
Dotted line a and strigula b represent bare electrode and determine phosphate buffer and 100.0 μ gL respectively in figure-1Indoles standard
The volt-ampere curve of product, figure dotted line c and solid line d represent modified electrode and determine phosphate buffer and 100.0 μ gL respectively-1Yin
The volt-ampere curve of diindyl standard items.
Fig. 3 is the curve map that phosphate buffer pH value of the present invention influences oxidation peak current.
■ represents the oxidation peak current of blood plasma in figure.
Fig. 4 is the curve map of electrode modification liquid product influence oxidation peak current of the present invention.
V represents electrode modification liquid product in figure.
Fig. 5 is canonical plotting of the oxidation peak current increment to blood plasma indoles concentration in the present invention.
C represents blood plasma indoles concentration in figure.
Fig. 6 is the dependency graph of blood plasma indoles detection method of the present invention and high performance liquid chromatography testing result.
Specific embodiment
Only by taking CHI852C electrochemical workstations as an example, it is purchased from Shanghai Chen Hua instrument has electrochemical workstation of the present invention
Limit company.
Embodiment 1：Detect the preparation method of the electrochemical sensor of blood plasma indoles
1) screen printing electrode of the present invention is prepared：
Screen printing electrode of the present invention be printed successively on polyethylene terephthalate (PET) substrate carbon slurry,
Silver/chlorination silver paste and insulation are starched.Specifically include following steps：
1. pet substrate is cleaned, carbon slurry is printed after drying on pet substrate, working electrode and auxiliary electrode is made, normal temperature is done
It is dry；
2. the silver paste containing silver chlorate is printed on above-mentioned pet substrate, reference electrode, air drying is made；
3. circular work region is avoided, insulation slurry is printed on above-mentioned pet substrate, wire is covered；
4. above-mentioned working electrode, auxiliary electrode and reference electrode form a circular work region, and each electrode passes through dielectric film
Under wire be connected with interface,
Then in 30~40 DEG C of drying, save backup.
2) silk-screen printing modified electrode of the present invention is prepared：
1. 0.5wt.% chitosan solutions：0.5g Chitosan powders are dissolved in 100mL 1.0% (v/v) acetic acid solution, 4 DEG C of guarantors
Deposit standby；
②1mg·mL-1The chitosan dispersion of multi-walled carbon nano-tubes：4.0mg functionalized multi-wall carbonnanotubes are added
In chitosan solution described in 4.0mL, ultrasonic 90min；
3. the shell of multi-walled carbon nano-tubes gathers described in the 5 μ L of the working electrode surface for printing electrode dropwise addition after being cleaned with pure water
Sugar dispersion liquid, spontaneously dries；
4. the screen printing electrode that working electrode surface is covered with modified coatings is obtained.
3) silk-screen printing modified electrode prepared by the present invention is placed in container and is saved backup.
Embodiment 2：Detect the application method of the electrochemical sensor of blood plasma indoles
1) analytically pure ether is purchased, for plasma sample pretreatment；
2) phosphate buffer is prepared：0.1molL is prepared respectively-1Sodium dihydrogen phosphate and disodium phosphate soln, then press
Certain volume is placed in container than mixed preparing pH=7.0 phosphate buffers；
3) 300 μ L plasma samples and diethyl ether solution described in 600 μ L are taken in test tube, and is shaked in shaking table with 250rpm
18min, subsequent 13300g, 4 DEG C of centrifugation 5min, finally take supernatant nitrogen drying at 30 DEG C；
4) by the sample after treatment, redissolved with 150 μ L ultra-pure waters；
5) isometric step 3 is taken respectively) solution and phosphate buffer that obtain, it is vortexed and mixes, obtain reaction mixing
Liquid；
6) screen printing electrode is connected with CHI852C electrochemical workstations, its parameter is set to：Low potential
0V, high potential 1.0V, current potential increment 0.005V, amplitude 0.1V, pulse width 0.05s, pulse period 0.2s, by step 4) obtain
Reaction mixture be added dropwise in the working region of the electrode, the oxidation peak current to indoles is directly determined, its oxidation peak
Electric current incrementss are within the specific limits good linear relationship with the concentration of indoles in blood plasma, so as to realize to blood plasma indoles
Direct quantitative is analyzed.
Embodiment 3
The present embodiment is to investigate the screen printing electrode front and rear electrochemical response to indoles of modification.Respectively using bare electrode and
Modified electrode determines indoles standard solution, as a result sees Fig. 2.Dotted line a and strigula b represent bare electrode and determine phosphoric acid respectively in figure
Salt buffer and 100.0 μ gL-1The volt-ampere curve of indoles standard items, figure dotted line c and solid line d represent modified electrode survey respectively
Determine phosphate buffer and 100.0 μ gL-1The volt-ampere curve of indoles standard items.Result shows that the electrochemical signals of gained are Yin
Diindyl is aoxidized and produced, additionally, the indoles electrochemical response signal that modified electrode is determined is significantly improved, and the peak shape of volt-ampere curve is bright
It is aobvious to improve.
Embodiment 4
The present embodiment is the oxidation peak current for investigating phosphate buffer pH value to indoles (oxidation peak current ■ is represented)
Influence.Result is shown in Fig. 3.At 6.0~7.0, the pH value with phosphate buffer increases pH, and oxidation peak current gradually increases；
In pH=7.0, oxidation peak current is maximum；
As pH ＞ 7.0, oxidation peak current declines rapidly.The pH value of phosphate buffer be indoles detection crucial effect because
Element, therefore the pH value of phosphate buffer elects 7.0 as.
Embodiment 5
This implementation is the influence of the volume to the oxidation peak current of indoles of electrode face finish liquid of investigating.Phosphate delays
Fliud flushing pH value is 7.0, and other experiment conditions are with embodiment 4.Decorating liquid volume is investigated in the range of 3~7 μ L to the oxidation peak
The influence of electric current, is as a result shown in Fig. 4.When modification liquid product reaches 5 μ L, electrochemical response signal is maximum.Excessive decorating liquid will
Cause reagent waste and reduce oxidation peak current, therefore decorating liquid volume selects 5 μ L, prepares modified electrode.
Embodiment 6
The present embodiment is to investigate the sensor to be used for quantitative phase between blood plasma indoles concentration and oxidation peak current increment
Guan Xing.By various concentrations (5.0~100.0 μ gL-1) indoles standard solution addition blank plasma, prepare a series of blood plasma samples
Product, under optimal testing conditions, are printed electrode using modification and indoles are measured.Result shows, oxidation peak current increment
With the indoles concentration for adding in 5.0~100.0 μ gL-1In the range of be in good linear relationship (Fig. 5), regression equation be Δ I (μ
A)=0.0232c (μ gL-1)+0.3005(R2=0.9915).When signal to noise ratio is 3 (S/N=3), its lowest detection is limited to 1.0
μg·L-1, sensitivity is higher.
Embodiment 7
The present embodiment is to investigate sensor of the present invention to be used to determine the precision of blood plasma indoles, is divided in blank plasma
Not Jia Ru concentration for (5.0,
25.0、75.0)μg·L-1Indoles standard solution, in a few days replication 5 times, same sample METHOD FOR CONTINUOUS DETERMINATION 5 days,
The various concentrations indoles coefficient of variation in a few days and in the daytime is calculated respectively, precision investigation is carried out, and the results are shown in Table 1.The coefficient of variation is most
Greatly 9.0%, show that the sensor in the present invention has good precision.
The precision of table 1.
Embodiment 8
The present embodiment is to investigate sensor of the present invention to be used to determine the rate of recovery of blood plasma indoles, is divided in blank plasma
The indoles standard solution of high, medium and low concentration is not added, determines the oxidation peak current of the indoles Plasma Before And After for adding each concentration,
Carry out recovery experiment.Relative recovery=(concentration of indoles-blank plasma indoles concentration in spiked plasma)/addition indoles standard
Concentration × 100% of product, the results are shown in Table 2, and the rate of recovery shows that the present invention has preferably accurate between 92.2.%~105.3%
Exactness.
The rate of recovery of table 2.
Embodiment 9
The present embodiment is to investigate sensor of the present invention to be used to determine the antijamming capability of blood plasma indoles.Bilirubin
It is most common endogenous interfering material in plasma sample with hemoglobin.Interference experiment is clinical according to American National
The interference test guide (CLSI-EP7-A) that the laboratory standard committee (NCCLS) formulates is carried out, that is, determine and do not add indoles standard
The blood plasma indoles concentration (X of productC) and add the blood plasma indoles concentration (X after indoles standard itemsT), interference value (=XT-XC) in this hair
Be in the range of the 1.96S (i.e. 95% confidence level) of bright method without interfering significantly with (represented with N), such as interference value more than 1.96S, then for
Interfere significantly with (represented with I).3 are the results are shown in Table, when bilirubin concentration is more than 25mgL-1It is more than with HC
0.43g·L-1When interfere significantly with indoles measure.Under normal circumstances, human blood mesobilirubin and HC are far below and are somebody's turn to do
Value, shows that the sensor prepared in the present invention has preferable antijamming capability.
The interference experiment of table 3.
Embodiment 10
The present embodiment is the correlation for investigating sensor of the present invention and Plasma By Hplc indoles.Point
Not with indoles content in 24 plasma samples of electrochemical sensor of the present invention and high effective liquid chromatography for measuring, figure is as a result seen
6.Result shows, sensor of the present invention determines blood plasma indoles result, has to existing method measurement result good related
Property (r=0.886, p=0.000), illustrate sensor of the present invention for blood plasma indoles assay, it is stabilization, reliable.
Claims (10)
1. it is a kind of detect blood plasma indoles electrochemical sensor, it is characterised in that：The electrochemical sensor includes silk-screen printing
Compound of the electrode containing working electrode surface is modified.
2. electrochemical sensor according to claim 1, wherein, the screen printing electrode includes a printed electrode
The conductor interface of one end on PET substrates, PET bases external insulation printed on chip and substrate, it is characterised in that described substrate
On be also printed with three electrodes, respectively carbon working electrode, Ag/AgCl reference electrodes and to electrode, three electrodes form one and justify
Shape working region, each electrode is connected by the wire printed under dielectric film with interface.
3. electrochemical sensor according to claim 1, wherein, the compound modification, it is characterised in that what it was prepared
Step and method of modifying：
1) 0.5wt.% chitosan solutions：0.5g Chitosan powders are dissolved in 100mL 1.0% (v/v) acetic acid solution, and 4 DEG C of preservations are standby
With；
2)1mg·mL-1The chitosan dispersion of multi-walled carbon nano-tubes：4.0mg functionalized multi-wall carbonnanotubes are added into 4.0mL steps
In the rapid chitosan solution for 1) obtaining, ultrasonic 90min, 4 DEG C save backup；
3) working electrode surface that prints electrode after pure water cleaning is added dropwise 5 μ L steps 2) dispersion liquid that obtains, spontaneously dry
It is standby；
4) screen printing electrode of modified coatings must be covered with to surface.
4. a kind of electrochemical sensor that blood plasma indoles is detected as described in claim 1-3, it is characterised in that including following specific
Step：
1) 300 μ L plasma samples are taken respectively and 600 μ L ether are placed in test tube, and 18min is shaked with 250rpm in shaking table, then
13300g, 4 DEG C of centrifugation 5min, finally take supernatant and are dried up in 30 DEG C of nitrogen；
2) by step 1) sample that obtains, is redissolved with 150 μ L ultra-pure waters；
3) same volume step 2 is taken) solution that obtains and phosphate buffer be vortexed and mix, and obtains reaction mixture；
4) the silk-screen printing modified electrode is connected with electrochemical workstation, by step 3) reaction mixture that obtains is added dropwise in institute
The working region of electrode is stated, the oxidation peak current of indoles is directly determined using voltammetry, so as to test plasma sample
In indoles quantified.
5. it is according to claim 4 detection blood plasma indoles electrochemical sensor, wherein, the ether, plasma sample, surpass
The volume ratio of pure water is 2:4:1.
6. it is according to claim 4 detection blood plasma indoles electrochemical sensor, wherein, the phosphate buffering liquid concentration
It is 0.1molL-1, pH=7.0.
7. it is according to claim 4 detection blood plasma indoles electrochemical sensor, wherein, the phosphate buffer by
0.1mol·L-1Sodium dihydrogen phosphate is prepared with disodium phosphate soln.
8. the electrochemical sensor of detection blood plasma indoles according to claim 4, wherein, the step 2) solution that obtains
It is 1 with phosphate buffer volume ratio:1.
9. it is according to claim 4 detection blood plasma indoles electrochemical sensor, wherein, reaction mixture be added dropwise described
The volume of the working region of electrode is 50 μ L.
10. it is according to claim 4 detection blood plasma indoles electrochemical sensor, wherein, electrochemical workstation parameter sets
It is set to：Low potential 0V, high potential+1.0V, current potential increment 0.005V, amplitude 0.1V, pulse width 0.05s, pulse period 0.2s.
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| CN109507260A (en) * | 2018-12-29 | 2019-03-22 | 中国科学院苏州生物医学工程技术研究所 | Electrochemical Detection chip, electrochemical sensor and its preparation method and application |
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| CN110441371A (en) * | 2019-08-22 | 2019-11-12 | 重庆医科大学 | A kind of highly sensitive indoles electrochemical detection method |
| CN112198201A (en) * | 2020-07-27 | 2021-01-08 | 重庆医科大学 | Indole electrochemical sensor constructed based on nitrogen-doped ordered mesoporous carbon modified electrode and application thereof |
| CN113702463A (en) * | 2021-08-31 | 2021-11-26 | 重庆医科大学国际体外诊断研究院 | Electrochemical method for rapidly detecting indoxyl sulfate in blood plasma |
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| CN109507260A (en) * | 2018-12-29 | 2019-03-22 | 中国科学院苏州生物医学工程技术研究所 | Electrochemical Detection chip, electrochemical sensor and its preparation method and application |
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| CN112198201A (en) * | 2020-07-27 | 2021-01-08 | 重庆医科大学 | Indole electrochemical sensor constructed based on nitrogen-doped ordered mesoporous carbon modified electrode and application thereof |
| CN113702463A (en) * | 2021-08-31 | 2021-11-26 | 重庆医科大学国际体外诊断研究院 | Electrochemical method for rapidly detecting indoxyl sulfate in blood plasma |
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