CN100552450C - Detect the method for the method of agricultural chemicals, biological microsensor and reduction current noise - Google Patents
Detect the method for the method of agricultural chemicals, biological microsensor and reduction current noise Download PDFInfo
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Abstract
The present invention relates to the method for a kind of Pesticides Testing method, biological microsensor and reduction current noise.The invention provides a kind of method and biological microsensor that detects agricultural chemicals, is to utilize the electric current of the electrode detection sample of surperficial immobilized enzyme, gold nano grain and organic catalyst to change, and the data of utilizing the temperature compensation adjustment to be detected.Biological microsensor of the present invention can reduce the interference of natural material in the detection limit of biology sensor and the environment, shortens detection time simultaneously, reaches the instant purpose that detects of terrain.
Description
Technical field
The present invention relates to the method for a kind of Pesticides Testing method, biological microsensor and reduction current noise.
Background technology
In order to increase output and to keep the attractive in appearance of crops, use a large amount of agricultural chemicals usually, wherein organophosphorus pesticide (organophosphorous compounds; OPCs) owing to have the advantage of low bioaccumulation and high Biodegradable, having replaced organo-chlorine pesticide at present becomes the pesticide variety that the most generally uses.Though organophosphorus pesticide has above-mentioned advantage, under a large amount of situations about using, may cause in soil, crop, surface water and industrial waste water residually, health and physical environment are caused sizable threat.Countries in the world are noted in detail to the control of organophosphorus pesticide, formulate quite strict control test at promulgation in potable water, food and the industrial waste water.
In case after organophosphorus entered in the biosome, it can follow cholinesterase (cholineesterase; ChE) activity site forms irreversible combination, and then suppresses this enzymatic activity, delays acetylcholine (acetyl choline in the biosome; ACh) hydrolysis rate disturbs neural transmission.According to the difference of toxicity, dosage and the duration of contact of agricultural chemicals itself, the symptom that is caused comprises: tired, nauseating, drowsiness, the dimness of vision, and when serious even can be dead.
Traditionally, chemical method (comprising: high performance liquid chromatography HPLC, vapor-phase chromatography GC, gas chromatography-mass spectrography GC-Mass, inductively coupled plasma mass spectrometry method ICP-Mass etc.) is mainly adopted in the analysis of agricultural chemicals.Though traditional laboratory chemical apparatuses analytical approach can provide accurate detection to environmental samples, but pre-treatment and analytical procedure are very tediously long, in addition instrument itself cost an arm and a leg, bulky, and need to operate through the personnel of professional training, it is too high to cause detecting cost, basically and do not meet the requirement of polluting the terrain at-once monitor.For being about to the agricultural product of listing, these traditional chemical analysis methods also can't provide any help for the instant detection of agricultural chemicals with screening.
In view of this, developing one can fast screening pollutant levels, has the microsensor (Micro-sensor) of portability, easy and simple to handle and cheap characteristic simultaneously concurrently, becomes the research and development emphasis of current detection monitoring technology.
Biology sensor (biosensor) can overcome the disappearance of traditional analysis method owing to have following many advantages, and dark tool development potentiality: (1) via the application of immobilization technology, biological elements is reusable, reduces cost; (2) biological identification element selectivity height can avoid non-object to disturb; (3) operation is simple and easy; (4) highly sensitive, required sample size is low; (5) reply fast, reduce analysis time; (6) digital signal output reaches microminiaturization, and is portable, can be used for on-the-spot the detection.
Because the difference of the transmittability of signal transmission component own, but the sensing range of each biology sensor is different.The detection limit of traditional electrochemica biological sensor can only reach the ppm level, is the poorest in all signal transmission components.Yet electrochemica biological sensor is because easy and simple to handle, equipment cost and detection are with low cost, still can do accurately at the sample that color and high turbidity are arranged simultaneously and detect, therefore be still development the earliest and the most perfect, no matter all have considerable result of study to propose both at home and abroad.Why the electrochemica biological microsensor can't reach the degree of generally using at present, and main difficulty comprises: oxidizing potential is too high during (1) operation, and easily the interfering material in the simultaneous oxidation environment causes noise to produce; (2) compare other forms of biology sensor detection time for long; (3) detection limit is too high, can't do detection at the pollutant of low concentration, and application surface is too narrow.If can overcome these shortcomings, can quicken the business-like speed of this type of biology sensor, enlarge the application surface that it detects in environmental pollution simultaneously.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of method that detects agricultural chemicals, comprise a sample is provided; This sample is contacted with at least one electrode; And detect the current signal that produces, wherein this electrode surface has enzyme, gold nano grain and organic catalyst to fix on it.
In the method for detection agricultural chemicals of the present invention, this enzyme is acetylcholinesterase or choline oxidase.
In the method for detection agricultural chemicals of the present invention, this organic catalyst is Prussian blue.
In the method for detection agricultural chemicals of the present invention, this electrode surface is to fix with the electropolymerization method.
In the method for detection agricultural chemicals of the present invention, this electropolymerization fixing means is that immobilized enzyme, gold nano grain and organic catalyst are on polypyrrole layer at electrode surface polymerization one deck polypyrrole.
In the method for detection agricultural chemicals of the present invention, the concentration of this gold nano grain is 0.5-2.0ppm.
In the method for detection agricultural chemicals of the present invention, this gold nano grain particle diameter is 16.5-20nm.
In the method for detection agricultural chemicals of the present invention, this electrode is a platinum electrode.
In the method for detection agricultural chemicals of the present invention, it also comprises makes this sample contact with a temperature inductor, detects this sample temperature.
In the method for detection agricultural chemicals of the present invention, this current signal carries out temperature compensation with sample temperature to be handled.
In the method for detection agricultural chemicals of the present invention, this agricultural chemicals is organophosphorus or carbamate.
In the method for detection agricultural chemicals of the present invention, this electrode and temperature inductor are arranged on the flat board.
Another object of the present invention is to provide a kind of biological microsensor, comprising: at least one electrode, in order to sensed current signal; One processing unit in order to receive handling this current signal, and is changed this current signal and is become a concentration data; And a display unit, in order to show this concentration data; Wherein this electrode surface is fixed with enzyme, gold nano grain and organic catalyst.
In the biological microsensor of the present invention, this enzyme is acetylcholinesterase or choline oxidase.
In the biological microsensor of the present invention, this organic catalyst is Prussian blue.
In the biological microsensor of the present invention, this electrode surface is to fix with the electropolymerization method.
In the biological microsensor of the present invention, this electropolymerization fixing means is that immobilized enzyme, gold nano grain and organic catalyst are on polypyrrole layer at electrode surface polymerization one deck polypyrrole.
In the biological microsensor of the present invention, this gold nano grain concentration is 0.5-2.0ppm.
In the biological microsensor of the present invention, this gold nano grain particle diameter is 16.5-20nm.
In the biological microsensor of the present invention, this electrode is a platinum electrode.
Biological microsensor of the present invention also comprises a temperature inductor, in order to detected temperatures and be sent to this processing unit.
In the biological microsensor of the present invention, this current signal carries out temperature compensation with this detected temperatures to be handled.
Biological microsensor of the present invention is used to detect agricultural chemicals.
In the biological microsensor of the present invention, this agricultural chemicals is organophosphorus or carbamate.
In the biological microsensor of the present invention, this electrode and temperature inductor are simultaneously on a flat board.
Biological microsensor of the present invention also can comprise at least one electrode, a temperature inductor, and a data processing display instrument, and wherein this electrode surface has enzyme, gold nano grain and organic catalyst to fix on it, in order to detect agricultural chemicals.
Another object of the present invention further provides a kind of method that reduces current noise, comprises gold nano grain and organic catalyst are fixed on the fixing electrode surface of an enzyme.
In the method for reduction current noise of the present invention, this organic catalyst is Prussian blue.
In the method for reduction current noise of the present invention, this gold nano grain particle diameter is 16.5-20nm.
In the method for reduction current noise of the present invention, this gold nano grain concentration is 0.5-2.0ppm.
In the method for reduction current noise of the present invention, this electrode surface is with electropolymerization method fixedly gold nano grain and organic catalyst.
In the method for reduction current noise of the present invention, this electropolymerization fixing means is at electrode surface polymerization one deck polypyrrole, and gold nano grain and organic catalyst are fixed on the polypyrrole layer.
In the method for reduction current noise of the present invention, this enzyme is acetylcholinesterase or choline oxidase.
The present invention except need are possessed the advantage of conventional biosensor, also wants to overcome envirment factor and disturbs in the exploitation of biological microsensor, keep biological identification element activity, shorten detection time, also want simultaneously to reduce detection limit, could enlarge range of application.Desire to reach these targets, the exploitation of whole microsensor needs to set up and comprises: platform technologys such as electrode preparation, biological identification element are fixed, current signal amplification, noise removing and signal digitalized, sensor microminiaturization.
Aspect the preparation of electrode, Material Selection comprises: glass, gold, platinum, palladium, graphite and carbon black, no matter electrode structure is that plate electrode, needle electrode (solid or hollow out) all can use.Treatment of electrode surfaces can acid, alkali, physical grinding or ultrasonic treatment, obtains clean electrode surface.Biology identification element fixing aspect, can use physically trapping or chemical covalency bond to reach fixing purpose, but in fixing process, need consider run off problem with inactivation of biological identification element.
In addition, shorten detection time, improve detection limit simultaneously, need by reducing the noise that envirment factor produced and amplifying output signal and reached.Except the use of electron mediator, in fixing process, adopt conducting polymer (for example polypyrrole and propane diamine) to carry out biological identification element and fix oxidizing potential value required in the time of also can suitably reducing detection, avoid the impurity in the environmental samples simultaneously oxidized, lower the degree of the interference of envirment factor.
In addition, the achievement in research of nano material shows that in a single day material enters nanometer-scale, no matter all can significantly promote at material characters such as intensity, electrical conductivity and the capacity of heat transmission.In the fixation procedure of biology identification element, use nano material, will help the amplification of sensor output signal, and then promote detection speed, accuracy and the sensitivity of microsensor.
Desire to make the user to be convenient to left-hand seat, can be used for field monitoring simultaneously, except instrument need itself were compact, the result of detection preferably can directly be presented on the panel of sensor, and this part then depends on signal digitalized and the sensor microminiaturization is reached.
The present invention attempts enzyme biologic sensor is combined with the notion of nanosecond science and technology, develop the agricultural chemicals in a kind of biological microsensor detection water, utilize the special nature of high-specific surface area that golden nanometer particle has and high electrical conductivity and make up organic catalyst, in order to reduce the oxidizing potential electron transfer, the output signal that reaches amplified current simultaneously produces with avoiding noise, promotes the S/N value of biological microsensor of the present invention.Therefore, biological microsensor of the present invention can reduce the interference of natural material in the detection limit of biology sensor and the environment, shortens detection time simultaneously, reaches the instant purpose that detects of terrain.
Description of drawings
Fig. 1 is a concrete example of biological microsensor of the present invention.
Fig. 2 illustrates that the background current of electrode system of the present invention (does not add H
2O
2) variation.
The H of batch interpolation 2 μ M in Fig. 3 illustrative system
2O
2The current signal output of solution changes.
Fig. 4 illustrates H
2O
2Output current value changes and H before and after adding
2O
2The variation of concentration.
It is preceding that Fig. 5 A～enzyme (not immobilized enzyme) is directly added in Fig. 5 E explanation, and organophosphorus pesticide is to the inhibition situation of enzymatic activity.Fig. 5 A is for immobilized enzyme not and do not have the blank test that agricultural chemicals adds; Fig. 5 B is for before the immobilized enzyme not, and the electric current that adds paraoxon concentration 0.47ppm changes; Fig. 5 C is for before the immobilized enzyme not, and the electric current that adds paraoxon concentration 4.7ppm changes; Fig. 5 D is for before the immobilized enzyme not, and the electric current that adds paraoxon concentration 47ppm changes; And Fig. 5 E is for before the immobilized enzyme not, and the electric current that adds paraoxon concentration 470ppm changes.
Fig. 6 explanation is when acetylcholinesterase (AChE), cholinesterase (ChE) and choline oxidase (ChO) addition (enzyme is fixed) when being 1mM, 0.004Unit (enzyme activity unit) and 0.2Unit, for the variation of different paraoxon concentration (47ppm, 4.7ppm, 0.47ppm) and sensitivity action time.
Fig. 7 is presented at fixedly under the paraoxon concentration (4.7ppm), and suspension enzyme (not immobilized enzyme) suppresses the relation of time and electric current inhibiting rate in difference.
Fig. 8 shows the electrode sensitivity of enzyme being fixed in front and back, platinum electrode surface with sol-gel (sol gel) method.Hurdle (a) uses the platinum electrode surface with the sol-gel process immobilized enzyme; Hurdle (b) is that the standard white gold electrode in the sample is not fixed, is suspended in to the use enzyme; Hurdle (c) is that 1 centimeter platinum electrode in the sample is not fixed, is suspended in to the use enzyme; Reach hurdle (d) for using, directly place water, be not fixed in the electrode of electrode surface with behind the sol-gel embedding enzyme.
Fig. 9 shows the relation of the current output signal of enzyme before and after electropolymerization is fixing.Hurdle (a) uses the not electrode of immobilized enzyme; Hurdle (b) uses fixedly, and pyrroles (pyrrole) monomer/enzyme acetylcholinesterase (AChE) is the electrode of 20 μ l/1000U with peroxidase (HRP); Hurdle (c) uses fixedly, and pyrrole monomer/enzyme AChE and ferrocene (ferrocene) are the electrode of 20 μ l/1000U; Hurdle (d) uses fixedly, and pyrrole monomer/enzyme AChE is the electrode of 0.013mg/1000U; And hurdle (e) uses fixedly, and pyrrole monomer/enzyme AChE is the electrode of 20 μ l/1000U.
Figure 10 is presented at organic catalyst (Prussian blue Fe (CN)
6 3-) have or not the oxidizing potential decline effect under existing; Solid line represents not have Fe (CN)
6 3-Dotted line is represented Fe (CN)
6 3-Exist.
Figure 11 shows the oxidation current value of ascorbic acid under the different impressed voltages.
Figure 12 is the electron scanning micrograph of the gold nano grain of the present invention's use.
Figure 13 A shows fixing or not immobilized enzyme and the electrode of nanogold particle and the relation of electrode sensitivity; The not electrode of immobilized enzyme and nanogold particle is used in square mark representative; The electrode of immobilized enzyme and gold nano grain is used in the triangular marker representative.When Figure 13 B showed the electrode that uses immobilized enzyme and nanogold particle, nanogold particle added the relation of concentration and electrode sensitivity.
Figure 14 shows the relation of the volume and the electrode sensitive degree of nanogold particle.
Figure 15 illustrates the enzymatic activity recovery that the biological microsensor of the present invention uses.
Figure 16 illustrates the variation of the biological microsensor of the present invention in the sensitivity of different detected temperatures bottom electrode.
Figure 17 shows that the biological microsensor of the present invention detects the variation situation of current output signal under different pH values.
Figure 18 A shows the activity relationship of methyl alcohol and the biological microsensor of the present invention; Figure 18 B shows the activity relationship of ethanol and the biological microsensor of the present invention; Figure 18 C shows the activity relationship of acetone and the biological microsensor of the present invention.
Figure 19 A shows the activity relationship of cadmium ion and the biological microsensor of the present invention; Figure 19 B shows the activity relationship of ferric ion and the biological microsensor of the present invention; Figure 19 C shows the activity relationship of copper ion and the biological microsensor of the present invention; Figure 19 D shows the activity relationship of lead ion and the biological microsensor of the present invention.
Figure 20 shows sulfate radical SO
4 2-Activity relationship with the biological microsensor of the present invention.
Figure 21 illustrates 25 ℃ of following paraoxon concentration and the relation of the electric current inhibiting rate of the biological microsensor of the present invention, 10 minutes detection times.
Figure 22 illustrates the range of linearity of biological microsensor electric current inhibiting rate of the present invention and paraoxon.
Figure 23 shows that biological microsensor of the present invention detects the data and the comparison of the tomato juice of tomato juice stoste and interpolation paraoxon agricultural chemicals.Hurdle (a) uses tomato juice stoste; Hurdle (b) uses the Tomato Juice of adding 30ppb paraoxon agricultural chemicals; Hurdle (c) uses the tomato juice stoste of 10 times of dilutions; And hurdle (d) uses the tomato juice stoste of the interpolation 30ppb paraoxon agricultural chemicals of 10 times of dilutions.
Embodiment
For above-mentioned purpose of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and cooperate appended graphicly, be described in detail below:
The formation of biological microsensor
As shown in Figure 1, of the present invention one concrete example comprises three electrodes 101,103 and 105, is respectively electrode, working electrode and reference electrode, and each working electrode is fixed with enzyme, gold nano grain and Prussian blue (Fe (CN)
6 3-), in order to measure the current signal S1 that produces in the sample, export the current signal that is detected to a processing unit 120.
As shown in Figure 1, biological microsensor of the present invention also comprises a temperature inductor 110, in order to detecting sample temperature, and the temperature signal S2 that detects is imported a processing unit 120.Changing into after according to following formula (4) current signal that detects being handled through following temperature compensation in this processing unit 120 is a concentration numerical value, and import a display unit 122, on this display unit 122, show contained pesticide concentration in the sample, promptly detect pesticide concentration in the sample.
The current output signal testing mechanism
The whole Electrochemical Detection mechanism of biological microsensor of the present invention can be divided into three steps, at first utilizes acetylcholinesterase (AChE) (1000Unit/mg solid; Sigma) to acetylcholine (ACh) (99%, Sigma) go hydrolytic action, the choline that is produced is again by choline oxidase (ChO) (100Unit/mg solid, Sigma) effect, produce hydrogen peroxide, with impressed voltage hydrogen peroxide is carried out oxidation more at last, make the reaction ejected electron, so can measure the variation of enzyme reaction process current output signal by pot.Whole enzyme reaction and current signal testing mechanism can be represented by following equation:
Because organophosphorus all can combine with the activity site on the acetylcholinesterase with the carbamate agricultural chemicals, cause the activity of enzyme to suppress, and then reduce the reaction rate of the successive reaction of above-listed formula (1), (2) and (3), and current output signal is diminished.
With the concentration relativeness of current output signal and inhibitor (agricultural chemicals to be measured), be defined as follows the electric current inhibiting rate (RI) of formula, then can pass through current output signal, confirm the concentration of organophosphorus in the analysis water-like according to formula (4).
DI wherein
1/ dt is the output current rate of change (or sensitivity) before enzyme is suppressed; DI
t/ dt is the electric current rate of change (or sensitivity) after enzyme suppresses through certain hour t.
The production method of gold nano grain
After the deionized water of getting 247.5ml boils, add 1% sodium citrate solution 15ml, boiled 5 minutes, add HAuCl
4Solution 2.5ml, solution gradually by faint yellow change grey, dark brown into after, continue 90 ℃ of heated and stirred 15 minutes, place room temperature to cool off solution, solution is met and is restored bolarious golden nanometer particle, is stored in 4 ℃ of refrigerators.As reductive agent, its carboxyl (COO-) can be reduced to the monovalence gold ion with the trivalent gold ion, generates acetone dicarboxylic acid (acetonedicarboxylate) simultaneously, is reduced into gold atom by the monovalence gold ion again with sodium citrate.The entire reaction formula is as follows:
If desire prepares the golden nanometer particle of different-grain diameter size, can reach by the ratio that changes tetra chlorauric acid and sodium citrate.The ratio of adjusting sodium citrate and tetra chlorauric acid is 7,1.75,3.75 or 14, prepares the gold nano grain that mean grain size is 25nm, 36.3nm, 16.5nm or 19.4nm respectively.
The enzyme fixing means
Before enzyme was fixing, electrode surface was respectively with ultrasonic concussion and red fuming nitric acid (RFNA) cleaning platinum electrode surface.Below with sol-gel fixing means (sol-gel) and electropolymerization method enzyme is fixed in the platinum electrode surface respectively.
(a) sol-gel fixing means (sol-gelimmobilization)
Get 10 μ L TEOS (tetraethyl orthosilicate, tetraethyl orthosilicate; Aldrich Chemicals), the HCl of 200 μ L deionized waters, 30 μ L ethanol and 1 μ L 0.1M in the plastic cement small test tube, with ultrasonic concussion one hour, placed 2-3 hour in room temperature, the pH value of sol-gel (sol gel) solution maintains 6.Getting the AChE of 0.1mg and ChO and solution of gold nanoparticles 1 μ L (control group does not then add) is dissolved in the PBS solution (phosphate buffer, phosphate buffersolution) of 100 μ L pH values 7.Get 25 μ L sol-gel (sol gel) solution and add in the aforesaid enzyme solutions, fully mix.Take out 50 μ L, insert in another little plastic cement test tube with platinum electrode, 4 ℃ air-dry 24 hours, fully clean the environment be stored in 4 ℃ of dryings again with PBS solution.
(b) electropolymerization fixing means (electropolymerization)-use polypyrrole (polypyrrole)
Utilize cyclic voltammetry (cyclic voltammetry; CV) between 0-1V, with the velocity sweeping of 10mV/sec, with the enzyme electropolymerization in the platinum electrode surface.As preparation layers, directly be attached to electrode surface prior to platinum electrode surface aggregate last layer polypyrrole (as shown in the formula (7)) to avoid enzyme.Carry out the enzyme electropolymerization with the condition of table 1 again.Aspect the preservation of electrode, need earlier with behind the deionized water rinsing, in it being stored in 4 ℃ 0.1M PBS.
Table 1: material and condition that the polypyrrole electropolymerization is adopted
| Parameter value | Electropolymerizatioconditions conditions |
| Electrode material | Platinum (Pt) |
| Sweep velocity | 10mV/sec |
| The electrolytic solution condition | 0.1M LiClO 4 |
| Monomer concentration | 0.1M pyrroles |
| Enzyme concentration | 0.8g/L |
| Thickness: preparation layers (Prelayer) | 0-1.0V twice scan period vs.Ag/AgCl |
| Polypyrrole-enzyme layer | 0-1.0V twice scan period vs.Ag/AgCl: |
| Temperature | |
| 22℃ |
The pot current output signal is tentatively tested
At the beginning of experiment is carried out earlier with platinum electrode as working electrode, via impressed voltage+700mV (that is H
2O
2Oxidizing potential), test potential meter current output signal is to H
2O
2The reaction result of concentration change as shown in Figure 2.Fig. 2 is that the background current value of system changes, and does not add H as can be seen from Figure in test sample book
2O
2The time system the background current value after 300 seconds, reach stationary value 0.
Fig. 3 is a batch H who adds 2 μ M
2O
2Solution is in test bottle, and the current signal of system is exported variation diagram.Can obviously find out by Fig. 3 along with H
2O
2The interpolation of solution, current value are a batch decline, show that pot can be to H in the sample
2O
2Concentration do to detect accurately.
With each interval H
2O
2Output current value changes and H before and after adding
2O
2Concentration mapping result as shown in Figure 4.The current output signal of native system and the H that added as seen from Figure 4
2O
2Concentration become a proportional relation, especially at H
2O
2Concentration is between 0.002-0.006mM the time, the H that therefore can be used for this experimental design and produced
2O
2Detection, and then suppress the concentration of organophosphorus pesticide in the confirmatory sample via enzymatic activity.
Organophosphorus pesticide is to the inhibition situation of suspended state enzymatic activity
Add the acetylcholine (acetylcholine of 0.1ml 500mM earlier; ACh) in the test bottle that contains 0.1M PBS and 0.1M KCl, then during changing, measuring current adds acetylcholinesterase (acetylcholinesterase; AChE) 3.75Unit and choline oxidase (choline oxidase; CHO) 2Unit, impressed voltage+700mV simultaneously adds the paraoxon (paraoxon belongs to a kind of of organophosphorus pesticide) of variable concentrations (0,0.47,4.7,47 and 470ppm) again, observes the variation of current output signal, and the result is shown in Fig. 5 A-Fig. 5 E.Can obviously find out when paraoxon concentration is promoted to 470ppm by Fig. 5 A-Fig. 5 E, current output signal just has than obvious variation, infer that its possible cause is too many for acetylcholinesterase (AChE) addition in the experiment, so that after adding the inhibitor paraoxon, remaining enzymatic activity still can make acetylcholine (ACh) reaction, so the phenomenon that potential change does not weaken.
Revise the addition of zymolyte and enzyme, AChE, ChE and ChO addition respectively are 1mM, 0.004Unit and 0.2Unit, detection limit can be dropped to 4.7ppm (as shown in Figure 6).The result carries out under fixing paraoxon concentration 4.7ppm according to this, and different paraoxon suppress the experiment of times to current output signal (inhibiting rate) influence, and the result as shown in Figure 7.Can obviously find out by Fig. 7, along with suppressing time lengthening, the degree that enzyme is suppressed by paraoxon is high more, so the inhibiting rate of current output signal is big more, that is current output signal is also closely related with the inhibition time except the influence that is subjected to inhibitor (paraoxon) concentration.
Activity performance after enzyme is fixing
According to above-mentioned enzyme fixing means, respectively with the sol-gel fixation and voltolisation is legal that enzyme is fixed on the electrode surface.Found that having the gel condensation course with the sol-gel process immobilized enzyme is difficult for controlling, dehydrate overlong time, the easy crisp enzyme of colloid easily runs off, shielding effect hinders shortcomings such as molecular diffusion and electron transport, after causing enzyme fixing, little a lot of before current output signal is more fixing, sensing sensitivity also with decline.As shown in Figure 8, with sol-gel process enzyme is fixed in platinum electrode surface (Fig. 8 (a)), compared to directly enzyme being suspended in the sample (Fig. 8 (b), (c)), electrode sensitivity descends about 4-10 doubly.Simultaneously if with enzyme with the sol-gel embedding after, directly place water sample, and be not fixed in electrode surface (Fig. 8 (d)), then because matter passes the existence of resistance, and the person is poorer to cause sensitivity to be directly fixed on the electrode surface than enzyme, almost do not have current signal to take place.
And with voltolisation legal-polypyrrole (polypyrrole) is fixed in the platinum electrode surface with enzyme, not only can be by conductive elements and the regulation and control of enzyme ratio, reach membranaceous fixing (filmimmobilization), avoid because of the enzyme fixed bed too thick, cause the mass transport resistance, simultaneously since the electropolymerization monomer this as conductive material, with electropolymerization fixedly the sensitivity of rear electrode be height than the suspended state enzyme on the contrary, though and enzymatic activity has a little inactivation in fixing process, the reactive residual rate is still up to 90%.Fig. 9 is that different pyrrole monomers/enzyme value electropolymerization is finished the after-current variation of output signals.As seen from Figure 9 when pyrrole monomer/when AChE enzyme amount is 0.013mg/1000U (Fig. 9 (d)), the electrode current output signal after enzyme is fixedly finished is about the twice of suspended state enzyme (Fig. 9 (a)).It mainly is because pyrroles itself is conductive material that this situation takes place.With PTC (Propionylthiocholine, propionylthiocholine) cover group (CACTA service srl.) is measured the activity of enzyme after fixing, find the remaining rate of enzymatic activity still up to (suspended state) before fixing 89%.
Noise is got rid of and the current output signal amplification
The sensitivity of biological microsensor is if will promote, and then the ratio of signal and noise (S/N) must increase.Reach this purpose, can set about from both direction, (1) avoids in the environmental samples other materials oxidized in testing process, reduces noise; (2) amplified current signal.In the process of exploitation, with the Prussian blue Fe of organic catalyst (CN)
6 3-Be fixed in the platinum electrode surface together with AChE and CHO enzyme, by Prussian blue (PB) self-catalyzed reaction with Prussia white (PW), can be with script oxidation H
2O
2Required impressed voltage was reduced to for about 0 (as shown in figure 10) by 700mV.Can reduce the application current potential is because Prussian blue (PB) provides a good catalysis role, makes H
2O
2Script needs the oxidation reaction of high potential (700mV) to convert the reduction reaction of electronegative potential to, and the driver of this reduction reaction is played the part of by the ortho states Prussia white (PW) that goes back of Prussian blue (PB).
White (PW) electro-catalysis (electrocatalytic) H in Prussia at first
2O
2Reduction, current potential (0mV) is used in the reduction that provides by electrode more afterwards, impels Prussian blue (PB) to be reduced into Prussia white (PW) once again, therefore obtains a reduction current signal.So can make electrode detection to signal be Prussian blue (PB) self-redox change of finishing but not the H that takes place in the solution
2O
2Redox reaction, and then promote electrode pair H
2O
2Sensitivity.
Therefore at this hardly under the situation of impressed voltage, originally may simultaneously oxidized material (as: humic acid, ascorbic acid, NH in the terrain testing process
4+-N etc.) no longer oxidized, can significantly reduce the generation of current noise.Figure 11 represents ascorbic acid oxidized situation under different impressed voltages.As shown in Figure 11, along with impressed voltage rises, because of the current value that Ascorbic Acid Oxidation produced is big more, and level off to 0 the time when impressed voltage, oxidation current just disappears, and in other words, this current noise will can not produce.
On the other hand, utilize good conduction of the golden nanometer particle of chemosynthesis and huge specific surface area characteristic (shown in the electron scanning photomicrograph of Figure 12), it is fixed on the platinum electrode surface together with enzyme and the method for organic catalyst with electropolymerization, find because the electric transmission resistance declines to a great extent and catalytic capability significantly rises, current output signal promotes a lot of relatively, that is the sensitivity of sensor lifting, make the sensor whole detection limit can drop to the ppb level.If consider the relation of detection time and detection limit, but then between work one is compromise, makes its sensitivity that not only detects can reach requirement, also can shorten detection time simultaneously.A noticeable especially place is arranged, and as Figure 13 A, when enzyme and golden nanometer particle were suspended state, golden nanometer particle added many more, and the sensitivity of electrode is also high more.Yet under fixing state, as Figure 13 B, the sensitivity meeting of sensor raises along with the interpolation of golden nanometer particle earlier, then if the excessive interpolation of golden nanometer particle, the sensitivity meeting of electrode descends thereupon, be preferably and contain the 0.5-2.0ppm gold nano grain in the electropolymerization solution, optimum addition is 0.7ppm.This phenomenon is mainly under the suspended state that the gold nano grain addition can be considered on a small quantity compared to the volume of test sample book, adds manyly more, and electric conductivity ought to be good more; And be under the state that film fixes when enzyme and golden nanometer particle, there is the proportioning an of the best in the amount of enzyme, organic catalyst, electropolymerization monomer and golden nanometer particle, when golden nanometer particle add excessive, not only the easy aggegation of particle disperses to be difficult for, and because crowding-out, cause the content of central enzyme of fixed bed and organic catalyst low excessively, make current output signal descend.Can find out in addition among Figure 13 A that if suitably add the golden nanometer particle amount, then the transducer sensitivity of stationary state will high (about 5 times) sensitivity (crest of stationary state curve) under suspended state far away.
The gold nano grain that synthesizes different volumes among the present invention in addition with different tetra chlorauric acids/sodium citrate proportioning, be fixed on the electrode surface, found that the current signal enlarge-effect just (as shown in figure 14) can occur when working as the golden nanometer particle diameter that added less than 20nm (about 16.5-20nm).
The enzyme reactivation
AChE can 2-PAM (pyridine-2-aldoxime chloromethanes, pyridine-2-aldoxime methochloride) be recovered its activity after being subjected to the organophosphorus pesticide inhibition.Its mechanism of action is to combine with the phosphoric acid of acetylcholinesterase surface phosphoric acidization, makes acetylcholinesterase recover active.Between positive charge on the nitrogen and glutamic acid (glutamateacid) Coulomb attraction power is arranged, make the oxygen atom of the very strong nucleophilic power of tool can attack phosphorus atoms, enzymatic activity thereby the recovery that combines with serine (serine).2-PAM with variable concentrations among the present invention acts on the electrode that is subjected to after organophosphorus pesticide suppresses, and recovery rate and the current output signal of measuring enzymatic activity change, and the result as shown in figure 15.Among Figure 15, A0 represents standard value; A1 represents that the 0.126ppm paraoxon handled 10 minutes, and A2 represents with 2-PAM reactivation 10 minutes, and A3 represents behind after the 2-PAM reactivation 30 minutes (test 1); B1 represents that paraoxon handled 10 minutes, and B2 represents that B3 represented after preparation on July 8, uses July 12 (test 2) with 2-PAM reactivation 15 minutes; C1 represents that paraoxon handled 10 minutes, and C2 represents with 2-PAM reactivation 10 minutes, and after C3 represented after the 2-PAM reactivation 30 minutes, C4 represented after preparation on July 12, uses July 16 (test 3); D1 represents that paraoxon handled 10 minutes, and D2 represents not use 2-PAM, and D3 represents with 2-PAM reactivation 10 minutes, and D4 represents behind after the 2-PAM reactivation 30 minutes (test 4).Suppress electrode later through 0.126ppm paraoxon solution as seen from Figure 15, can within 30 minutes, the effect by 2-PAM it be recovered about 70% activity, find that through the continuous four times test of inhibition-recovery enzymatic activity does not obviously descend, agent is suitable as enzymatic activity with 2-PAM in demonstration, and electrode has very big reusable potentiality.
The environmental interference factor is inquired into
May cause enzyme deactivation at the interference factor that may exist in the environment, or exist easily oxidized material to cause current noise to produce.The present invention is directed to temperature, pH, heavy metal and organic solvent is inquired into the influence of enzymatic activity.Figure 16 is illustrated in the variation of enzymatic activity under the different temperatures, and temperature and 25 ℃ poor (T-25) are measured in abscissa representative, and ordinate changes with electric current represents inhibiting rate.When measuring temperature between 10-40 ℃ when (abscissa numerical value-15～15), enzymatic activity rises along with temperature and improves as seen from Figure 16, show when sensor when terrain uses, temperature compensation is necessary.This research shows at the enzymatic activity under the different temperatures and inquires into, and selected 25 ℃ is reference point, measures the variation situation of inhibiting rate under the different temperatures, between 10-40 ℃ scope, finds that the enzymatic activity performance is a linear relationship (as shown in figure 16) under the different temperatures.
Figure 17 shows the variation situation of current output signal under the different pH values.Current output signal has the situation of obvious rising to take place between pH8-9 as seen from Figure 17.Why have this situation to be because when pH is too high, R-choline (R-Choline) can cause current output signal to become rapidly greatly automatically by chemical hydrolysis, causes erroneous judgement.The scope of application of hence one can see that this electrode is only under pH levels off to neutral situation.
Figure 18 and Figure 19 represent the influence to enzymatic activity of organic solvent and heavy metal respectively.No matter can find out that by Figure 18 A-Figure 18 C which kind of organic solvent all more or less has inhibition to enzymatic activity.At under the present water quality standard of releasing (concentration shown in black is rectangular), each organic solvent is all high to the maximum inhibition of enzyme.The most normal working concentration of acetone (Figure 18 C) is only about 0.2% more fortunately, and acetone does not almost have any active inhibition to enzyme in the case, shows that acetone is that the agricultural chemicals extraction measures better suited organic solvent.Can find out that then each heavy metal species is all influential to enzymatic activity in Figure 19 A-Figure 19 C, wherein serious to the inhibition of enzymatic activity with copper ion (Figure 19 C), when copper ion concentration reached 1ppm in the water sample, enzymatic activity is only surplus made an appointment with half.As for plumbous (Figure 19 D), iron (Figure 19 B), and the influence of cadmium (Figure 19 A) ions enzyme activity then obviously less, the order of influence degree is copper＞cadmium＞iron＞lead.When electrode when real field uses, the situation of contained heavy metal must be noted in the water.
As for SO
4 2-Influence to enzymatic activity can be represented by Figure 20.As seen from Figure 20 under the existing water quality standard of releasing, the existence of sulfate radical to enzyme almost without any influence.
The relational expression of fixation measuring electric current inhibiting rate and organophosphorus concentration under the time with can use
Scope
Traditionally, the detection limit of electrochemica biological sensor often falls within the ppm grade.So high detection limit causes this type of biology sensor only can be used for the measurement of high concentration, can't provide any help for the monitoring of on-the-spot pollutant.By the foundation of above-mentioned many platform technologys, the biology sensor detection limit of this research institute exploitation can extend to the ppb grade on extremely short detection time.So low detection limit is much smaller than the current law control test, thereby can be used for the usefulness of terrain pollutant monitoring.Figure 21 is 25 ℃ of following organophosphorus concentration and the graph of a relation of electric current inhibiting rate, 10 minutes detection times.Can find by Figure 21 that originally the electric current inhibiting rate increases along with organophosphorus concentration and be linear and rise, but surpass 130ppb when organophosphorus pesticide concentration, just no longer change near the electric current inhibiting rate increases to 70%.The quenchable enzyme of this phenomenon hint (1) organophosphorus pesticide (or organophosphorus pesticide can touch enzymatic activity position) only accounts for 70% of whole immobilized enzymes; Remaining immobilized enzyme of 30% may be because influence too dark or stereo omnibearing causes the organophosphorus pesticide can't be close; (2) organophosphorus pesticide of 130ppb just is enough to suppress the enzymatic activity position that all can contact, thereby causes the electric current inhibiting rate also no longer to raise under high organophosphorus concentration.
The range of linearity among Figure 21 (picture place) drawn again be shown among Figure 22.The scope that can measure of the organophosphorus pesticide of biological microsensor of the present invention is 10-130ppb as seen from Figure 22.But this range of application can be enlarged by increasing the enzyme fixed amount.Yet organic catalyst, gold nano grain, pyrrole monomer need be confirmed once more at different electrode size (surface area) with the best proportioning of enzyme at this moment.
The temperature compensation of electrode performance
The temperature compensation test is carried out in performance under different temperatures at electrode.With 25 ℃ be reference point, cooperate electric current inhibiting rate and the organophosphorus pesticide relation of gained among Figure 22, select the standard solution of known organophosphorus concentration and measure, carry out two stage temperature compensation with measurement result.Phase one is the temperature compensation of single concentration; Subordinate phase then is the temperature compensation of full concentration range (0-100ppb).The organophosphorus concentration of measuring comprises: 20,40,60,80 and 100ppb; Temperature range then contains: 10,18,25,32 and 40 ℃, the result is as shown in table 2.P1 is original (not temperature compensated) measurement result by Figure 22 relational expression conversion gained in the table 2; P2 is the result through single concentration-temperature compensation back gained; P3 then is the result through full concentration range temperature compensation gained.With the actual measured results is benchmark, will be summarized in table 3 at the temperature compensation equation of each organophosphorus concentration and gamut organophosphorus pesticide gained.When carrying out the temperature correction of full concentration range, when being lower than 100ppb, 1c draws P3 ' by formula, draws Coeff. by formula 1b again, draws P3 by formula 1a at last; When 100ppb was above, 2c drew P3 ' by formula, drew Coeff. by formula 2b again, drew P3 by formula 2a at last.(standard of comparison solution concentration and P2) as seen from Table 2, except high concentration organophosphorus (100ppb), by the revised result of single concentration-temperature compensation equation, regardless of environment temperature, its SD (standard deviation) all can be less than 2%; If carry out temperature compensation (standard of comparison solution concentration and P3) by full concentration range equation, can find out that working as environment temperature departs from 25 ℃ more, compensation effect is poor more.When environment temperature during between 18-32 ℃, the result after the full organophosphorus concentration compensation, its SD＜7%; When environment temperature is low to moderate 10 ℃ or high during to 40 ℃, its SD is sometimes up to 15%.Really test when carrying out authentic sample, can pass through electric current inhibiting rate and concentration relationship formula and gamut temperature compensation equation, obtain increased organic phosphorus content in the sample.If sample temperature is too high or too low, exceed temperature compensation range, row measurement again after suggestion should be risen again.
Table 2: raw data that each organophosphorus (paraoxon) concentration condition of different temperatures bottom electrode is measured and the result after the temperature compensation
Table 3: the temperature compensation equation of single concentration and full concentration range
The current output signal digitizing
By a series of experimental result, set up the relational expression of fixation measuring electric current inhibiting rate and organophosphorus concentration under the time.With the burned chip of this relational expression, the connection of setting up the signal acquisition system, finish signal digitalized, as the digital signal transmission system of biological microsensor.Basically the exploitation of this digital signal transmission system mainly is divided into soft, hardware two parts and carries out.Hardware components includes: hand held pot, signal transmssion line (RS232 winding displacement), signal analysis and register (microprocessor) etc.And software mainly is to use LabVIEW (Laboratory Virtual Instrument EngineeringWorkbench) packaged program language to come the work such as signal acquisition, transmission, analysis and record between control hardware.LabVIEW is the program language of a kind of image-type of being developed in 1986 by National Instrument company, its main application be can fully integrated control communication interface, for example GPIB, VXI, PXI, RS232, and support data to present (data presentation), data storing (data storage), data analysis (data analysis), data acquisition (data acquisition), environment control functions such as (serial instrument control).Because modular instrument Express VIs can make prototype and test mixing Design of Signal and produce the test procedure sign indicating number automatically among the LabVIEW; Can guarantee that Real-Time Desktop PC function will be any based on PC (PC-based) test macro energy optimization system degree of stability and performance; PDA data acquisition usefulness and DMM support fast, set up customized Portable data acquisition system; Bluetooth support-utilization wireless blue tooth science and technology is done communication with other equipment; 50 kinds of brand-new mathematical function function-utilizations increase LAPACK/BLAS basic function storehouse, increase precision and speed and reach as high as 200%; Hyperthread (Hyper-Threading) science and technology increases and is up to 100% system's implementation capacity.In the process that detects, except the concentration that can obtain pollutant, also can comprise at testing conditions: inhibition time, oxidizing potential, electric current record frequency change, and are quite convenient.
Authentic sample detects
In order to confirm the practicality of the biological microsensor that this research institute develops, make a preliminary test at commercially available tomato juice, the result is as shown in figure 23.In experimentation, tomato juice stoste is carried out the measurement of organophosphorus pesticide in advance, then in stoste, mix the paraoxon agricultural chemicals of (spike) 30ppb, observe sensor and whether can make measurement accurately this.In another experiment, then, carry out organophosphorus pesticide and measure earlier with ten times of tomato juice stoste dilutions, then similarly in this dilution, mix the paraoxon agricultural chemicals of 30ppb, observe the measurement situation of sensor.The whole agricultural chemicals inhibition time is 10 minutes in the experiment.Can know by Figure 23 and to find out, the organophosphorus pesticide concentration of tomato juice stoste is 25.6ppb, after mixing 30ppb paraoxon agricultural chemicals, measured value becomes 54.9ppb, and another the experiment in, dilute ten times after, pesticide concentration has been lower than measurable range in the dilution, after then mixing 30ppb again, the organophosphorus pesticide in the dilution becomes again and can measure its value for 39.6ppb.Thus the result as can be known the biological microsensor of this research institute exploitation can measure the residual quantity of organophosphorus pesticide in the fruit juice exactly, interfering material in the while fruit juice, for example: sugar, pigment, suspended solid and antioxidant etc. can't have any influence to measurement result.Show that this system can be used for the measurement of authentic sample.
The industry utilizability
The present invention is in conjunction with biotechnology and nanosecond science and technology, exploitation is based on the electrochemica biological microsensor of enzyme reaction, can do quick and sensitive detection at the pollutant in the terrain water environment, chemical analysis method with respect to the traditional experiment chamber, not only can reach the economical purpose that saves time, on the convenience of using, more significantly promote simultaneously.
With the electropolymerization fixing means with enzyme, be fixed on the electrode surface together with organic catalyst and golden nanometer particle, because the existence of organic catalyst can reduce and detect the required magnitude of voltage that applies when carrying out, thereby significantly dwindle the oxidized noise that produces of other materials in the environmental samples.In addition, by the high-specific surface area of golden nanometer particle and the speciality of high conductivity, therefore the current signal that scalable enzyme reaction produced can significantly promote the sensitivity of biological microsensor, improves detection limit simultaneously, shortens detection time.Set up current output signal and the analyte concentration relation between the two according to result of experiment, cooperate the foundation of current signal output digitization system, the biological microsensor of the stream of developing will have the characteristics of sensitivity, quick, accurate, with low cost and easy left-hand seat concurrently.
The biological microsensor weight that the present invention produces is less than 1 kilogram, can be in 10 minutes detection time, accurately the agricultural chemicals in the detection water sample is to 10ppb, compare with traditional biology sensor, biology sensor of the present invention not only volume and weight is less, detection limit two orders of magnitude that descend, and shorten detection time, and detect cost and only need about 10 yuan, on the application surface of pollution detection, will significantly promote.
The above only is preferred embodiment of the present invention; so it is not in order to limit scope of the present invention; any personnel that are familiar with this technology; without departing from the spirit and scope of the present invention; can do further improvement and variation on this basis, so the scope that claims were defined that protection scope of the present invention is worked as with the application is as the criterion.
Being simply described as follows of symbol in the accompanying drawing:
101: electrode
103: electrode
105: electrode
110: temperature inductor
120: processing unit
122: display unit
S1: current signal
S2: temperature signal
Claims (23)
1. a method that detects agricultural chemicals is characterized in that, comprising:
One fluid sample is provided;
This sample is contacted with at least one electrode, and wherein this electrode surface has enzyme, gold nano grain and organic catalyst to fix on it; And
Detection is by the current signal of this electrode output, wherein this electrode surface be with the electropolymerization method at electrode surface polymerization one deck polypyrrole, and enzyme, gold nano grain and organic catalyst are fixed on this polypyrrole layer with the electropolymerization method,
This agricultural chemicals is organophosphorus or carbamate.
2. the method for detection agricultural chemicals according to claim 1 is characterized in that, this enzyme is acetylcholinesterase or choline oxidase.
3. the method for detection agricultural chemicals according to claim 1 is characterized in that, this organic catalyst is Prussian blue.
4. according to the method for each described detection agricultural chemicals in the claim 1～3, it is characterized in that the concentration of this gold nano grain is 0.5-2.0ppm.
5. the method for detection agricultural chemicals according to claim 1 is characterized in that, this gold nano grain particle diameter is 16.5-20nm.
6. the method for detection agricultural chemicals according to claim 1 is characterized in that, this electrode is a platinum electrode.
7. the method for detection agricultural chemicals according to claim 1 is characterized in that, it also comprises makes this sample contact with a temperature inductor, detects this sample temperature.
8. the method for detection agricultural chemicals according to claim 7 is characterized in that, this current signal carries out temperature compensation with sample temperature to be handled.
9. the method for detection agricultural chemicals according to claim 7 is characterized in that, this electrode and temperature inductor are arranged on the flat board.
10. a biological microsensor is characterized in that, comprising:
At least one electrode is in order to sensed current signal;
One processing unit in order to receive handling this current signal, and is changed this current signal and is become a concentration data; And
One display unit is in order to show this concentration data;
Wherein this electrode surface at this electrode surface polymerization one deck polypyrrole, and makes enzyme, gold nano grain and organic catalyst be fixed on this polypyrrole layer with the electropolymerization method with the electropolymerization method;
This biology microsensor is used to detect agricultural chemicals, and this agricultural chemicals is organophosphorus or carbamate.
11. biological microsensor according to claim 10 is characterized in that, this enzyme is acetylcholinesterase or choline oxidase.
12. biological microsensor according to claim 10 is characterized in that, this organic catalyst is Prussian blue.
13., it is characterized in that this gold nano grain concentration is 0.5-2.0ppm according to each described biological microsensor in the claim 10～12.
14. biological microsensor according to claim 10 is characterized in that, this gold nano grain particle diameter is 16.5-20nm.
15. biological microsensor according to claim 10 is characterized in that, this electrode is a platinum electrode.
16. biological microsensor according to claim 10 is characterized in that it also comprises a temperature inductor, in order to detected temperatures and be sent to this processing unit.
17. biological microsensor according to claim 16 is characterized in that, this current signal carries out temperature compensation with this detected temperatures to be handled.
18. biological microsensor according to claim 16 is characterized in that, this electrode and temperature inductor are simultaneously on a flat board.
19. method that reduces the current noise of biological microsensor as claimed in claim 10, it is characterized in that, this method comprises with the electropolymerization method at electrode surface polymerization one deck polypyrrole, and enzyme, gold nano grain and organic catalyst is fixed on this polypyrrole layer with the electropolymerization method.
20. the method for reduction current noise according to claim 19 is characterized in that, this organic catalyst is Prussian blue.
21. the method for reduction current noise according to claim 19 is characterized in that, this gold nano grain particle diameter is 16.5-20nm.
22. the method for reduction current noise according to claim 19 is characterized in that, this gold nano grain concentration is 0.5-2.0ppm.
23. the method for reduction current noise according to claim 19 is characterized in that, this enzyme is acetylcholinesterase or choline oxidase.
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| CN102375006A (en) * | 2010-08-13 | 2012-03-14 | 华东师范大学 | Au-TiO2-AChE composite system, its preparation method and its application |
| CN102621321A (en) * | 2012-03-30 | 2012-08-01 | 山东理工大学 | Construction method of sensitive interface of immunosensor for detecting pesticide residues |
| CN109342537B (en) * | 2018-12-13 | 2020-12-15 | 上海大学 | Method for detecting bifenthrin residue in vegetable by using 430 stainless steel wire bundle electrode |
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| CN2528010Y (en) * | 2002-04-02 | 2002-12-25 | 泰博科技股份有限公司 | Biological sensor |
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| CN1224160A (en) * | 1998-11-19 | 1999-07-28 | 山东农业大学 | Method for fast testing farm chemicals contamination in vegetables and fruits |
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