TWI301541B - Method for detecting pesticides ,bio-microsensor,and metood for reducing current noise - Google Patents

Description

1301541 t < IX, invention description: [Technical field of invention] The present invention relates to a pesticide detection method and a biological micro-sensor. [Prior Art] In order to increase the yield and maintain the aesthetics of crops, a large amount of pesticides are usually applied, and organophosphorous compounds (OPCs) have been replaced by organochlorines due to their low bioaccumulation and high biodegradability. Pesticides have become the most commonly used pesticide species. Although organophosphorus pesticides have the above advantages, they may cause residues in soil, crops, surface water and industrial wastewater in the case of heavy use, posing considerable threat to human health and the natural environment. Countries around the world have paid close attention to the regulation of organophosphorus pesticides, and have imposed stricter regulatory standards on drinking water, food and industrial wastewater. Once the organic phosphorus enters the organism, it forms an irreversible bond with the active site of choline esterase (ChE), thereby inhibiting the activity of the enzyme and delaying the in vivo acetylcholine (ACh) test. The rate of hydrolysis, which interferes with nerve transmission. Depending on the toxicity, dose, and exposure time of the pesticide itself, the symptoms include: fatigue, nausea, sleepiness, blurred vision, and even death if severe. Traditionally, pesticides have been analyzed primarily by chemical methods (including HPLC, GC, GC-Mass, ICP-mass, etc.). Although the traditional laboratory chemical instrument analysis method can provide accurate detection of environmental samples, the pre-processing and analysis steps are extremely lengthy, and the instrument itself is expensive, bulky, and requires professionally trained personnel to operate, resulting in cost of testing. High, basically does not meet the requirements of real-time monitoring of pollution. For the upcoming agricultural products, these traditional chemical analysis methods do not provide any assistance for the immediate detection and screening of pesticides. 0296-A21230TWF(N2);345;jessica 5 1301541 With this clock, we developed a micro-manufacturer (Μι(10)·加斯) that can quickly check the concentration of pollutants while also being portable, operating limbs and cheap. Cheng: The research and development focus of current detection and monitoring technology. ... biosensor (bi〇sensor) can overcome the lack of unified analysis methods due to the following advantages: (1) through the application of limb technology, bio-components can be reused and reduced costs; (2) Biometric identification components are highly specific, avoiding non-standard interference; (3) easy to operate; (4) high sensitivity, low sample volume required; (5) fast response, reducing analysis time; (6) digital signal output, miniaturization Easy to carry and can be used for on-site detection. The range of the biosensors varies depending on the transmission capability of the signal transmission components themselves. The traditional electrochemical biosensor (4) has a measurement limit of only PPm and is the worst of all signal transmission components. However, due to its simple operation, low equipment cost and low detection cost, electrochemical biosensors can still be accurate for samples with color and high turbidity, so it is still the earliest and most perfect, both at home and abroad. Phase # more research results are presented. At present, the main reason for the difficulty of the electrochemical bio-sensing enthalpy is that it is difficult to achieve widespread use, including the excessive oxidation potential of the erbium, and the interference in the oxidizing environment, which leads to the production of cockroaches. (2) Detecting The measurement time is longer than other forms of biosensors. (3) The limit is too high to detect low concentrations of contaminants. The application surface is too narrow. The right 9 overcomes these shortcomings and will accelerate such organisms. The speed of sensor commercialization, while expanding its application in environmental pollution (4). SUMMARY OF THE INVENTION In view of the above, the present invention is directed to providing a method for detecting pesticides, including providing the same; ^ , the sample is contacted by at least one electrode; and the detected electricity* 纟 ^ β ^ surface has an enzyme, a gold nanoparticle and an inorganic catalyst solid 0296-A21230TWF (N2); 345; jessica 1301541. Another object of the present invention is to provide a biological micro-sensor comprising at least one electrode, a temperature sensor, and a data processing display device, wherein the electrode is fixed on the surface with an enzyme, a gold nanoparticle and an inorganic catalyst. Used to detect pesticides. Another object of the present invention is to provide a method of reducing current noise, comprising immobilizing a gold nanoparticle and an inorganic catalyst on an electrode surface to which an enzyme is immobilized.

*In the development of biological micro-sensors, in addition to the advantages of traditional biosensors, it is necessary to overcome the environmental factor interference, and keep the biometrics % i cattle = shortened _s! Can reduce the detection limit, in order to expand the power,: injury, = two: =: whole:: sensor development needs to be established: number digitization, sensing: two; Taiwan: signal amplification, noise _ signal in the electrode In terms of preparation, the choice of materials includes glass, gold, white, and treatment. The cadaver treatment can be used for acid, alkali, physical grinding or ultrasonic physical electrode surfaces. In terms of fixing the biometric component, it is necessary to consider the problem of loss and deactivation of the biometric component. - In the mouth of the strong, in addition to shortening the detection time, the noise generated by the environmental factors and the amplification of the round: the detection limit ' needs to be achieved by reducing the use of the medium. In addition to the electronic medium and propylenediamine), the bio-identified element solid polymer (for example, the poly-batch oxidation potential value, avoiding the environmental exposure too much), can appropriately reduce the degree of interference required by the sub-detection. Oxidized, reduced environmental factors - Jessica 〇 296-A21230TWF (N2); 345 7 1301541 In addition, the research results of nanomaterials show that the material is in the nanometer specifications, regardless of the properties of the material such as strength, electrical conductivity and thermal conductivity. The large material of the object identification component will help the output of the detector to amplify, thereby improving the detection speed and accuracy of the micro-sensor. ', for the user to get started At the same time, it can be used for on-site monitoring. In addition to the lightness and shortness of the instrument itself, the results of the debt test can be directly displayed on the panel of the sensor. This part is achieved by digitalization of the signal and miniaturization of the sensor. The invention attempts to combine the enzyme biosensor with the concept of nanotechnology to develop a biological micro-sensor to measure the pesticide in water, and to use the high specific surface area of the gold nanoparticle to be highly conductive. The special properties of electricity and the combination of inorganic catalysts to reduce the electron transfer of the oxidation potential, while at the same time achieving the output signal of the amplified current and avoiding the generation of noise, and improving the biological micro-sensor of the present invention. Sensing can reduce the detection limit of the biosensor and the interference from the iron material in the environment, and shorten the time to achieve the purpose of real-time system f. The above objects, features and advantages of the present invention can be more clearly understood. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the preferred embodiments will be described in detail with reference to the accompanying drawings. [Embodiment] The configuration of the biological micro-sensor is as shown in FIG. 1F. The electrodes 〇1 103 and 1G5 ' are the counter electrode, the jade electrode and the reference electrode, respectively, and each work is fixed with enzymes, gold nanoparticles and Prussian blue (cn) wide, and the flow generated in the sample. Signal si, outputting the detected current signal to a processing unit 120 〇0296-A21230TWF(N2); 345;]essica 8 1301541 As shown in FIG. 1, the biological micro-sensor of the present invention further includes a Degree sensor 110 for detecting the temperature of the sample, and the temperature detection signal S2 inputted a processing unit 120. The processing unit 120 converts the detected current signal into a concentration value by the following temperature compensation process according to the following formula (4), and inputs it into a display unit 122, and displays the sample in the display unit 122. The concentration of the pesticide contained in the sample is the amount of pesticide detected in the sample. Current Output Signal Detection Mechanism The entire electrochemical detection mechanism of the biological micro-sensor of the present invention can be divided into three steps. First, the acetaminophen esterase (AChE) (1000 units/mg solid; Sigma) is used for the acetamidine. The biliary test (ACh) (99%, Sigam) is hydrolyzed, and the resulting biliary test is followed by the action of cholesteryl oxidase (Ch〇) (100 unit/mg solid, Sigma) to produce hydrogen peroxide, and finally the applied voltage. Oxidation of hydrogen peroxide causes the reaction to emit electrons, so that the change in the current output signal of the enzyme reaction process can be measured by a potentiometer. The whole enzyme reaction and current signal detection mechanism can be expressed by the following equation: (1) CH3COO(CH2)2N+ (CH3)3 acetylcholine 醯S CH3COOH + H0(CH2)2N+ (CH3)3 醯 醯 test H2° Choline HO(CH2)2N+(CH3)3+20 Choline Alkaline Oxidase->2H202 +hooc(ch2)n+(ch3)3 2 H20 (2) Betaine h2〇2 Applied Voltage->02+2H ++2e' (3) Since both organophosphorus and carbamate pesticides are combined with the active sites on the acetylcholinesterase, the activity of the enzyme is inhibited, thereby reducing the above formula (1), (2) And (3) the reaction rate of the continuous reaction, and the current output signal is made smaller. 0296-A21230TWF(N2);345;jessica 9 1301541 f Kang compares the current output signal with the concentration of the inhibitor (pesticide to be tested), and defines the current inhibition rate (RI) of the following equation, which can be output by the current signal. Determine the concentration of organic fill in the analytical water sample according to formula (4). RI(%)= diydt-d^ /dt /dt xlOO% (4) where dh/dt is the rate of change (or sensitivity) of the output current before the enzyme is not inhibited; dlt/dt is the inhibition of the enzyme after a certain time t Current rate (or sensitivity). The production method of the golden rice granules is boiled in 247.5 ml of deionized water, 15 ml of 1% sodium citrate solution is added, boiled for 5 minutes, and force is added into 2.5 ml of HAuCU solution, and the solution gradually changes from pale yellow to gray, dark brown. Thereafter, the mixture was heated and stirred at 90 ° C for 15 minutes, and the solution was cooled at room temperature, and the solution phase was reduced to dark red gold nanoparticles, and stored in a refrigerator at 4 ° C. With sodium citrate as a reducing agent, the carboxyl group (COCT) reduces trivalent gold ions to monovalent gold ions, and simultaneously forms acetone dicarboxylate, which is then reduced to gold atoms by monovalent gold ions. The overall reaction formula is as follows:

Au3+ + HO(COO~)3 + 〇H~ —Au + 0(C00~)2 + C02 + H20 ( 5 )

Au+ ———> Au2+ —^—^Au3+ -> Au (6) If different kinds of gold nanoparticles of different particle sizes are to be prepared, it can be achieved by changing the ratio of tetrachloroauric acid to sodium citrate. The ratio of sodium citrate to tetrachloroauric acid was adjusted to 7, 1.75, 3.75 or 14, and gold nanoparticles having an average particle diameter of 25 nm, 36·3 ηηη, 16.5 nm or 19.4 nm were prepared, respectively. Enzyme immobilization method 0296-A21230TWF(N2);345;jessica 10 1301541 Before the enzyme was immobilized, the surface of the electrode was ultrasonically oscillated and concentrated to clear the surface of the platinum electrode. The enzyme was immobilized on the surface of the platinum electrode by a sol-gel method and an electropolymerization method, respectively. (a) Sol-gel immobilization: Take 10pL TEOS (tetraethyl orthosilicate; Aldrich Chemicals), 200pL deionized water, 30μί ethanol, and ΙμΙνΟ.ΙΜ in HC1 in a plastic test tube and vortex for one hour. After standing at room temperature for 2-3 hours, the pH of the sol gel solution was maintained at 6. O.lmg of AChE and ChO and gold nanoparticle solution lpL (not added in the control group) were dissolved in 100 μL of pH 7 PBS (phosphate buffer solution) solution. A 25 pL sol gel solution was added to the aforementioned enzyme solution and mixed well. 50 μM was taken out, placed in another small plastic test tube together with the platinum electrode, air-dried at 4 ° C for 24 hours, thoroughly washed with a PBS solution, and stored in a dry environment at 4 ° C. (b) Electropolymerization - Polypyrrole is used to electropolymerize enzymes in white gold by cyclic voltammetry (CV) at 0-1 V at 10 mV/sec. Electrode surface. A layer of polypyrrole (p〇lypyrr〇le) (see equation (7) below) is polymerized on the surface of the platinum electrode to avoid direct attachment of the enzyme to the electrode surface. Further, the electropolymerization of the enzyme was carried out under the conditions shown in Table 1. For electrode storage, rinse with deionized water and store in 4 μC of 0.1 M PBS.

(pyrrole) (polypyrrole) 0296-A21 230TWF(N2);345;jessica 11 !3〇1541 λ Table 1 Polypyrrole electropolymerization iTWT, substance and condition of ~

Material i-drawing speed Electrolyte condition 3 body concentration $-concentration Thickness: Pre-layer (Prelayer) O--enzyme layer i degree

Ag/AgCl-UV two-phase period vs. Ag/AgCl: film B-2TC^~--------- Potentiometer current output signal preliminary test At the beginning of the experiment, the platinum electrode was used as the working electrode. The result of the test of the potentiometer current output signal on the change of the H2〇2 wave amplitude by the externally applied voltage 〇7 2 TI, that is, the H 2 Q 2 a potential) is as shown in Fig. 2 _ ^ The corpse is not broken. Figure 2 shows the background of the system. The value of the electric k value changes. It can be seen from the figure that when the test sample is not added to Η"2, the back π current value of the system reaches a stable value after about 300 seconds. Figure 3 shows the change of the current signal output of the system in the test bottle by adding 2μΜ of ΗιΟ, 六6, ##, 丨 J叱〇2. From Fig. 3, it can be seen that the left side of the mountain is #, 丄". It can be seen that with the addition of the H2〇2 solution, the current value drops in batches, indicating that the potentiometer can be exposed by /, and read-ahead. For the concentration of h2〇2 in the sample, the change of the output current value before and after the addition of each interval h2〇2 and the H2 plot result are shown in Fig. 4. From Figure 4, we can see that the 屮太金优%, mouth" shows that the current rotation of the 糸 system is proportional to the concentration of the added 〇2, especially in H〇, 曲戒0.002~〇 · _ Μ between, therefore can be used in the design of this experiment 2 produced 2 = in J ^ 2 〇 9 0296-A21230TWF (N2); 345; jessica 1301541 t » detection, and then confirmed by enzyme activity inhibition, confirm the sample The concentration of organophosphorus pesticides 0 The inhibition of the activity of suspended phosphorus enzymes by organophosphorus pesticides first added π 0.1 ml 500 mM acetylcholine (ACh) in test bottles containing 0.1 M PBS and 0.1 M KC1, and then tested. During the current change, add acetylcholinesterase (AChE) 3.75 unit and choline oxidase (CHO) 2 unit, and add voltage + 700mV, then add different concentrations (0,0·47,4.7 , 47 and 470 ppm) of paraxon (a type of organic pesticide), observe changes in the current output signal, the results are shown in Figure 5A-5E. It can be clearly seen from Fig. 5A-5E that the current output signal changes significantly until the concentration of Barlason is increased to 470 ppm. It is speculated that the possible reason is that the amount of acetylcholinesterase (AChE) added in the experiment is too So much that after the addition of the inhibitor balason, the residual enzyme activity can still react with acetylcholine (ACh), so the potential change does not decrease. The amount of enzyme substrate and enzyme added was modified. The AChE, ChE and ChO additions were ImM, 0.004 unit and 0.2 unit, respectively, which reduced the detection limit to 4.7 ppm (as shown in Figure 6). Based on this result, an experiment was conducted on the influence of different Barlason suppression time on the current output signal (inhibition rate) at a fixed blatson concentration of 4.7 ppm, and the results are shown in Fig. 7. It can be clearly seen from Fig. 7 that as the inhibition time is prolonged, the degree of inhibition of the enzyme by Balason is higher, so the inhibition rate of the current output signal is larger, that is, the current output signal is not affected by the inhibitor (Balazon). In addition to the effect of concentration, it is also closely related to the inhibition time. Activity after immobilization of enzymes 0296-A21230TWF (N2); 345; jessica 1301541 Human method ΓΓΐ 述 enzyme immobilization method, respectively, using sol (10) fine running method and electropolymerization 2 to apply enzyme I to the electrode surface. The result is (4) The enzyme has gelatinization and condensation, and g J/mai u疋 crispy yeast is a drought control. The dehydration and drying time is too long, and the colloid is easy to cause enzyme solidification: the 'blocking effect hinders molecular diffusion and electron transfer. Disadvantages, the guide: After the solid-state, the current output signal is smaller than before it is fixed. For example, the s 闰 巫 巫 巫 巫 金 电极 电极 电极 电极 m m m 以 以 以 以 以 以 以 以 sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol sol ), the electrode is sensitive

(10) (4) After embedding, directly set Φ times. At the same time, if the enzyme is dissolved in Figure 8 (5), the water sample is not fixed on the electrode surface (Fig. (d)) 'Because the left side of the quality of the resistance is six, the original enzyme is directly fixed to the hair. The extreme surface is worse - almost no current signal occurs. 4 Hi polymerization method side ° (four) Na. 1e) Fixing the enzyme to the platinum electrode Surface = only the fineness of the conductive monomer and the enzyme can be adjusted to achieve a film-like solid mnnob mountain. n), to avoid _ phase layer is too thick, and because the electropolymerization monomer is too helmeted, the resistance of transmission, ^ ^ ^, , ^ ^ ^ substance, after electro-polymerization fixed, although the electricity is right 埤 4, 壬The γ-enzyme is back, and the activity of the enzyme is inactivated during the fixation process, but the activity residual rate is still as high as 9〇%. First

Pyrrole monomer/enzyme value is taken from the person's gate ratio. From Fig. 9, it can be seen that the current output signal changes after 卩^^^. 0.013mg/U)00U (Fig. 9(4)) H) The amount of the early/AChE enzyme is about twice that of the suspended enzyme (the current output of the electrode of the 9th 彳4 is m). This happens mainly because pyrrole itself is a PTC (proplony pool lochollne) kit (CACTAServlce test beta: leave the activity of 'activity' found that the residual activity of the enzyme remains as high as unc 296-A21230TWF (N2) ); 345; jessica 14 1301541 noise elimination and current output signal amplification (if the sensitivity of the lake detector is increased, the ratio of signal to noise) will be large. To achieve this, you can start from two directions. = Other substances in the sample are oxidized during the surface measurement process to reduce noise; (7) Enlarge m tiger. During the development process, the inorganic catalyst Prussian blue, together with AChE and (10) enzymes, is on the surface of the platinum electrode. The self-catalytic reaction between PB and Pw can reduce the required voltage of the original oxidation h2〇2 from ·mv to about 0 (as shown in Figure 1). Prussian Blue (PB) provides a good catalytic role for H2o2's original, higher potential (7QQmV) oxidation reaction to a low potential reduction reaction. The driver of this original reaction is Prussian Blue (PB) reduction. (pw) ^ First Prussian white (PW) electrocatalytic (electr〇catalytic) H2〇2 reduction, and then by the reduction application potential (〇mV) provided by the bungee, prompted Prussian blue (PB) to be re-introduced into Prussian white ( PW), thus obtaining a -reduction current signal. This allows the signal detected by the pole to be a self-oxidative reduction of the surface-modified Prussian blue (p B) rather than the H2〇2 redox reaction occurring in the solution, thereby enhancing The sensitivity of the electrode to H2〇2. Therefore, in the case where almost no voltage is applied here, the substance which is oxidized (such as humic acid, ascorbic acid, ΝΗ4+_Ν, etc.) may be guilty during the detection process. Further deuteration can greatly reduce the generation of current noise. Figure 11 shows the oxidation of ascorbic acid at different applied voltages. From Figure 11, it can be seen that the current value due to oxidation of ascorbic acid increases with the addition of electricity. The bigger the value, the more the oxidation current disappears when the external electric power is close to 0. In other words, this current noise will not be generated. On the other hand, the chemically synthesized gold nanoparticles are excellent in electrical conductivity and giant. Large 0296- *A21230TWF(N2); 345; jessica 15 1301541 specific surface area characteristics (as shown in the electron scanning photomicrograph of Fig. 12), immobilized on the platinum electrode (4) together with the inorganic catalyst and the inorganic catalyst by electropolymerization It is found that due to the significant drop in electron transmission resistance and the significant increase in catalytic capacity, the current output signal is relatively increased, that is, the sensitivity of the sensor can be lowered to ppb and level. If the measurement is between m and the price, the stomach The system can be used as a compromise between the two, so that not only the sensitivity of the detection can be met, but also the preparation time can be shortened. There is a particularly noteworthy place. As shown in the figure, when both the enzyme and the gold nanoparticles are suspended, the more the gold nanoparticles are added, the higher the sensitivity of the electrodes. However, in the case of a fixed shape, ^13B, the sensitivity of the sensor will first increase by π with the addition of the gold nanoparticles, and then if the gold nanoparticles are excessively added, the sensitivity of the electrode will be lower. Jia is an electropolymerization solution containing 〇5_2 〇ppm gold nanoparticles, the best addition is 〇.7Ppm. This phenomenon is mainly in the state of suspension. The addition of yttrium in the Jinnai granules can be regarded as a small amount compared with the volume of the test sample. The more the addition, the better the conductivity should be; and when the enzyme and the Jinnai particles are fixed in the film. There is an optimal ratio of the amount of enzyme, inorganic catalyst, electropolymerized monomer and gold nanoparticle. When the gold nanoparticle is added excessively, not only the particles are easy to aggregate and disperse, but also because: the squeezing effect leads to the fixed layer. The content of the enzyme 舆 inorganic catalyst is too low, so that the current output signal drops. In addition, it can be seen from the 13th shot that if the amount of full-grain particles is appropriately added, the sensitivity of the sensor in the fixed state will be much higher (about 5 times) in the suspension state (the peak of the fixed state curve). In the present invention, different volume of gold nanoparticles are synthesized by different ratios of tetrachloroauric acid/sodium citrate, and fixed on the surface of the electrode, and it is found that when the diameter of the added gold nanoparticles is less than 2 〇nm ( At about 165_2Gnm), the current signal amplification effect will appear (as shown in Figure 14). 0296-A21230TWF(N2);345;jessica 1301541 Enzyme reactivation After AChE is inhibited by organic pesticides, its activity can be restored by 2-PAM (pyridine-2-aldoximemethochloride). Its mechanism of action is to combine with phosphoric acid phosphorylated on the surface of acetylcholinesterase to alter the activity of acetylcholine-S enzyme. The positive charge on the nitrogen has a coulomb attraction, so that a strong nucleophilic oxygen atom can attack the phosphorus atom bound to the serine acid (4), and the enzyme activity is restored. In the present invention, the electrodes of different concentrations of 2-PAM are used as poems for the poems of the agricultural poems (4), and the recovery rate of the enzyme activity and the current output signal are measured. The results are shown in Fig. 15. It can be seen from the figure that after 0.126 Ppm [electrode after the relaxation of the solution, it can be recovered for about 3 minutes by the action of 2·ΡΑΜ, and the enzyme activity is found after the inhibition_recovery test. There was no significant decrease, and it was shown that (4) was the active agent of the enzyme, and the electrode had the potential to greatly repeat the enthalpy. Environmental Interference Factor Discussion For possible interference factors in the environment, current noise is generated in substances that are easily oxidized. This; =, production 1 storage weight = and the effect of organic solvents on enzyme activity. Second; degree two: the change in the activity of the element, the abscissa represents the temperature of the measurement 5. Two differences (Ding-25), the ordinate is the difference between current 栾 and U 詈 mn Ζ. Table does not inhibit the rate. From Fig. W, it can be seen that the muscle amplitude in Luo test is between 10-40oC (the cross-seat J has an increase in the field measurement and increases, showing when the sensor ^(4)~5), the enzyme activity is warm. This study is aimed at different temperatures (4) when the temperature compensation is necessary

For the reference point, the different temperature-sound performances were discussed. The range of 25〇C was selected and the temperature W of different temperatures was found, as shown in the figure of H)-40〇C). · Performance in a linear relationship (eg, 16 0296-A21230TWF(N2); 345; Jessica 1301541 Figure 17 shows the change in current output signal at different pH values::, out 2. The H" current output signal has a significant rise The reason why the situation is that the younger brother has the situation of the court is because when the ρΗ is too high, the bio-chemical will be automatically hydrolyzed, resulting in a rapid increase in the current output signal, double, wide: The material is applicable only to Qiu. Approaching neutral feelings; ^. Judge. The effects of the solvent on the 18th and 19th points are shown. It can be seen from the 18C chart of the first team that no matter what kind of organic ^ =; rarely: systemic. ·^^ 不辰度), the inhibition rate of the activity of each organic solvent on the enzyme The most common use concentration of acetone (the map) is only about %2%, and in this case ^ =

Si enzyme has no activity inhibition. It shows that acetone is suitable for pesticide extraction measurement: =:> In the 19A_19C chart, it can be seen that various heavy metals have an effect on the enzyme activity= Among them, copper ions (Fig. 19C) are serious for the enzyme. When the copper ion concentration in the water sample reaches 1%, the enzyme activity is only about = As for the wrong (19D), iron (Traditional force -) _ just map), and the road (the younger brother 19A, the son of the enzyme live (four) shirt is obviously more embarrassing, the order of influence is the age of Lulu. In the case of transporting heavy metals in the water, it is necessary to pay attention to the situation. As for so?· The effect on the activity of the enzyme can be represented by the second map. From the third 汕 == see the discharge water standard, sulfate The existence of the enzyme has almost no speculation 3: the current inhibition group and the organic phosphorus concentration Lai system and the applicable range of transmission, charging, the detection limit of the children's chemical biosensor often falls in ppm, etc. This detection limit results in the use of such biosensors only for high concentrations. 'The monitoring of on-site contaminants does not provide any (4) assistance. By 0296-A21230TWF(N2);345;jessica 18 1301541

f V two sets::? Li, the organism developed by this research is the limit of sensor detection. It can be extended to ppb level. So low

It is less than the current regulations/standards, so it can be used for the monitoring of local pollutants. 21 The graph shows the relationship between the concentration of organic phosphorus and the current inhibition rate at 2% of ❹jk. From Fig. 21, it can be seen that the current inhibition rate initially increases linearly with the increase of organic Wei', but when the organic drug concentration exceeds i3Qppb, the current inhibition rate increases to 70% and does not change. This phenomenon implies that (1) there are (10) pesticides that inhibit _ (or the active site of enzymes that can be contacted by organic pesticides) account for only 70% of all immobilized enzymes; the remaining 3Q% of immobilized enzymes may be affected by too deep or stereoscopic orientation. Organic pesticides cannot be approached; (2) 13_ organic pesticides are sufficient to inhibit all active enzyme sites, and thus the current inhibition rate does not increase at high organic phosphorus concentrations. The linear range (at the circle) in Fig. 21 is redrawn as shown in Fig. 22. From Fig. 22, it can be seen that the biomicrosensor of the present invention has a pesticide-measurable range of 10-130 ppm. This range of application can be expanded by increasing the amount of enzyme immobilized. However, the optimum ratio of inorganic catalyst, gold nanoparticles, pyrr〇ie monomer and enzyme needs to be confirmed again for different electrode sizes (surface areas). Temperature compensation of electrode performance Temperature compensation test for the performance of the electrode at different temperatures. Taking 25〇c as the reference point, the relationship between the current inhibition rate and the organic dish-receiving rate, which is transmitted in the figure 22, is selected as the standard solution of the known organically determined 1 degree, to measure the gentleman's fruit for two stages. Temperature compensation. The first stage is temperature compensation for a single concentration; the second stage is temperature compensation for the full concentration range (〇 -1 ooppb). The measured organic fill concentration includes ··· 20, 40, 60, 80 and lOOppb; the temperature range covers ···1〇, 18, 25, 32 0296-A21230TWF(N2);345;]essica 19 1301541

Ϊ I and 40QC, the results are shown in Table 2. In Table 2, Ρ1 is the original (not temperature compensated) measurement result obtained by the conversion of Fig. 25; P2 is the result obtained after the single concentration temperature compensation; P3 is the result obtained by the temperature compensation in the full concentration range. . Based on the actual measurement results, the temperature compensation equations obtained for each organophosphorus concentration and the full range of organophosphorus pesticides are summarized in Table 3. It can be seen from Table 2 (compare the standard solution concentration with P2), except for the high concentration of organic phosphorus (100 ppb), the SD (standard deviation) is corrected by the single concentration temperature compensation equation, regardless of the ambient temperature. It can be less than 2%; if the temperature compensation is performed by a single full concentration range equation (comparing the standard solution concentration with P3), it can be seen that the worse the ambient temperature is, the worse the compensation effect is when it deviates from 25 QC. When the ambient temperature is between 18-32 QC, the result of compensation for all organic phosphorus concentration is SD<7%; when the ambient temperature is as low as 10 T or as high as 40 °C, the SD sometimes reaches 15%. When the true sample is tested, the content of organic phosphorus in the sample can be obtained by the relationship between the current inhibition rate and the concentration and the full range temperature compensation equation. If the sample temperature is too high or too low and exceeds the temperature compensation range, it is recommended to return to temperature before measuring. Table 2 Raw materials measured by different concentrations of organic phosphorus (Balazon) at different temperatures and temperature compensation results 20ppb paraoxon 40ppb paraoxon 60ppb paraoxon 80ppb paraoxon lOOppb paraoxon °c PI P2 P3 PI P2 P3 PI P2 P3 PI P2 P3 PI P2 P3 10 17.6 20.2 23.8 29.2 40.2 40.9 36.5 59.6 51.8 55.1 80.0 79.5 59.4 87.7 85.9 18 19.1 20.3 21.6 35.2 40.3 40.6 51.1 61.9 59.4 68.7 80.3 80.2 84.1 97.3 98.4 25 20.0 20.0 20.0 40.2 40.2 40.2 59.5 59.6 59.6 80.8 80.8 80.8 100 100 100 32 21.5 20.3 19.3 45.5 40.3 40.1 69.2 58.5 60.7 91.7 80.0 80.2 105 92.7 92.6 40 22.7 20.2 18.4 51.2 40.2 39.8 84.2 61.1 64.7 105 80.1 80.4 118 89.9 90.3 20 0296-A21230TWF(N2);345;jessica 1301541 Table 3 Single concentration and Temperature compensation equation for full concentration range (PI) Temperature correction (Ρ2) Temperature and concentration repair iE(P3) 20ppb P1=0.1703(T-25)+20.18 Ρ2=Ρ1-[0.1703(Τ-25)] P3 =Pl-[Coeff.(T-25)] Coeff = Ω -Π 9Q97 40 ppb P 1=0.733 8(T-25)+40.26 Ρ2=Ρ1-[0.7338(Τ-25)] P3=P1+0.2927( T- 60 ppb P1=1.5369(T-25)+60.1 Ρ2=Ρ1- [1.5369(Τ-25)] 25)/1+0.0267(T-25) (excluding lOOppm) 80ppb Ρ1=1.6597(Τ-25)+80.26 Ρ2=Ρ1-[1.6597(Τ-25)] P3=Pl -[Coeff.(T-25)] Coeff =0.0216P3-0.1039

P3=P1+0.1039(T-25)/1+0.0216(Τ-25) (Including IOOdditD Current Output Signal Digitization A series of experimental results are used to establish the relationship between the current inhibition rate and the organic fill concentration at a fixed measurement time. The correlation is sintered on the wafer, the connection of the signal acquisition system is established, and the signal digitization is completed, which is used as a digital signal transmission system of the biological micro-sensor. Basically, the development of the digital signal transmission system is mainly divided into soft and hard. The body part is carried out. The hardware part includes: palm-type potentiometer, signal transmission line (RS232 cable), signal analysis and recorder (microprocessor), etc. The software mainly uses LabVIEW (Laboratory Virtual Instrument Engineering Workbench) The programming language is used to control the signal acquisition, transmission, analysis and recording between hardware. LabVIEW is an image-based programming language developed by National Instrument in 1986. Its main purpose is to fully integrate control. Communication interfaces such as GPIB, VXI, PXI, RS232, and data presentation, data storage Data analysis, data acquisition, and serial instrument control, because the modular instrument Express Vis in LabVIEW will enable prototyping and testing of mixed-signal design and automatic generation of automated test programs. Code; ensures that the Real-Time Desktop PC function will optimize system stability and performance in any 0296-A21230TWF(N2);345;jessica 21 1301541 t \ PC-based test system; fast PDA data capture performance and DMM Support, establish a customized portable data capture system; Bluetooth support - use wireless Bluetooth technology to communicate with other devices; 50 new mathematical function - use the LAPACK / BLAS basic library to increase accuracy Degrees and speeds up to 200%; Hyper-Threading technology to increase system execution by up to 100%. In addition to the concentration of contaminants available for detection, it can also be used for detection conditions, including: suppression It is quite convenient to change the time, oxidation potential, and current recording frequency. > Real sample detection In order to confirm the biological micro-sensing developed by this research The practicality of the device was preliminarily tested for the commercially available juice. The results are shown in Figure 23. During the experiment, the tomato juice stock was firstly tested for organophosphorus pesticides, and then in the stock solution spike 3 Oppb. Pesticides, observe whether the sensor can make accurate measurements. In another experiment, the tomato juice stock solution was first diluted ten times for organophosphorus pesticide measurement, and then the spray 3 Oppb of the bala pine pesticide was similarly observed in the dilution, and the measurement of the sensor was observed. The overall pesticide inhibition time in the experiment was 10 minutes. > It can be clearly seen from Fig. 23 that the concentration of organophosphorus pesticide in the tomato juice stock solution is 25.6 ppb, and the value becomes 54.9 ppb after spike 30 ppb bala pine pesticide, and in another experiment, after ten times dilution The concentration of the pesticide in the diluent is lower than the measurable range, and after the spike 30ppb, the organophosphorus pesticide in the diluent becomes measurable value of 39.6ppb. The results show that the bio-micro-sensor developed by the research institute can accurately measure the residual amount of organophosphorus pesticides in the juice, and the interfering substances in the juice, such as sugar, color, suspended solids and antioxidants, etc. Will not have any effect on the measurement results. This system is shown to be used for the measurement of real samples. 0296-A21230TWF(N2);345;jessica 22 1301541 Industrial Applicability+The present invention combines biotechnology with nanotechnology to develop an enzyme reaction based on biochemical sensing, which can be targeted at the local water environment. The rapid and sensitive detection of pollutants can save time and money compared to the chemical analysis methods of traditional laboratories, and the convenience of use is greatly enhanced. The polymerization method is used to fix the enzyme together with the inorganic catalyst and the gold nanoparticle factory on the surface of the electrode. Due to the presence of the inorganic catalyst, the voltage value required for detection can be reduced, thereby greatly reducing Other substances in the environmental sample = the noise generated by the chemical. In addition, the high specific surface area of the gold nanoparticles and the specificity of the Γ7' package can amplify the current signal generated by the enzyme reaction, thereby greatly increasing the sensitivity of the biological micro-sensor and improving the detection limit. Shorten the detection time: According to the experimental results, the relationship between the current output signal and the analyte concentration is established. With the establishment of the current signal output digital system, the developed biological micro-sensor will be both sensitive and fast. Accurate, low cost and easy to use. The volume of the biological micro-sensor produced in this volume is less than 丨 kg, and the pesticide in the water sample can be accurately measured below the detection time within 1 〇 minutes. Compared with the traditional biosensor, The biosensor of the invention not only has a volume and weight which is two orders of magnitude lower than the limit of _ _, the detection __ is short, and the side cost only needs about 10 it, and the application of the loss detection detection will be greatly improved. Although the present invention has been disclosed in the above preferred embodiments, it is not intended to limit the invention, and the invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application attached. 〇296~A21230TWF(N2);345;jessica 23 1301541 [Simple description of the drawing] Fig. 1 is a specific embodiment of the biological micro-sensor of the present invention. Figure 2 illustrates the change in background current (no H202 added) of the electrode system of the present invention. Figure 3 illustrates the change in current signal output for a batch of 2μΜ H202 solution added to the system. Figure 4 illustrates the change in output current value and H202 concentration before and after H202 addition. Figure 5 illustrates the inhibition of the activity of organophosphorus pesticides on enzymes before the addition of enzymes (unfixed enzymes). Figure 5A shows the blank test for unfixed enzymes and no pesticides added; Figure 5B shows the change in current with a concentration of balazone of 0.47 ppm before unfixed enzymes; and Figure 5C shows the concentration of balamant before adding unfixed enzymes. The change in current at ppm; the 5D plot shows the change in current at a concentration of 47 ppm for the balason before the enzyme is immobilized; and the change in the current at which the concentration of the balason is 470 ppm before the unfixed enzyme. Figure 6 shows that when the amount of acetylcholinesterase (AChE), cholinesterase (ChE) and choline oxidase (ChO) added (enzyme is not fixed) is 1 mM, 0.004 unit and 0·2 丨 unit, For different concentrations of balamon (47 ppm, 4.7 ppm, 0.47 ppm) and changes in sensitivity of the action time. Figure 7 shows the relationship between suspending enzymes (unfixed enzymes) at different inhibition times and current inhibition rates at a fixed balason concentration (〇.47 ppm). Fig. 8 shows the electrode sensitivity before and after immobilizing the enzyme on the surface of the platinum electrode by a sol gel method. Bar (a) uses a sol gel method to immobilize the surface of the platinum electrode; column (b) is a standard platinum electrode that is not fixed and suspended in the sample; and column (c) is unfixed and suspended in the enzyme. 1 cm of platinum electrode in the sample; and column (d) is used to embed the enzyme in sol gel, directly placed in water, 0296-A21230TWF (N2); 345; jessica 24 1^01541 does not fix it The electrode on the surface of the electrode. ^ Figure 9 shows the current output signal of the enzyme before and after electropolymerization, f ° block (4) using the electrode of the unfixed enzyme; resting (8) using the fixed bismuth (four) dish monomer / enzyme acetylcholinesterase (AChE) and Peroxidase (HRp) is an electrode called 2/1〇_'s (c) using a fixed pyrrole (pyrr〇le) monomer/enzyme AChE and ferrocene for 2〇μ1/1〇〇〇υ electrode delay (4) using a fixed batch of (four) Ting (four) monomer / enzyme AChE is 〇.013 called /1〇〇〇1; the electrode; and block (4) using a fixed lot (Pyrr〇le) monomer / enzyme AChE is 20pl / 1000u electrode. The figure ίο shows the effect of the oxidative packing decrease in the presence or absence of the inorganic catalyst (Prussian blue Fe(CN)63_); the solid line indicates no Fe(CN) wide; the broken line indicates the presence of Fe(CN)63·. Figure 11 shows the oxidation current values of ascorbic acid at different applied voltages. 12 is an electron scanning micrograph of the gold nanoparticles used in the present invention. Figure 13A shows the relationship between the electrode and electrode sensitivity of fixed or unfixed enzymes and nanogold particles; the square mark represents the use of ungranulated electrodes; the three (four) mark represents the appointment of „与金奈米'=2 pole. The graph shows the relationship between the concentration of nanogold particles added and the sensitivity of the electrode when using an electrode with immobilized enzyme and nano-gold particles. Figure 14 shows the relationship between the volume of nano-gold particles and the sensitivity of the electrode. The micro-twister makes the enzyme activity resilience. Figure 16 illustrates the change of the sensitivity of the bio-sensing device of the present invention at different temperature measurement. Figure 17 shows the biological micro-sensor of the present invention detecting at different pH values. Measurement, change of current output signal. Figure 18A shows the relationship between methanol and the enzyme activity of the biological microsensor of the present invention; the figure shows the relationship between ethanol and the enzyme activity of the biological microsensor of the present invention 0296-A21230TWF ( N2); 345;] essica 25 1301541 Department, brother 18 C picture shows the C-class disk system. Called this (four) biological micro-μ (four) material activity of the relationship between the activity of the relationship between the 19th map and the biological micro-invention Detector relationship 4 shows the relationship between iron ions and the bio-microsensor of the present invention. '帛19C shows the relationship between copper ions and the fermentability of the bio-microsensor of the present invention.: The coffee chart shows the wrong ions and the present Inventive bio-microsensor== Sexuality: Figure shows sulphate called. Enzyme activity of the bio-microsensor of the present invention is illustrated in Figure 21 _ time 1G minutes 25 ° C under the concentration of balason and the present invention

The relationship between the current suppression rate of the bio-micro sensor. X Figure 22 illustrates the current suppression ratio of the bio-microsensor of the present invention and the linear range of Barneson. ^ Figure 23 shows the data and comparison of the biological microsensor of the present invention for detecting tomato juice stock solution and adding tomato juice of bala pine pesticide. Block (a) use tomato juice stock; column (b) use tomato juice with spike 30ppb bala pine pesticide; column (c) use diluted tomato juice stock; and column (d) use 10 times diluted spike 30ppb bala pine pesticide tomato juice stock solution. [Description of main components] 101~electrode 120~processing unit 103~electrode 122~display unit 105~electrode S1~current 5 tiger 0296-A21230TWF(N2);345;jessica 20 1301541 110~temperature sensor S2~temperature signal

0296-A21230TWF(N2);345;jessica 27

Claims (1)

1301541 The pesticide is organophosphorus or carbamate. 12. The method of detecting a pesticide according to claim 9, wherein the electrode and the temperature sensor are disposed on a flat plate. 13· a biological micro-sensor, comprising: at least one electrode for detecting a current signal; a processing unit for receiving and processing the current signal, and converting the current signal; becoming a concentration data; and displaying a unit for displaying the concentration data; • wherein the surface of the electrode is immobilized with an enzyme, a gold nanoparticle, and an inorganic catalyst. 14. The biological microsensor of claim 13, wherein the enzyme is acetylcholinesterase or choline sterolase. The biological micro-sensor of claim 13, wherein the inorganic catalyst is Prussian Blue (Fe(CN) 63-). The biomicrosensing according to any one of claims 13, 14 or 15, wherein the electrode surface is fixed by an electropolymerization method. ^ I.7. The biological micro-sensor according to claim 16, wherein the turtle is a method for collecting polypyrrole on the surface of the electrode, immobilizing the enzyme, Chennai. The rice granules and the inorganic catalyst are on the polypyrrole layer. 18. The biological micro-sensor of claim 17, wherein the gold nanoparticles have a concentration of 〇.5_2.〇ppm. 19. The biological micro-sensor of claim 2, wherein the gold nanoparticles have a particle size of 15.5-2 〇 nm. λ 20. The biological micro-sensor of claim 13, wherein the electrode is a platinum electrode. 21) The biological micro-sensor as described in claim 13 of the patent application, further comprising a -Mongo sensor for detecting temperature and transmitting to the processing unit. The bio-micro sensor of claim 21, wherein the current signal is temperature-compensated by the detected temperature. 23·If the patent application scope is claimed, the method is as follows. The biological micro-sensor of claim 13 is for detecting a pesticide. The biological micro-sensor of claim 23, wherein the drug is organic phosphorus or carbamate. The biological micro-sensor of claim 21, wherein the electrode is on a flat plate at the same time as the temperature sensor. Tim 26 - A method for reducing current noise, comprising fixing gold nanoparticles and inorganic ruthenium on an electrode surface, wherein the surface of the electrode is immobilized by an enzyme. 27. The method of reducing current noise according to claim 26, wherein the inorganic catalyst is Prussian Blue (Fe(CN)63_). , 28) If the method of reducing current noise is applied in the 26th article of the patent application, the particle size of the Chennai particles is 16.5-20mn. 29) The method for reducing current noise as described in claim 26, wherein the concentration of the gold nanoparticles is 0.5-2.0 Ppm. 彳, r —·such as the reduction of current noise in the scope of claim 26 The method is to fix the gold nanoparticles and the inorganic catalyst by electropolymerization. 方法 The method for reducing current noise according to the item in the range of item %, ', ^ electric hard. The fixing method is to collect a layer on the surface of the electrode (poly ^rmle)" Fixes the gold nanoparticles and the inorganic catalyst on the layers of the poly-port. If the current noise is reduced as described in claim 26, the enzyme is acetylcholinesterase or choline oxidation. Enzyme. ... law, 0296-A21230TWF(N2);345;jessica