TWI302198B - - Google Patents

Description

1302198 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a method for simplifying optimization in the development of a chemical sensor system, and more particularly to a simplified biosensor system development process. The method of optimizing the program. [Prior Art] The sensor with pH change as the detection reference has become the 4th-ring in the development of sensor technology. This thief measures the H-side job index to sense the proton or hydrogen in the chemical or enzyme reaction. The concentration of root ions changes. In some dye-sensitive dye molecules (such as bromophenol blue [br〇m〇phen〇lblue], bromocresol pUrpie], thymol blue [thym〇1 Wue], 9-(4-diethylamino -2-octadecyloxy-styryl).acridine, carboxy-seminaphtharhodafluor], aminofluorescein and fluorescein], high fluorescence concentration and high fluorescence yield due to fluorescence yellow at argon ion laser wavelength of 488 nm The well-known optical property 'is a fluorescent probe suitable for the above-mentioned sensor. In addition, since fluorescent yellow has been commercially used in dextran derivatives having different molecular weights, it is possible to reduce the problem of leaks >4 which are conventionally stored in sol-gel or polymer embedding. lowest. The visual 'model simulation' has become a useful tool to describe the phenomena in the experiment and to estimate the behavior of the survey system in different situations. A reliable mathematical model not only helps to confirm the correctness of the observation 1302198, but also to clarify the possible reaction mechanisms that exist in the system. What's more, in the development of sensors, such models can also be used to simplify the optimization process. Although the design and application of the above sensors have been extensively studied, the mathematical model that can be used to predict system 1) 11 value changes and result signals remains to be developed. SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for simplifying the optimization process in the development of a chemical sensor system. Another object of the present invention is to provide a method of simplifying the optimization process in the development of a biosensor system. The present invention provides a method for simplifying an optimization procedure in a development of a biosensor system, wherein the biosensor system has a buffer solution, a reactant and an enzyme, and the biosensor is changed in pH. Detection benchmark. First, it is estimated that all components of the biosensor system have a fucking ability to the buffering strength of the buffer solution, and the part of the composition is derived from the interaction of the substance and the enzyme. Next, the hetero-buffer strength mode 4 calculates the buffer strength, and the distribution of the lining ability is known under the combination of various reactant concentrations and different yang values. When the biosensor system reaches equilibrium, the buffering capacity is integrated to know the final pH of the biosensor system. [Embodiment] 1302198 The present invention discloses a method for simplifying the optimization procedure in the development of a biosensor system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In accordance with a preferred embodiment of the present invention, the following is detailed. 1. Principle 1.2 Linear buffer strength model 〇inear buffer capacity model) The slope of the titration curve is related to the change in pH in the solution. Please refer to the following reaction formula (1). The buffer capacity of the solution (10) is inversely proportional to the slope of the titration curve:

dpH dpH

Among them, CA and CB are strong acid/strong alkali concentrations added to the system. In fact, the buffer strength can be obtained by various methods (such as general titration curve model, linear buffer capacity model, non-linear symbolic buffer capacity model, and nonlinearity). The non-linear tableau_method based buffer capacity model. If only the acid-base chemical equilibrium is considered, the linear buffer strength model represented by a series of reaction formulas is sufficient to describe the observed buffer capacity related data. Nonlinearity refers to the results of other reactions in the system (such as complex reaction and precipitation reaction), which are also taken into consideration. At this time, the parameters related to the reaction equilibrium in the model are nonlinear. In contrast, the linear buffer strength The model has the advantages of fast calculation, extremely accurate 1302198* (impiementati〇n) and fault tolerance (10), and (4), and easy to apply to the new model. It is based on this model to calculate the buffer strength of the solution. 7 with / single proton (aqueous), w double proton, For example, the weak acid of the three protons is calculated as the following equation. · (2) (3) β = 2303χΙΗΊχίι + Σ^^^ 1 b erm x = C aK a --!_ i[H+]+ Ka )2

Term 2 =CaKaX

(4) (5) where ca and Ka are each a weak acid concentration and dissociation constant, and cut mi, term2, and term3 represent a single proton, a double proton, and a triple mass, respectively. The buffer with a total buffer strength equal to the contribution of the three constituents is treated as a single protonic acid. Compared with the Qianding curve method, the linear buffer strength model does not take into account the variation factors related to the added amount, which is a method for facilitating the execution of computer programs. ', 1.2 Buffering capacity in biosensors 1302198 As can be seen from equations (2) to (5) above, the pH of the solution and the possible components present in the > trough are 'two buffering capacities. The main parameters. In this example, the urea lysis, creatinine deiminase (CD) and acetylcholinesterase (AChE) are taken as examples to illustrate the appropriateness and applicability of the linear buffer strength model. . Please refer to the following equation, which is the hydrolysis reaction catalyzed by urea decomposition, creatinine deimin and acetylcholine ester. (English nouns in the chemical formula have been translated in the text): (A)

II urease H2N-C-NH2 3H20 Urea 2ΝΗ: + HCCV+ Ohf (B)

o HN

Creatinine deiminase HfsT CH3 Creatinine H20

NH4+ + 〇H- N-Methylhydantoin N _ Cf (C) ^ AChE H3C-c-〇-(ch2)j~n (Chya h2〇 H+

Choline

Acetylcholine in the system of urea decomposition, 'ammonium ions, carbonate ions and hydroxide ions are released from the system after hydrolysis reaction. Similarly, in the hydrolysis reaction catalyzed by creatinine deimin, the presence of amine salt ions and hydroxide ions can also be observed, the only difference being that the hydrolysis system will produce N-mercaptoacetone 1302198 (N -methylhydantoin). Different from the above two reactions, acetylcholine is decomposed by acetaminophen, which produces choline, acetic acid and hydrogen protons. Referring to Figure 1, the water, i, 3-, [3, bis[hydroxymethyl]methylamino]propane; BTP] (hereinafter referred to as BTP) buffer solution is shown. And the dissociation constant of each reactant/product in the above three formulas. Since these components all affect the buffering capacity of the solution, it is necessary to take into account its utility. However, it is worth noting that in the present invention, acetylcholine and urea are not included in the model. This is due to the fact that acetylcholine does not have a dissociation center, so it is assumed that it does not produce a dissociation reaction. ,In addition, the dissociation constant of urea is -2.54, (U, 14.43 and 18.30 (calculated using ACD/pKa ν7·02 software purchased from Advance Chemistry Development, Canada), since these dissociation constants have exceeded the normal pH range. (ΡΗ 1-14) is to ignore its contribution to the buffering capacity of the solution. In calculating the buffering capacity, it is assumed that the moiré of the product follows a stoichiometric relationship. The present invention is based on a linear buffer strength model and a predetermined amount of product. A program (MATLAB) was developed to calculate the buffering capacity of different biosensor systems, and the 3D distribution of buffer strength was obtained under different reactant concentrations and different pH values. Figure 2A~C A 3D distribution of buffer strength at a concentration of 1 mM BTP buffer at different pH values and different reactants (urea, creatinine and acetylcholine, respectively), wherein the pH is set at 5 Up to 10 to meet most biological systems; the concentration of the reactants is set to 1 μΜ to 10 1302198 M. The simulation results show that in the urea decomposition system, when the pH is 6.4, the buffer strength 0.06 dM/dpH; at a pH of 9.2, the buffer strength is 0.14 dM/dpH. In the creatinine deiminol-creatinine system, when the pH is 5, the buffer strength is 〇·〇6 dM/ dpH; and when the pH is increased to 9·3', the intensity of the acuity also rises to 0.12 dM/dpH. It is different from the above. In the acetylcholine decomposition system, when the pH is greater than 6, the buffer strength is almost maintained. No change, this may be due to the dissociation constant of acetic acid and biliary test (4.76 and 13.9 each) has deviated from the measured pH range (pH 5 -10) 〇 in the pH-based biosensor The proton or hydroxide ion is generated from an enzyme reaction, and the amount of these ions should be related to the modified reactant concentration. Since these ions will resist the total buffer capacity, the sol-condensation can be changed. The pH value in the gel matrix. In view of this, when the system reaches equilibrium, the integral of the buffering capacity obtained from the pH value should be equal to the molar concentration of the proton or hydroxide ion in the following equation (6). The reactants are incorporated into a BTP buffer solution containing different concentrations and initial pH, systemic The final pH value can be known.

Ca or CB = j β dpH 1·3 FITC-Emitted fluorescence of FITC-dextran Fluorescent yellow is mainly composed of cation (FH/), neutral (FH2), monovalent 1302198 anion _ Or a dissociated form such as a dianion (F2·) is present in the optical properties of the fluorescein and the pH is closely related to the acidity of the biosensor as described above. After the concentration, the molar concentration ratio of each of the dissociated I of the fluorescent yellow is obtained by the amount of f and the dissociation constant. Equations (7) and (8)__(9) The mass balance equation and the dissociation equation.

~ΛρΗ) = cfh + (PH) + c+ c-yang (pH>c(4) FH;-)fh2+h+ PK' = 2.08 fh2 ^FH^H+ PKa2 = 4.31 fit - + F2- + H+ Team 3: = 6.43 (7) (8) (9) (10)

Figure 3 shows the calculated Mohr concentration ratio at different pH values. Since fluorescein yellow has three _ constants, it is a mode in which the distribution follows a typical three-proton weak acid. For biological or biomedical systems, biological activity is depleted in low pH (pH < 5) and high pH (PH > 9) environments, depending on the pH range. Located between 5_9. In this pH fine towel 'fluorescent yellow main fine-valent anion and divalent anion dissociation form is present in the solution; neutral type accounts for less than 2%%; as for the cation, when the pH is greater than 5 The effect can be ignored.

In 1995, Sjoback et al. confirmed the dissociation equilibrium of fluorescein yellow, and determined the absorption and fluorescence characteristics of each dissociation type, so that the distribution of each dissociation state at different pH values (indicated by ~ (office)) was Calculate to get 12 l3 〇 2i9g know. What is more, after obtaining the above distribution, the fluorescence intensity of the fluorescent yellow at different excitation wavelengths, different laser wavelengths and different pH values (represented by /(H, 〆/), respectively) can be obtained by the following equation (11) - (15) derived. , , pH )^K{[Term FH. + Term FH 2 + Term F//. ] x Φ ^ _ (Aex )I FH-{λ em)

+ r^F2_x〇/2.(^)V(^)} (11) (12) (13) (14) (15)

Term : c + {ρΗ)ε + {λ )φ^ FH3 玛 FH, ->FH2 FH2->FH-

Termfh2 ·· cfh2 (pH)εfh2 rew (handy from) τ^ηηρ2_\ 〇ρ2.{ρΗ)ερ2.{λ6Χ) where & is the instrument parameter, in order to simplify the calculation, this parameter can be set to j.

~ (Secret is the concentration of a specific dissociation type (X species) of fluorescein at different pH values; heart (allow) and 〇> ((夂) each is the Mohr concentration of the specific dissociation type Absorbance (M'm-i) and fluorescence yield. The gas percentage is the firefly produced by the first special dissociation type (X!) converted to the second specific dissociation type (3⁄4). Light yield; G(;L) represents the laser obtained after normalization of the specific pattern; as for u, the excitation wavelength (nm) 〇 Figure 4 shows the optical properties and dissociation constant of the fluorescent yellow. At a pH of 13 1302198 At f, 'there is a relatively low molar concentration absorption and a negligible Mohr concentration ratio due to the cation dissociation between Wei and the towel. It is: the neglect of the fluorescence intensity, and the health-listening The condition of the dissociative ion dissociation type. (4) To minimize the leakage effect of the conventional sol-gel embedding 'fluorescent yellow-derivative' FITc___, observation of the type of biological sensing [FITC_« "There is a similarity to the fluorescence of yellow, the nature of the study, these properties include absorption and excitation spectra, in addition to fluorescent κ and FITC-glucan The dissociation constants are similar. For these various reasons, the optical parameters of Fluorescent Yellow are used to present biosensor systems with FITC-Glucan as the embedding material, and the accompanying simulation process. A flow chart showing the development of a biosensor model developed using FITC_glucan as a pH probe. The decomposition of urea embedded in a sol-gel can be obtained by the steps described in the figure. Fluorescence performance of enzyme biosensor, acetylcholine biosensor. 2. Experimental details 2. 1 GC/MS analysis using a Hewlett-Packard 5973 mass spectrometer (GC/MSD) Hewlett-Packard 6890 Gas Chromatography Analyzer for the determination of organic compounds decomposed from acetylcholine. As a result, the column used contains a stationary phase of polystyrene-divinylbenzene. 0.316 mm LD·, J & w Scientific) 30_m GS-Q. Nitrogen is used as a gas with a flow rate of 13.3 ml/min. The injection port temperature of the mass spectrometer is set to 1302198 degrees. 230 degrees, the temperature of the quadrupole is set to 150 degrees. The phenomenon occurs in the ion impact (EI) mode (70 eV). The voltage and amplification of the electron multiplier tube are based on the built-in value of the instrument. The mass spectrometer is calibrated with electrons of tetrafluorotributylamine. The impact debris is the reference, and the charge-to-mass ratio is 69, 219, and 502. In the full scan scan mode (f^ii scan acquisition mode), the mass spectrometer scans from 5 to 500 atomic mass units. The gradient change of the column temperature is set at 42. (: Maintain for 4 minutes, then increase by 8 per minute. (: The speed until the temperature reaches 240. (: Stop. The system temperature takes about 28/75 minutes per operation. 2.2 Liquid Chromatography Mass Spectrometer Analysis and LC/MS analysis and NMR Spectroscopy uses a vacuum pumping tube, a P4000 quadruple grading pump, a UV6000LP diode-array UV detector, and Liquid chromatography mass spectrometry experiments were performed with a TSP LC system equipped with an electrospray ionization source (ESI, Finnigan MAT) and a Finnigan LCQDU0 quadrupole ion trap mass spectrometer on XCALIBUR 1.2 software. The sputter ionization is set in positive ion mode, the electrospray voltage is 4500 volts, and the mass spectrum obtained is the charge to mass ratio of 80 to 1000. In this experiment, methanol is used as a carrier solution at a concentration of A 0.5 mM BTP (loading ratio of 283.3) solution was used as a standard within the quantification. The 1 Η and 13 C NMR spectra were obtained from Varian Unity Inova 500-MHz. Spectrometer measured, and 15 8 using the four-base stone as the internal standard. 3. Results and discussion 3.1 pH determination. In the absence of enzymes, the biological sensory benefits embedded in the system can be used to make pH changes, and because enzymes are not present in such systems' are chemical sensors that measure pH changes in this type of system. Figure 6 shows the experimental series (open squares) from the sensor array and The calculated fluorescence intensity (curve) at different pH values. It can be seen from the figure that when the pH value is upward, the 'Wei New series increases; when the pH reaches 8.5', the increase of the fluorescence intensity tends to be gentle. The results of the simulation are quite consistent with the actual results 'the glory intensity derived from the aforementioned theory' is quite suitable for describing the behavior of the sensing H array. The advantage of the model simulation is that the dissociation patterns can be distinguished from the pH value. The contribution 'and the possible mechanism of the ray-forming. Figure 7 shows the contribution of the respective dissociation patterns of fluorescein to the fluorescence intensity at different pH values. The results show that the pH is between 3 and 6 Anion solution is proud The main contributor to the intensity; and when the pH range is 6 to 9, the glory intensity is mainly controlled by the second ship. The results also prove that the 刼 ion dissociation type and the neutral dissociation type The contribution of the light dragon 磬 磬 , 〇 〇 〇 〇 3.2 3.2 3.2 3.2 3.2 3.2 3.2 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感 敏感The optimization program of the item. Figure 8 shows the simulation results of the biosensing of the urea-decomposing enzyme type, in which the urea decomposition reaction is carried out in a BTP buffer solution (concentration of imM) of different pH values, and the results show that the initial pH value of the system has a very good analysis result. The large effect, that is, when the initial pH is low, a larger dynamic range (dynaiJc range) and a more southerly sensitivity are obtained. Figure 9 shows the simulation results of a urea-decomposing enzyme type biosensor, in which the urea decomposition reaction is carried out in different concentrations of BTP buffer solution (selected pH value of 6 〇), and the results show that the concentration of the selected buffer solution is not Will affect the dynamic range, and it has a significant impact on system sensitivity. When the concentration of the selected buffer solution is low, the force of the new ship is also low, and the sensitivity of the 纟(10) measurement is increased. Waicerz et al. also obtained similar results on electrochemical biosensors in 1995. Buffer solutions in the system when urea was measured using a pH membrane electrode-based u_e biosensor. A decrease in pH and concentration results in an increase in sensitivity. What's more, even if the pH of the buffer solution decreases, the dynamic range of the system increases, and the value of the buffer solution has a great influence on the dynamic range of urea detection. In addition to the above model applicable to BTP buffer solution systems, the present invention also investigates its suitability for use in other buffer solution systems including tris(hydromethyl)-aminomethane-HCl; Hereinafter, •s HC1], 2-(N-morphin)ethylsulfate [2-〇sj_morph〇lin〇) 17 1302198 ethanesulfonic acid; hereinafter referred to as MES], sputum [imidazole], N-(2-hydroxy-B Piperazine-N'-(4-butanesulfonic acid) [N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid); hereinafter referred to as HEPES] and buffering system such as linoleic acid, Figure 10 The dissociation constants and pH ranges of these buffer solutions are also listed. Figure 11A to E show the simulation results of a urea-decomposing enzyme type biosensor in which the urea decomposition reaction is carried out in different buffer solution systems. At the time of the simulation, the concentration of the buffer solution was 1 mM, the pH was 5 to 10, and the urinary concentration was between 0 and 100 mM. The results show that the 3D distribution of MES, imidazole and phosphate buffer solution is similar to the 3D distribution of BTP buffer solution. The distribution of Tris-HCl and HEPES is different from the above buffer solution. 3.3 Urea determination In the urea hydrolysis reaction of urea decomposition catalysis, according to the stoichiometric relationship, it can be known that 1 mole of urea can produce iMo's bicarbonate ions, hydroxide ions, and 2 moir amine salt ions. Where the hydroxide ion increases the pH of the sol-gel. The hydrogen carbonate ion and the amine salt ion are the main components of the total buffering capacity of the solution and are resistant to an increase in pH. When the above reaction formula reaches a chemical equilibrium state, the final pH of the system can be recognized by the FITC_glucan embedded in the sol-gel and expressed as fluorescence intensity. As for the sol-gel matrix, there may be some ungelatinized hydroxyl group IX si_〇H, ξ represents a bond with the other phase, ϋ) This oxy group can affect the total buffering capacity of the solution, With the invention (surface deprotonation

18 1302198 constant of silica ; (pKas= 6·8) 14). Figure 12 shows the results of the fluorescence intensity obtained by the model calculation (solid line) and the experimental results (open circles) detected by the biosensor, wherein the urea concentration is 1 μΜ to 1Μ, and the reaction time is 1 minute. As can be seen from the figure, the simulation results are in agreement with the experimental results, and there is only a slight difference in the low concentration region, indicating that the model disclosed in the present invention is suitable for predicting biosensor behaviors using FITC-glucan to detect valence changes. . 3.4 Determination of creatinine acid Similar to the above reaction system, creatinine acid is hydrolyzed by creatinine dehydroacetate to produce amine salt ions and hydroxide ions. However, since creatinine has a dissociation constant (value of 4.83) which is close to the measured pH range (pH 5 - 1 〇), it is also considered to have an effect on the buffering capacity. Figure 13 shows the results of the fluorescence intensity obtained by the model calculation (solid line) and the experimental results obtained by using the biosensor's debt (open circles), wherein the urea concentration is 1 μΜ to 1 M, and the reaction time is 1 minute. As can be seen from the figure, there is a considerable difference between the simulation results and the experimental results. There are two possible reasons for the speculation, that is, the dissociation constant used is not correct or the system has not reached equilibrium. Since the dissociation constant of creatinine and _methylhydantoin _methylhydant 〇in) is predicted by the aforementioned software, its correctness needs to be further confirmed. After investigation into the database (ACD/pKa DB v7.〇25 Advance Chemistry Development, Inc., Canada), it was found that the dissociation constant of creatinine and N-methylhydantoin was divided into shun Yang (Failey, CF; B_). , E^ ship 1302198 1933, 102, 767) and 9.09 (Campi, E; 〇stac〇H, G;

Vanni, A. Gazz·C7w.m.侃 1965, 95, 796). Since the above dissociation constant is similar to the dissociation constant used in the model simulation, the difference between the pseudo value and the actual value may not be due to the fact that the dissociation constant is not correct. The sol-gel technique of embedding the above enzyme may delay the diffusion of the analyte or product in the porous structure, and the activity of the enzyme caused by the release of the solvent (such as methanol) or the aging of the glutinous gum may be reduced. The rate of enzyme reaction observed in the sol-gel system is slower than that observed in aqueous solutions. Compared with the reaction rate of urea decomposition and acetylcholine ester (3545 S·1 and 6807 S·1, respectively), creatinine acid has a lower reaction rate (668 s·1) due to creatinine acid deacetylation. Upon hydrolysis, the reaction system may not have reached equilibrium. In order to prove this hypothesis, a higher concentration of creatinine deacetylate (丨u/mL) was prepared in a pH of 6.0, 1 mM Βτρ buffer solution to catalyze different concentrations (0.02 to 20 mM) of creatinine The decomposition, during which a pH electrode is used to monitor the change in pH. This method directly records the change in pH of the system'. The error in the signal delivered from the pH to the fluorescence intensity via FITC-glucomann is minimized. Figure 14 shows the change in pH of the solution in the creatinine acid hydrolysis system. The results show that when creatinine is added to the reaction tube, the pH of the solution gradually increases. However, at 1 minute, some reactions have not reached equilibrium, especially in the higher concentrations of creatinine (1 and 20 mM) systems. From the relationship between the pH value and the fluorescence intensity shown in Fig. 13, it can be seen that in the system for decomposing creatinine using creatinine to deacetylate, the pH recorded at 10 1302198 minutes can be converted into fluorescence intensity. Referring to Figure 15, the simulation results (solidline) and the experimental results (solid squares) are consistent, meaning that the difference between the above simulation results and the experimental results is due to the system reaction has not yet reached equilibrium. In view of this, the reaction time of the creatinine-depleted biosensor was corrected to 3 〇 minutes, and the result was plotted as a hollow circle in Fig. 15. As shown, the above results are consistent with the analog values, indicating that the creatinine system takes longer to reach equilibrium. 3.5 醯 醯 验 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱 虱It is a near-valued estimate and it is usually slightly lower than expected. This intrinsic limitation cannot be manifested only by the calibration curve; and 'when acetylcholine is used in the determination of acetylcholine, this limitation is not considered in biosensors. . Unlike the above, the present invention simulates the theoretical value of the concentration input into the model, which can be used to clarify this limitation. Like the urea decomposition and creatinine deacetylation system, a similar method can be used to calculate the strength of the acetylcholine type biosensor, where the concentration of acetylcholine is 1 μΜ to 1 M. When acetylcholine is decomposed, protons are generated. Therefore, when the concentration of acetylcholine increases, the FT value decreases, and an inverted sigmoidal curve as shown in Fig. 16 is formed. Please refer to Figure 16 for the reaction 21 21 2198 curve (open circle) detected by the biosensor and the experimental result (dummy line) obtained by using the model calculation. Among them, in the model simulation, 100% of the acetylcholine system with an initial pH of 8.5 was applied to the model. As can be seen from the figure, when the concentration of acetylcholine is 0.9 - 4 mM, the simulated value is highly consistent with the experimental results. However, when the concentration of acetaminophen falls outside this range, there is a significant difference between the simulated value and the experimental results. . This phenomenon may be caused by a decrease in the positive value of the night and the concentration of the test. It is well known in the art that when 1 mM of acetylcholine is formulated in a Tris-HC1 buffer solution having a concentration of 10 mM and a pH of 8 Torr, the pH will drop by one unit after 10 hours. Figure 17 shows the change in pH in the solution over time. The 5 mM acetaminophen system was prepared at 浓度τρ buffer/glutle solution at a concentration of 丨 mM and pH 8.5 (this is the use The biosensor measures the stock solution of acetylcholine. As shown, within 1 minute, the pH dropped significantly from 8.5 to 7.6, after which the pH value continued to drop slowly to I] (1 (8) minutes). These results indicate that the initial pH value of the biosensor is different from the initial pH used for model simulation. Since the choline solution used in the present invention is freshly prepared and used within 10 to 20 minutes, the initial pH value of 7.5 is selected as the initial pH value for subsequent model simulation. Another difference between the experimental results and the simulated system (which occurs at a high concentration of the sputum test) may be due to changes in the concentration of the acetaminophen. As previously described, 'chlorinated B' is a highly absorbing compound that rapidly absorbs the moisture of the air towel during the preparation of the process towel. Under this premise, the concentration of the prepared sputum test is actually lower than the biliary value; another possibility that causes the difference is the automatic decomposition of acetylcholine, which is worth 22's. Decomposition does not change the pH. Since the automatic decomposition process of acetylcholine is still unknown, the reduction rate of acetammetry is obtained by comparing the simulated value at pH 7.5 with the experimental results. Please refer to the 18th. When the actual concentration of B is the 7G% of the expected concentration, the simulated curve (solid line) and the experimental result (: heart circle) are in good agreement. These results show that the model disclosed in the present invention not only clarifies the inherent limitations of the directional hygroscopic compounds (such as initial pH variation or reset change) that are not considered in conventional biosensor systems, and This limitation provides an explanation of the effects of biosensors. The present invention has been described above with reference to the preferred embodiments thereof, and other equivalent modifications and modifications may be made without departing from the spirit and scope of the invention. The scope of patent protection of the present invention is to be determined by the scope of the claims, the drawings and the equivalents thereof. BRIEF DESCRIPTION OF THE DRAWINGS The above and other advantages of the invention will be readily understood by the following detailed description in conjunction with the accompanying drawings in which: Figure 1 shows water, BTP buffer solution, and reaction formula (A) The dissociation constants of the reactants/products in (B) and (C). Figure 2A shows the BTP buffer solution at a concentration of 1 mM, which is 3d in the buffer strength at 1302198 with the same pH and different urea concentrations. Figure 2B shows the BTP buffer solution at a concentration of 1 mM, which is a 3D plot of buffer strength at different pH values and different creatinine acid concentrations. Figure 1 shows the CTP buffer solution with a concentration of 1 mM, which is a 3D plot of buffer strength at different pH values and different concentrations of acetylcholine. Figure 3 shows the calculated Mohr concentration ratio at different pH values. Figure 4 shows the optical properties and dissociation constants of fluorescent yellow. Figure 5 shows a development flow chart of the biosensor model developed using FITC-glucan as a pH probe. Figure 6 shows the experimental data (open squares) from the sensor array and the calculated fluorescence intensity (curve) at different pH values. Figure 7 shows the contribution of the respective dissociation states of fluorescein to fluorescence intensity at different pH values. Figure 8 shows the results of a biosensor of a urea-decomposing enzyme type in which the urea decomposition reaction is carried out in a BTP buffer solution (concentration: 1 mM) of different pH values. ®9 shows the simulated results of a biosensor of a urea-decomposing enzyme type' wherein the urea decomposition reaction is carried out in different concentrations of BTP buffer solution (selected pH value of 〇). Figure 10 shows the dissociation constants and pH ranges of buffer systems such as Tris-HCl, MES, imidazole, HEPES, hydrazine and phosphate. Figure 11A shows the simulation results of a urea-decomposing enzyme type biosensor, in which the urea decomposition reaction is carried out in a Tris-HCl buffer solution system for 0 (s 1302198. Figure 11 shows the urea-decomposing enzyme type biosensing The simulation result of the device 'where the urea decomposition reaction is carried out in the MES buffer solution system. Fig. 11C shows the simulation, and the fruit of the urea decomposing enzyme type biosensor, wherein the urea decomposition reaction is in the imidazole buffer solution system. The simulation results of the biosensor of the urea-decomposing enzyme type are shown in Fig. 11D, and the urea decomposition of the towel is carried out in the HEPES buffer solution. Figure 11 shows the biosensor of the urea decomposition enzyme type. The simulation results of the reactor, in which the urea decomposition reaction is carried out in a phosphate buffer solution system. Figure 12 shows the results of the work intensity (solid line) obtained by using the model calculation and the experiment using the biosensor detection. Results (open circles), wherein the urea concentration is ΙμΜ to 1Μ, and the reaction time is 1G minutes. Figure 13 shows the results of the camp light intensity obtained by the model calculation ( Line), and using the detection results obtained by the biosensor ^ concentric circles), where the urea concentration of 1 μΜ to 1M, the reaction time was 1 () min. Figure 14 shows the change in pH of the solution in the creatinine acid hydrolysis system. It shows that when creatinine is added to the reaction tube, the solution value will gradually increase. Figure 15 shows that the simulation results (_ line) and the experimental results (solid squares) have a degree of _hetery, which means that the difference between the pre-frequency quasi-results and the experimental results is due to the fact that the system response has not yet reached equilibrium. Figure 16 shows the S-plastic curve at which the FT value decreases as the concentration of the acetaminophen increases.曰 Figure 17 shows the change in pH in the solution over time. The 50 mM acetaminophen system was prepared in a BTP buffer solution with a concentration of 1 _ and a cation of μ 25 1302198. Figure 18 shows that when the actual concentration of acetylcholine is 70% of the desired concentration, the simulation curve (solid line) and the experimental result (open circle) are highly consistent.

Claims (1)

1302198 h, the scope of patent application ^ Simplified chemical sensor system developed "Best in the chemical sensor system has at least have an impact on the pusher her break _ degree with the intensity of the pool to calculate the buffer strength, and in different counter The distribution of the shore material and the complex value of the different values of ρ 〜 认 认 认 认 认 认 认 认 认 认 认 认 认 认 认 认 认 认 认 认 认 认 侍 侍 侍 侍 侍 侍 侍 侍 侍 侍 侍 侍 侍 侍 侍 侍 侍 侍 侍 侍 侍^ρΗ值? This force is accumulated. 2. For example, the scope of the patent scope is included - the detection probe. Go to 'where the chemical sensor system is more 3. For example, the dye of the second paragraph of the patent application scope The detection probe is a fluorescent light. 4. According to the fluorescein 〇 of the third application, wherein the fluorescent dye is fluorescent yellow 5', as in the third application of the patent scope. Sugar. '' /, the fluorescent dye is FITC-Port 6. The distribution of the detection probe is as in the second paragraph of the patent application. The determination includes the calculation of the different types of the 1302198. Following the second slave method, it also includes calculating the performance of the axonometric probe. Intensity 8. If the method of applying for the second slave is applied, it also includes the intensity of the performance of the probe when the concentration of different reactants is obtained. 9. - Simplified biological sensing a method of optimizing a program (_miZatiGn), wherein the biosensor system has at least one Buffer solution, at least - reactant and at least - enzyme (5), whether to limit one), and the biosensor is based on the detection benchmark, the method is estimated in the biosensor system All components which have an influencing ability to the buffering strength of the buffer solution, and the interaction between the sputum and/or the substance and the enzyme; the towel's product is recorded from the reverse _ age _ age material to the _ intensity, And the same kind of parameter cylinder, the distribution and mouth of the same reaction Knowing the buffering capacity and the fractional capacity, the buffering capacity is set, wherein the pH is set to 5, wherein the reactant concentration is set to be the urea, and the method is the method of the ninth aspect of the patent application. The method of claim 9 is in the range of 1 μΜ to 0.1 Μ. 12. The method of claim 9 of the scope of the patent application is that the enzyme is decomposed by urea. The method of claim 9, wherein the reactant is creatinine, the enzyme is creatinine deimin. 14. The method of claim 9, wherein the enzyme is acetylcholine when the reactant is choline. 15. The method of claim 9, wherein the buffer solution is 丨, 3_2 f [l?3-bis[tris(hydroxymethyl) methylamino]propane; BTP]. The method of claim 9, wherein the biological sensor system further comprises a detection probe. = The method of applying to turn over Item 9, wherein the _probe is a light-emitting light, wherein the fluorescent dye is ΠΤΟ, and the calculation includes the calculation of the different types of the detection probe. Method 17 of the patent scope ® Polymerase. 19. The method of claim 16 of the scope of the patent application. 2〇. The method of applying for the scope of patents, item 16 performance intensity. • If you apply for the scope of the patent, the 16th item, the sound of the Japanese singer, the singer, the singer, the singer, the singer, the performance of the test probe.