JPH01501618A - Ionophores and ion-selective membranes containing them - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Description of ionophores and ion-selective membranes containing them background ion selective electrode Ion selective electrodes respond preferentially or selectively to timed ionic species in the liquid.

They are often used in potentiometric measurements of the activity of ions in liquid samples . Potentiometric measurements measure the voltage between two electrodes that form an electrochemical cell in contact with a liquid. Measure the difference in position. The half-cell potential of one electrode - the reference electrode - is essentially constant; That of the other electrode - the indicator electrode - varies with the ionic activity of the liquid being analyzed. Ru. The potential across the pole is proportional to the logarithm of the activity of the ion to which the ion-selective electrode responds. Ru. The Nernst equation describes this logarithmic relationship. The potential difference is like an electrometer 13 potential M measuring devices! It can be measured.

Several types of ion-selective electrodes are available and widely used, for example in research studies. Includes conventional glass electrodes for pH measurements. Glass electrode is alkali ion silicic acid Based on salt composition. The electrode for pHfM measurement is made of lithium silicate glass or Can be made from borosilicate glass, which is permeable to hydrogen ions (H+) but not permeable to anions or other cations, selected for H+ When a thin layer of optically permeable glass is placed between two solutions of different H+ concentrations, H+ Ions move across the glass from a highly concentrated solution to a low H+ concentrated solution. the Such a transfer brings about the transfer of positive ions to the solution of low H concentration and the opposite side of the glass. A reference electrode independent of m PH that leaves negative ions and generates a potential across the glass. When immersed in a solution, a circuit is created and the potential difference can be measured.

Another type of ion-selective electrode uses a liquid ion exchanger and uses Acezaka cellulose. Alternatively, an inert polymer such as a polyvinyl chloride film may be used. be done. An important example of this type of electrode is the calcium salt of the diester of oil-soluble phosphoric acid. This is the basic C6 Kanoichi response electrode.

U.S. Patent 3,429,785 g 3,438,886, g and 3.445 ．． 365 selectively responds to divalent cations (e.g. Ca"+, MIIr+). W made of a porous inert material filled with an ion-exchanged organic liquid that A 1% ion selective electrode with I4 is described.

Neutral carrier-based sensors for monovalent divalent and divalent thiones are ion exchange Analogous to body-based electrodes, both contain ion exchange sites, especially media a negative mobile site or carrier derived from a substance Contains two negative fixation sites that result from hydrolysis of the substance.

When constructing neutral carriers, they can be closed or open chains, but are generally , is a hydrophobic complex forming agent with cations. Such compounds are r', Na'g, etc. selective extraction, and therefore selective permeability, to On is a type of carrier that would otherwise not dissolve in the membrane phase as a simple inorganic salt. An example of this is palinomycin, which is an electrode that selectively responds to Can be used.

Electrodes for measuring the H content of liquid samples have been described by others. They are Commonly, ion-selective components (ionophores (i・1su5, , )) and their ion plastics with solvent/sonicator compounds capable of dissolving the selective components. Contains a membrane. In addition to glasses selectively permeable to H+ ions, oxidative phosphorus Oxidation uncoupling agents such as fat-compatible carriers 2 and hydrogen ions such as lipophilic tertiary amines Onohoa has been used.

Simon et al. This electrode contains 5.5 and 12 decylamine) as a hydrogen ionophore. It has been reported that A-H changes between the two groups give a well-defined response. but.

At least two important drawbacks are that, for proper fermentation, the agricultural industry must use dissolved carbon dioxide. arises from the fact that it must include First, the electrode performance (i.e., C The oil content) is severely affected by changes in temperature and pressure. Second, manufacturing Mass production is extremely difficult as it must be carried out under carefully controlled conditions. Moreover, dissolved CO1 diffuses away during electrode storage.

Other types of ionophores act as uncouplers of oxidative phosphorylation in mitochondria. An example of 0 where 0 is described by Finklestein.　B<ec屓m1c a Bi*pλy Pa C-At ta1205: 1-6 (1970) e 2 + 4-dinitrophenol 3-chlorophenylhydrazone mesoki Weakly acidic uncoupling agents such as salonitrile act as H+ ion carriers. those However, as membrane components incorporated into ion-selective electrodes, certain water unsuitable due to their solubility (i.e. they do not remain bound to and are removed from the membrane) leached out).

Grain et al. described a pH 7 cer that is claimed to be suitable for long-term intravascular implantation. ing. That pH sensing! ! What is it?

The material is made permeable to ions through the attachment of a hydrophobic and lipophilic specific H+ ion carrier. In the thin film of the lastomer polymer, the carrier used is p-octadecyloxychloride. Lolo-722hydrazonemenoxalonitrile (OCPH, which is a weakly modified deconjugate Pharmacology - A high molecular weight homologous series of chlorophenylhydrazone menoxalonitrile ), Butin and the Ministry of Joint Research have shown that the OCPH molecule is a type of glass). Although acting as a mobile H-carrier, the H-responsive properties are obtained using a polymer matrix specifically engineered for the purpose. It states that it is only possible to U.S. Patent 43,743,588 (1973) to, H, Leblanc, J,! , Bran9 et al.'s J Juran JeA limd hat ・LJI soil 0:644-647<1916), can be used with OCPH molecules The production of polymers is described by Guorn. US I#7g63,1 89, 662 (1965) jH, A, Guorn 2 and E.

P, Goldperg's Palywcmr Latsara, 7: 569- 572 (1969).

US 5th license 3.691.047 (1972) Kss, Ross 2 and Martin describe an ion-sensing membrane for potentiometric electrodes. The membrane is a gelled mixture (2#), in which the solid phase is a polymer (e.g. cellulose trioxide). acetate), and the liquid phase is an organic ion exchanger A substance dissolved in an organic solvent (e.g. Calcium (bis-dibromo) dissolved in dioctylphenylphosphonate (rail-phosphate) t). The ion sensing electrode is A membrane whose main component is an organic ion exchanger dissolved in a non-volatile organic solvent. be exposed.

Rice 13il Buttocks II! F4, 214.968 (1980) 2, Bhattadaria described a dry-operable ion-selective electrode for measuring the ionic content of liquids. ing. The electrode consists of a dry internal reference electrode in contact with a hydrophobic ion-selective membrane. It is said to be a thing. Its internal reference electrode is a dry metal/metal salt reference half cell. Redox or W, linear redox couple reference electrode, suitable for liquid samples. getting wet. The ion-selective membrane contains an ion carrier (e.g. palinomycin). It is dissolved in a carrier solvent dispersed in a hydrophobic binder.

U.S. Patent 4,184.936 (1980') Kg, Ball and Pabaoglu describes an apparatus for measuring the ionic activity of liquid samples. The device is an internal reference Temporarily placed on top of the element (made of an electrolyte-containing material, metal salt 2 and metal layer) and a support. It has been described as having an ion-selective membrane that has been reversed. The two electrodes of the device are It is said to be solid and preferably linear.

In U.S. Pat. No. 4,053,381 (1977), % I. Nbu Vno et al. describes another device for measuring the ionic activity of components of the device Some electrodes.

the internal reference electrode comprises several layers and at least one ion-selective electrode; The layers are a metal layer, a layer of an insoluble salt of the metal, and preferably dried. The claimed device, which includes an electrolyte-containing layer, includes a solid electrode.

Currently, there are many types of ion selection available in the liquid ionic content 611 feet! very exists. However, the poles have restrictions on the selection of these ions. these rules About iM Tomishio about membranes composed of specially designed polymer matrices It is necessary to perform pre-neutralization with base to improve membrane sensitivity and reduce response time. Utilization of ionophores; the need for storage under controlled conditions +2 and.

In addition, currently used for pH measurement, including: 1 EIP sensitivity 2 and reduced reliability; Ion-selective electrodes require relatively large samples (i.e. 1. 0 or more) and are made of costly glass; It cannot be incorporated into electrodes suitable for automatic processing of very small sample volumes.

Samples that are smaller than those currently available (e.g. microliter size) ) can be used to accurately and quickly measure the ion content of There are It is also possible to analyze smaller samples than is possible with currently available electrodes. Accurate and quick differential measurement techniques for other constituents The technique of differential potential M measurement involves immersion in bath liquids of different activities separated by a salt bridge. It depends on the potential difference between two equal electrochemical half cells. These two The half-cells together constitute one half-cell. In this case, there are two cows! The activity of the pond t (aθ is fixed at 2 (reference), while the other activity (-) (sample) is Vary so that the 0 start f of the density battery is defined as: T Here, -7-59,1mV, (2 at 298°K, about 3-1) can be.

E, - reference half-cell reference f Et-Sample half-cell #affairs! E0 - standard reference electrode potential 72-Molkas constant ms 8.314 Holt Kuhlen/”K T - absolute temperature, to 1 悴 - Charge on ion F - Faraday constant 796.493 Ku ψ aI2 and phantom - ion activity of reference sample amount In the case of a cation such as potassium (CK+), the half-cell a is the reference half-cell. When defined as a cell and assigned zero potential, the -v of the cell f& nf is a@) If it is 6%, it is positive, and if o<at, it is negative.The 0 clause is fixed. Therefore, the equation for the battery potential contains only one arithmetic number (a,), and E,... If we measure 1g, the equation can be solved for season.

The differential measurement wave ff is sodium 9m (Na　), potassium tK+).

Including other ions such as calcium (Cs'') and chloride <Ct-), Used to indirectly measure the concentration or activity of components of biological fluids other than H It's been coming. Furthermore, such techniques often utilize biosensors or enzyme electrodes. However, they are sensitive to the production of biological catalytic reactions or cosubstrates. Contains a futuristic catalyst (eg, immobilized enzyme, cells, tissue layer) attached to the poles.

The concentration of enzyme or substrate can be determined using differential measurement techniques, e.g. ! # The cumulative reaction results in the formation of an acid or base, the ionization of that acid or base is This results in the release and uptake of H2 and a change in the pH of the solution, +concentration or p The measured change in H , urea, etc.) can be the basis for stoichiometric measurements of concentrations.

In the case of differential pH measurements, each half-cell contains hydrogen ions of 0 half-cells containing the H electrode. The activity is fixed, and that of a sample varies depending on the pH of the sample. by electrometer The #fPIf measured across the cell is then calculated as the phantom hydrogen ion activity. It can be used for.

In the case of differential measurements requiring enzymes, the number of cells is divided into one half cell or both. Place the sample into both half-cells, or add the sample to one half. to the cell and the calibrator to the other half cell.

As a result, when the enzyme reacts with the substrate in the sample, there will be two increases or decreases in pH. i The magnitude of the change is directly proportional to the amount of substrate in the sample. Similarly, the substrate can be fermented. The enzyme can be used to measure the enzyme activity of a given sample. can.

For example, Elsin and co-workers used hydrogen ion glass electrodes to detect glucose in solution. , used to create an enzyme-pHi pole for measuring urine concentration 3 and penicillin. #Elementary electricity It describes the development of poles. The # elements used in this measurement are each t-glucose oxidizer. 12 and penicillinase. ; Luzon, H1 et al., Ei ocki information atEtopkyaicaAeta, 320:529 53 4 (1973)a Mosca and co-workers also investigated the concentration of glucose by differential pH measurements. Describes measurements. The technique involves the catalysis of hekin kinase between glucose and ATP. It is based on the measurement of pH changes caused by medium reactions. they are two 1- Describe two systems that are said to be useful for measuring pH differences between aqueous samples. I'm looking forward to it. The concentration of glucose is determined by the formula derived by those authors. Differential measurement of pH for measuring whole blood S and glucose in blood serum and its An automated system then performs this glue same equipment and differential pH method on serum, plasma 2 for use in determining lipase activity in biological fluids such as and duodenal secretions. It has been described. C11vtcal ChaqIIatr of Kariotsuta F. et al. I, 3 1 1 257 260 (1985) e The precision of this differential pH measurement technique Densification is for the measurement of urea, creatinine-72 and glucose in blood aS and whole blood. It is said to serve as a substrate. #Element 9 Rease, RiV Atinine Iminohydrola 2 and hexokinase, respectively, are used for these measurements. These three The concentration of the substrate for is based on the observed change in AH in the solution after two reactions. It is calculated as follows.

U.S. Pat. No. 4,353,867 (1982) states that rutsana is a biochemical solution. Substances such as glucose, urea, 2 and enzymes in (e.g. dish fluids, serum, urine) It describes the equipment and method for measuring. The alternative is to use differential pH measurement. Therefore, in that case, two glass PH@ electrodes are placed in separate solutions, The pH change in the solution after the drug is added is measured, and the concentration of the substance of interest is determined by the observed pH. Calculated from change. The device has a sample cuvette, two glass capillary types 1 a cell, a means for measuring the pH at the two electrodes, and a means for determining the pH II of the cell; electronic means for calculating the concentration of a substance.

Electrochemical immunosensors can be used as potentiometric or amperometric measurements. It may be written in either way.

Potentiometric immunosensors can be used to measure the strength of antibodies or antigens. Can be done. They may be described as either M-contact or indirect, membrane or It can be either a solid electrode. N-contact potentiometric immunosensor for antigen measurement An example of this is described by Yamamoto et al. Antibody, anti-hcG, titaniwam It is fixed on θ. Place the anti-hcG tjl and reference electrode in a buffer solution. Then, antigen and ICG are added, and as the antigen binds to the immobilized antibody, the two The potential difference between the electrodes of is varied at until equilibrium is reached. The Hiraga electrician determines the antigen concentration. Directly proportional. C11nical Chemistry, 26 :1569-1572 (1980), 1! The exact nature of the topological response is not fully known. Not understood but generally recognized to involve neutralization or redistribution of surface charges has been done.

Another example of a direct potentiometric immunosensor for measuring antibodies is the Keating 2 and It is stated in Rechnitz. This type of immunosensor uses marker ions. The background potential is fixed by KFAt, so that the potential change is proportional to the antibody concentration. respond to specific antibodies in such a way that Immunosessor for measuring digoxin antibodies is written 'E? ), in which case digoxin is the marker ion carrier molecule benzo Binds to -15-Kracun-5. The resulting complex is polyvinyl chloride be incorporated inside. Antibody-selective potentiometer electrodes for key measurements also It is described in U.S. Pat. child Constant background levels of marker ions are a significant limitation to routine clinical use of the method. Therefore, biological samples must be dialyzed before analysis. There is no such thing.

Intercoagulant potentiometric immunosensors are @concentrative and generate enzyme-substrate reactions. In addition to using electrochemical sensors to measure substances, #primeimmunoassayand They are similar. Both homogeneous and heterogeneous potentiometric enzyme immunoassays have been described. 0 For example, Boachio-2 and co-workers found that measurements of estradiol were unequal. A qualitative potentiometric enzyme-linked immunoassay is described. C11sic of Boachio et al. al Ckemia・Agta*] 13 # 175-182 (1981) , antibodies against estradiol are immobilized onto a porous gelatin membrane. the membrane is stored together with peroxidase-labeled estradiol and free estradiol. Warm up. After washing, the membrane is fixed onto an iodide sensing electrode. Peroxidase activity is It is measured in the presence of hydrogen oxide and iodine ions. Iodide selectivity at polar potential is a function of estradiol concentration.

A homogeneous potentiometric enzyme immunoassay for human IIGK was performed by Onong 2 and Lech. stated by Nitsu.

The method is catalyzed by chloroperoxidase conjugated to the IIG antibody. . To prevent CO2 production caused by beta-ketoadipic acid using IIGK. Based on.

7 Onong 2 and Rechnitz, G., Asalytieal Chemi-1 Day = 56: 2586 2590 (1984) * This enzyme - 1t ( When incubating the 4 complex together with a sample containing the antigen (7yG) before reaction with the #primesubstrate. In this case, the potentiometric CO! Observation of CO2 emissions measured with gas sensing electrodes The speed at which The decrease in activity is proportional to the IgG concentration in the sample.

An amperometric enzyme immunocessor is an amperometric sensor, usually a 6R cumulative electrode, that ! It is similar to a potentiometric immunosensor except that it is used to measure elementary activity. be. for example.

Aizawa, Mori Saiki, 2 and Suzuki are tumor antigen alpha fetoprotein (AFF). ) describes an amperometric immunosensor for the measurement of Aizawa et al.'s 4th challenge 1ヱ4ChimicaActa, 15:6-66-67 (19 anti-AFP is porous Covalently immobilized on the plasma membrane. Membranes were treated with catalase-labeled AFPs Keep warm together with FP.

After competitive binding, the membrane was measured for catalase activity and amperometric measurement of oxygen after addition of hydrogen peroxide. Inspect according to the specifications. In a similar manner, Boachio and co-workers developed hepatitis B surface antimicrobials. describes the original amperometric enzyme immunoassay for the determination of l1lI. C of Boachio et al. l1nical Cketmiatrym25: 318-321 (1979 ).

The limitations of the amperometric sensor for t# elemental synthesis are the necessity of enzyme complex synthesis and the need for enzyme electrodes. The first problem is that currently available current measuring sensors are expensive.

”11Ω1 and L The present invention uses an ion-selective electrode to determine the ion content or activity of a sample. This is a sensor that measures the potential difference in the concentration of other components in the sample. This processor's input Ionophores for use in ion-selective membranes are also an object of the present invention. this sensor is the hydrogen ion content of the biological fluid, i.e. the pH; 11 Fl: 2 and.

By differential tH determination or immunoassay techniques. 8: Other components of the physical liquid (e.g. , glucose, urea, triglycerides, uric acid, aspartate aminotransfer Erase CA! ;T), alanine aminotransferase (ALT).

amylase, creatine kinase (CX), alkaline phosphatase, etc. It is particularly suitable for #1 rapid measurement of #1 enzymes and drugs. it's automatic Particularly useful for standardized handling or processing.

The sensor consists of ion-selective electrodes, which are held in a frame. and between them a porous material (e.g. made by Borex Chichnorrhea). porous polyethylene), such as those manufactured by This porous material is When diverted to the electrode, it provides a means for ionic flow between the electrodes.

These ion-selective electrodes are used to detect ions or other substances whose concentration is to be measured. Membranes that are permselective and reference! Consists of poles.

The membrane does not require pre-MJi before use.

Selective permeability is a combination of an ion-selective compound (or ionophore) and a thermoplastic resin or consists of plastic materials, all of which are soluble in organic solvents. Moreover, the membrane It can also be enriched in plasticizer '1: 0, e.g. hydrogen ion active ~IJ of the sample The ion-selective component for the sensor that can be used to add the following general formula: A compound having formula 9, R1, R, 2 and R1 are independently selected and each is 1] halogen; 2) the number of carbon atoms is 4 184! l alkyl group; 3) halogen-substituted alkyl group; 4) alkoxy group ;5) Halogen-substituted Al ;oxy group, 6) -Co, represented by H':! -ReR4 is an alkyl with a carbon purple color of 1-18@; also 1 nia) -COR.

a keto group represented by and R1 is selected from the groups defined for R4; or can be 8) a hydrogen atom;

In one embodiment of the invention, the membrane has an organic plastic matrix, polyvinyl chloride <pvc), it is an ion-selective color compound 2-ttadecyl oxy-5-carbethoxyphenylhydrazone mesooxalonitrile, This ion-selective compound, PVc%2 and 2-nitrophenyl in this embodiment 2 The softening agent %, which is octyl ether, is the solvent tetrahydrofuran (THF). There are 9 Raku among them. These ingredients make up the membrane formulation.

In a preferred embodiment, the membrane comprises 10-40 inches of PVC by weight; The thickness is about 1 mil! In a preferred embodiment #2, the membrane weighs 20% by weight. - Made of 35 inch PVC and approximately 3-15 mils thick.

The device described is used as a component of the inventive ion-selective electrode. it is , U.S. Patent No. 750,525, June 27, 1985. -2 and a partial continuation of U.S. Patent No. 750,525 filed in parallel. It is possible to insert hair into the sensor requested by m Wear. It can also be incorporated into commercially available electrode bodies.

The sensor of the invention is also an internal reference! ! has an element or substance whose concentration can be measured Contains known concentrations of sample components.

Brief description of the drawing Figure 1 is a perspective view of the top of a sensor with an ion-selective electrode and a handle. has a magnetic stripe (at stripe) on which data is recorded.

Figure 2 is a perspective view of the underside of the sensor with an ion-selective electrode and a handle; It has 81 stripes on which data is recorded.

Figure 3 shows how to measure the hydrogen ion activity (pf) of a sample or the concentration of other ions in the sample. 1 is a perspective view showing the individual components of a sensor that can be used to

FIG. 4 is a schematic representation of the ion selective electrode arrangement.

Figure 5 shows an uninvented ion-selective element inserted into a commercially available barrel-type electrode. shows.

Figure 6 shows the method used to measure the activity or fkK of a component of a sample by differential measurement techniques. 2 is a perspective view showing the individual components of a sensor that can be

BEST MODE FOR CARRYING OUT THE INVENTION The sensor that is the subject of the invention is a sensor that measures the ionic content of a sample or the concentration of other components of the sample. Used for phase difference measurements. It is the hydrogen ion activity of biological fluids (e.g. blood) (or pH) or other ions and in biological fluids. Other components (for example, glucose, urea, triglycerides, uric acid% enzyme) For example, aspartate aminotransferase (A, S7'), alanine amino Transferase (AZ7'), amylase, talealanine kinase (CK) , alkaline phosphatase S and drugs). trial selective for the raw material components and incorporated into the ion-selective membrane of this type of sensor. The ionophores that can be used are also the subject of the present invention.

This sensor will now be further described with reference to the cause.

Figures 1 to 3 are ion selection! If @ and data are recorded, it will have a stripe. It shows a sensor with a hand. The sensors shown in these diagrams are 1ite containing hydrogen ions has a pH of 1 or f! of other ions in the sample. measure degree It can be used to These figures are used for 11,111 samples of hydrogen ion content. Referenced here when describing the sensor used. However, this sensor Of course, it can also be used for the measurement of other components as well.

The sensor 30 connects the upper portion 12 and the lower portion 14 within the frame or body 10. It consists of a retained ion selective electrode. Furthermore, the sensor 30 has a magnetic strip on the bottom surface. It has a grip 5 with eight handles.

The upper part 12 of the frame 1.0 has four openings 4 and three grooves 6 arranged between them. have. 4 is located between the four openings 07 and those openings. The upper part 12 and the lower part 14, with three grooves (not shown), are connected to the opening 4 and the open lower part. are in line, and the groove 6 in the upper part is in line with the groove in the lower part. It is in. As a result, openings 4 and 7 form four openings 11, and #16 in the upper part The grooves in the lower part define the spaces between three of the openings 11. Upper part 12 has a small hole 15 located between the openings 40, and the hole 15 allows 9 Qi to pass through. The selection electrode is placed inside the opening 11 and is made of a cylindrical or rod-shaped porous material 17. means of ionic flow between the electrodes when the sample is applied to the electrodes. It is placed in the space between 11 and 11. The upper portion 12 and the lower portion 14 are porous materials. on the body 17 or between or within the frame or the two parts of the body 10. To prevent the sample from leaking, for example, by applying ultrasonic WI contact under pressure. It will be sealed.

As shown in Figure 3, the sensor 30 has four positions in which ion selective electrodes can be placed. I'm begging. In general.

Although there are two and one ion-selective electrodes at each of these positions, these can be used in various combinations depending on the analytical information desired. 2.3 or Can be used with 4 electrodes. Therefore, the groove 6 in the upper part 12 and the lower part 1 The groove position fIi in 4 is necessary for the analysis of contribution, and it changes to 2shi. For example, the groove 6 in the upper part 12 can be located in the lower part of the stem as shown in FIG. The groove in 14 is formed when the upper portion 12 and the lower portion 14 are joined. 9, positioned so that a space is defined between the three electrodes. Or again, two The grooves 6 in the upper part are placed parallel to each other and the grooves in the lower part 14 are correspondingly arranged. can be located.

The porous rod 17 is either hydrophilic and electrically conductive or hydrophobic and non-t4. A pair! very The use of conductive or non-conductive substances between determined. The various combinations of J are best described by example.

In the first example, as shown in Figure 48, three membranes are active, and these three membranes are active. All sides contain the same membrane.

Positions &a and b are the known concentrations of the ions to be measured, and positions me are the ions of interest. No Mishono 1f'? I have a sample that contains It is generated between a solution located in the middle of a One mf can be used to calibrate the sensor, and then use the ramp to calibrate b and eK2. The concentration of the sample is determined from the 1 mf generated between the solutions. Furthermore, a and b Since the ion concentration in is known, we can calculate the value of a and b using the pre-measured value. Calculate what the potential difference between them should be and compare this value with the measured potential difference, or or calculate the concentration for 6 and determine how this varies from the known concentration. is possible. In terms of clinical chemistry, the solution in a is then used as a reference standard. used.

This a,i,a configuration may also be used in a manner that allows repeated samples to be analyzed. Can be done. This is shown at 2 in FIG. In this case, the same sample is placed at position G. Place it on C! ! , place the reference solution at b, using the pre-measured slope.

Two values can be determined simultaneously for the same sample.

It is also possible to measure two analytical components simultaneously.

This is shown in Figure 41. For example, a and d are for one analytical component. Selective counterfeiting S&C can be selective membranes for different analytes. Ru. Useful combinations are Natriwam/Calicum, pH/Calcium, Glucose/Urine 2 and urea/creatiny/. In these cases, Calibration data is required.

The enzyme/substrate sensor can be configured in two ways, as shown in Figure 4dKE. this In case 1, a, i, c, 2 and d can all be the same membrane 1, for example, They can all be used at pH 91J. However, 6 and d have a pH of 11 In addition to % immobilized enzyme, the unknown analyte sample concentration was added to both - and d. , a known concentration of the same analytical component 11+ is added to - and b. The enzyme in G and d is the substrate All the membranes act as Assuming equality, the 1xf occurring between a and b (reference solution) will detect the electrode. It can be used to measure. The calibration data obtained for a and b are then C It can be used to calculate the sample placement of the instep of and d.

The enzyme/substrate sensor can be configured in two pieces, as shown in Figure 4kKB. this If a, h2 and 6 are all the same membranes), for example, they are all pH It is for measurement. However, a, ni 2 and C are in addition to pH expansion.

The substrate concentrations in σ and b, including the immobilized substrate, are different S).

The substrate concentration in C can be the same as in G or b by 1% or different. be able to. An unknown concentration of the analyte enzyme is added to G2 and d to be measured. ! Add a known concentration of enzyme to G and b. The enzymes in a% &, 2 and C are a, h2 It acts on the substrate in the water and causes a pH change that is proportional to the enzyme concentration in the sample. vinegar occurs between a and & (reference solution), assuming all membranes are the same. -/ can be used to calibrate the electrode. The calibration data obtained for a and b is It may then be used to calculate the enzyme sample concentration in C.

Figure 4kKB can be immobilized in enzyme/substrate sensors such as Examples of substrates include amino acids such as L-alanine 2 and L-aspartate, Atin 2 and starch. Each of these is an alanine aminotransferer enzyme, aspartate amino-transferase, taleatin kinase, S and amylase.

It can be used for measurements in samples. As described in Example 8, 2) Figure 1 An enzyme/substrate such as 4kVc2 can also be used to measure the creatinine concentration of a sample. It can be used to define

If the kinetic rattst * glhod If used, this methodology will eliminate any temperature effects on the enzymatic reaction rate. It also makes it possible to “correct” (-earth data ast) the results. Standardize the temperature effect in the C-Nernst equation for point or equilibrium measurements (sor rinse). alitad) can be done.

The porosity 1117 between the electrodes can be electrically conductive or non-conductive. However, the use of conductive or non-conductive materials between a pair of t-electrodes is prohibited. Determined by analysis.

Porous materials include nylon, polypropylene, polyethylene, and polyvinylidene fluoride. olide, ethylene-vinyl acetate, styrene-acrylonitrile, or can be of a microporous plastic such as polytetrafluoroethylene.

This porous material can also be a roll of filter paper or a vertical fiber. It can be cellulosic in nature like a bar bundle. in one embodiment 2, this porous material is made from ultra-high molecular weight microporous polyethylene (Polex Tech). (Nology, Inc., Fairburn, GA). This substance has its properties is hydrophobic so that it does not get wet with water.

This porous material allows it to absorb a solution of surfactant (approximately 0.1% to 0.6% by volume). It can be made water-resistant by immersion in it. was found to be useful The surfactant is a nonionic surfactant, such as Triton Octylphenoxypolyethoxyethano, a surfactant containing Polyoxylene ethylene ethers such as Polyol 5, Briley (Bde 5j) 35.

Polyoxyethylene sorbitan derivatives such as Ly-720, 3M Fluorad Fluoroaliphatic polymeric esters such as FC-171%2 and A surfactant such as Alosurf 66PE-12 from Su Chemical Co., Ltd. a Nionic and thionic surfactants can also be used. Wetting agent or surfactant The porous material is non-conductive. If there is no surfactant between the two t-poles, Of course, it results in non-conductivity.

The ion selective electrode has an ion selective a18 and a reference electrode 22 which is a material of % silver/silver chloride. Consists of. The ion selective membrane 18 is made of an ion selective compound (ionophore), thermoplastic Made of resins and plasticizers, they are all soluble in plasticizers. the membrane is held in place within the ion selection ta by retaining means such as retainer ring 24. Retained.

In this way, between this retainer means and the lower part of the sensor body, there is no possibility of leakage from counterfeiting. There is no mechanical seal. The weight of the sensor! ! characteristics are incorporated into it The ion-selective membrane used in the sensor can be used for preconditioning (e.g. does not require immersion into a solution of ions to be processed.

In the case of hydrogen ion selective membranes, the ion selective compound incorporated into the membrane has the general formula 2 in this compound having 2, RI, R* 2 and R1 are the same or different It can represent the components of a compound.

R1, R2,2 and R1 are 1) halogen, 2) alkyl having 4-18 carbon atoms group, 3) halogen-substituted alkyl group, 4) alkoxy group, 5) halogen-substituted alkyl group xy group, 6)-〇〇tR, where R4 also has 1-18 carbon atoms; represented by an acid group that is alkyl, or 7) -COE, and R1 is R , or 8) a keto group selected from the groups defined for , or 8) A hydrogen atom.

Preferred derivatives of this general formula for loading as components of the hydrogen ion g supra include: nothing: a, 2-)! JFluoromethyl-4-octadecyloxy722hydrazone- Meno beef salonitrile phenylhydrazone-menoxalonitrile c, 2-oc Tadecyloxy-5-carpethoxyphenyd, 2-year-old tadecyloxy-5-carpetoxyphenide 4-ph Luorophenylhydrazo/-menoxalonitrile The ion-selective compound is carpetoxyphenylhydrazone menoxalonitrile. Some membranes indicate that they exhibit a response time of 1 minute or less and produce a stable electrical potential; It shows a slope of the order of 759 mV per one order of magnitude change in hydrogen ions, and ! It is particularly useful in that it does not require any. Why is that kind of membrane thin? It is not clear whether it exhibits superior performance characteristics, but it is likely that Attributable to the presence of an electron-withdrawing ester group and its proton at the meta position The protons may be clearly more acidic and therefore more easily be exchanged.

Uninvented ion selection in contrast to the ion selective compounds mentioned above! 18 is also a plus It consists of a 1M thick or thermoplastic fat and, as an optional ingredient, a plasticizer. used plastics that can be used to form films by solvent casting. The plastic can be any type of plastic. For example, the plastic may be polyvinyl chloride, Must be polyvinyl acetate, silicone rubber or cellulose acetate Can be done. This membrane component serves the purpose of providing a bulk material and forming it into a membrane, and also contains ions. It acts as a matrix into which selective compounds are incorporated. Furthermore, it is a hydrophobic substance. In one embodiment, it is used in the The thermoplastic resin used is polyvinyl chloride. Other things you can use are , heptalulose acetate, polyvinyl acetate, 2 and silicone rubber. .

Plasticizers, which are optional components of membranes, can facilitate formulation or manufacturing of membrane formulations. and for the general purpose of improving membrane permeability; any non-volatile substance may be used; Plasticizers also contribute to the dissolution of ionophores. It can be used alone or in conjunction with e.g. can be one or more of the following: 7 talates, adipes sebacate, aliphatic 2 and aromatic ethers, aliphatic 2 and aromatic phosphates aliphatic 2 and aromatic esters, 2 and nitrated aliphatic 2 and nitrated In one embodiment of the invention, the plasticizer is 2-nitro Phenyl octyl etherte&le.

The components of ion selection [11 g can be present in various amounts. plastic used The weight of the membrane is approximately 10-30 mm, and one embodiment requires approximately 28 mm. The amount is %. The ion-selective compound should be F11 to FJ10 by weight of the membrane. and generally weighs from about 3 inches to about 6 inches. The plasticizer is approximately 5% by weight of the membrane. It can be from 0 inches to about 80 inches and generally comprises about 70% by weight.

The ion-specific membrane 18 incorporated into the ion-selective electrode of the invention generally It should be of greater thickness, preferably from about 3 mils to about 15 mils thick. Ru.

The ion-specific membranes of the present invention can be coated using at least two different methods: solvent casting2 and dip coating. , it can be created by

The solvent casting process is illustrated in Example 2 and generally includes the following steps: 1. a) Nitrated phenolic alkyl for producing ether h) conversion of the nitro group to the 7-mino group, g) precipitation of the hydrochloride of this amine, 2 and d) hydrogen ions by conversion of hydrochloride to menoxalonitrile conductor. Production of specific compounds 8 2. Ion-specific compounds, plasticizers 2 and plastics in volatile (organic) solvents 3. Removal of volatile solvents (e.g. by evaporation).

After the membrane has been formed, it can be shaped into the appropriate form (e.g. by cutting. ), the electrode body of the present invention or the Orion electrode body (Orion ・Electrodes such as Research Co., Ltd., Ry Bridge, MA, Catalog Analyzer 950015) FIG. 5 shows the structure of the invention held in the electrode by an O-ring 63. An Orion electrode body 61 with an ion-selective membrane 62 is shown. No. 200-Ring (not shown) prevents sample leakage around the membrane.

Ion-selective membranes can also be formed by dipping 5F coating or concatenated coating. can. There are two ways to do this.

Nylon mesh 3 or polyester mesh 3 (for example, 7" 3 55. Tect, Elmsford.

(NY) in a solvent bath (e.g., 4-methyl-2-pentanone). This is an analogy to remove air trapped within the mesh. It can be done ultrasonically using a Brannon ultrasonic cleaner, and The time required is approximately 10-15 seconds. While still wet with solvent, remove the mesh i1! After placing it in the formulation and removing it, scrape off the excess formulation. compounding The material-containing mesh is then dried, for example at room temperature or in hot air (e.g. For example, it can be carried out at 35-50°C.

The internal reference material consists of a solution of the salt of the ion to be measured. If the reference electrode is silver/ In the case of @silver ide, the counterion consists of chloride. like agar or agarose A gelling agent can be used to fix the reference material in place.

In the case of pH hexanocer, the reference substance is a buffer solution.

In one embodiment, the pH reference substance is 50m! 7 glycerin, pH'1. Water consisting of 4 phosphate buffered acupuncture water (Sigma Chemical Co., St. Louis, No. 4) It consists of so*, g of solution and 2.09 agar. Heat this mixture to make agar Melt it and then put it into the recess at the rear of the membrane. After a while 1 Usually 5-10 minutes Afterwards, the mixture gels.

For the Kaliwam sensor, the aqueous solution was mixed with 0.2M KCL in 50j. For the sodium 2 and chloride sensors, the aqueous solution was 0.2M Na It is CL.

To modify the sensor of Figures 1-3 to measure other ions, at least one membrane component It is necessary to change the internal reference material and denature it to contain the ions to be measured. For example, for the measurement of Calicum, the membrane is palinomycin.

klawan ether derivatives of benzo-15-klawan-5 or some other Internal reference materials, including potassium-specific ionophoric carriers such as neutral carriers, are Constructed from potassium chloride solution, with or without a gelling agent.

Figure 1-3 How to modify the sensor to measure sodium worms.

The membrane is made of monensin, a klawan ether derivative of benzo-12-kura9-5, or is supported by several other sodium-neutral carriers (e.g. A9Ialytic of Simon et al. al Ckgwh, 51:351-353 (1979). Internal reference materials include sodium chloride, including ionophores for ions.

To modify the sensor of Figures 1-3 to measure chloride.

The membrane is made of an ionophore for chlorides such as the quaternary ammonium salt Aliquat 336. The internal reference substance contains chloride ion.

As mentioned above, the sensor 30 is a magnetic stripe 81! It has a handle 5 with t. . This stripe is similar to the stripe commonly found on credit cards. It is very similar to V2, which holds the data recorded on the stripe during manufacturing. There is. The data includes, for example, the latest information on the type of exam, the closing date of the test card ( azpirafio%date) 2 and 1 Housekeeping (λosaa-Ke eping) - includes data, etc.

The magnetic stripe's independent functionality makes it a disposable tape designed for one-time use. This is to prevent the reuse of stock cards. This means that the test card has been removed from the instrument. This is achieved by destroying the encoded information when

The error checker a mentioned above will reject the card if you try to use it again. Extinct.

Figure 6 shows a diagram that can be used to measure the activity or composition of sample components by differential measurement techniques. The sensor of the present invention is shown in FIG. Measurable biological fluids (e.g. Blood, serum, plasma, urea, saliva, brain marrow? lI); for example, glucose gas, urea, triglycerides, creatinine, lipase, uric acid, 2 and other enzymes (For example, alanine aminotransferase (SGFTt or ALT>+A Lucky phosphatase (AZ, P) i creatinine kinase aspartate Contains aminotransferase (saor or AST).

Sensor 70 consists of an ion-selective electrode that includes an upper portion 42 and a lower portion 44. It is held within a frame or body 40. The upper part 42 of the frame 40 has four openings 3 4 and three grooves 36 located between the opening. The lower part 44 of the frame 40 has four pieces. It has an opening 35 and three tiles located between the openings. Upper part 42 and lower part 44, the opening 34 and the opening 35 are aligned in a line, and $36 of the upper part 42 and the lower part 4 It is in a relationship that the grooves of No. 4 are lined up in a line. As a result, opening 34 and opening 35 are different from opening 39. The groove 36 in the upper part 42 and the groove in the lower part 44 are formed between the three openings 39. defines the space of Upper portion 42 has eyelets 43 located between openings 340. Ru. Hole 43 allows air to enter space 41.

The ion selective electrode is placed within the opening 39 and is supported by a porous material that is cylindrical or rod-shaped. are connected. The porous material 46 is placed between the electrodes when a sample is applied. This cylindrical or rod-shaped porous material 46 provides a means for ionic flow. It is placed in the space 41 between 90 and 90.

Regarding the sensors in Figure 1-3, there are 3 sensors, 1 fewer than the 4 shown in Figure 6. Alternatively, 4@more ion selective electrodes can be used. Also, as mentioned above 2shi.

The porous material between the electrodes can be hydrophilic or hydrophobic.1 This type of analysis Determining properties 1 For example, an enzyme substrate can be measured using the sensor shown in Figure 6 and If a reference sample is to be used, its arrangement is as shown in Figure 4dK. can be done.

These ion-selective electrodes include a layer 48 on which an enzyme or substrate is immobilized; an ion-selective membrane; 50; membrane 52 placed between the two; internal reference substance 54; 2 and reference electrode 56; ．． That's what happens. An additional membrane 58 may be present near layer 48.

In the case of a urea electrode, the ion-selective membrane is used for H+ or ammonium ions (NH6 +) can be selective. H-selective membrane is placed on top for the 2B sensor. Made as described. In the case of ammonium-selective membranes, the ionophore Ammonium-selective substances such as G enonactin (%O%actss*) Ru. A retainer means such as retainer ring 60 in the ion selective membrane 1 ion selective electrode. is held in place by the

11148 has at least one enzyme or substrate immobilized thereon 0 layer 48 may be, for example, a nitrocellulose film (e.g. , Type AfflOO, Quereichel & Schnill, Inc., Keene, NE,) This R4 can also be nylon, cellulose, cellulose acetate. substrates, glass fibers, or solid substrates for immobilizing enzymes or engineered substrates. It may be made of any porous material that can serve as a material.

Enzymes or substrates can be physically retained (e.g. adsorption onto a solid phase) or chemically (e.g. adsorbed onto a solid phase). For example, it can be immobilized by covalent bonding or cross-linking).

Layer 48 is separated from ion-selective layer 50 by membrane 52, which membrane is generally thin (e.g. less than 20 microns thick) and e.g. dialysis membranes (e.g. Spectrum Tra Paul 2, Spectrum Medical Industries, Los Angeles The membrane 52 can be

For example, by preventing components of the ion selective membrane 50 from migrating into the layer 4. The immobilization of #8 serves the purpose of preserving the activity of the #transfer, which prevents the denaturation or Causes inactivation.

Membrane 58 is optional and its presence determines which component of the sample is to be measured. , set/lta): how the component is to be measured. Therefore, it is measured. i) The function of [58 is to physically retain the enzyme or substrate in layer 48 ( immobilization) and/or serve as a diffusion barrier for the substrate. For example, to measure the immobilized enzyme group 5UAK in one layer 48.

For dynamic measurements, it may be desirable to perform dynamic measurements rather than endpoint measurements. In order to expand the range of [41 properties], elementary reactions must be diffusion limited (diffsa ios Netted). In this case, the membrane 58 is serve a purpose. However, if endpoint measurements are desired, wavenumber barriers are not required. and membrane 58 is not necessary; membrane 58 can serve as a diffusion@wall for the substrate. can.

Regenerated cellulose (dialysis membrane) or: other ultra-reactive membranes or reverse permeation@substances can be done.

Internal reference substance 54 consists of a buffer solution. - In the second embodiment, it has a pH of 7. , 4 @ salt buffer peak water element solution (consisting of filli%g no1%tio) Ru. In a preferred embodiment 2, it is a phosphate buffer at pH'1.4 of about 5017. It is a gel consisting of synergic water, about 50 g of glycerin S, and 2.0 g of agarose. Ru.

The reference electrode 56 is an electrode with a fixed potential, so that the sample solution can be changed or Fluctuations in the liquid junction potential are also caused when the calibration solution changes it. Not shown.

The key considerations for satisfactory reference electrode performance are:

They are reversibility a (in an electrochemical sense), reproducibility 2, and stability. three types of references Illumination electrode systems are currently in use.

Metal amalgams such as saturated calomel electrodes (SEC); quinhydrone or phenate Redox couple like Lee7 erocyanide electrode! silver-silver chloride reference electrode In the present invention, there are three types of metal/halogenated metal electrodes.

The 85@ electrode is typically a silver/silver chloride button (hstt6).

The subject matter of the invention is illustrated by the following examples.

They should not be considered to imply Pi'Il limits.

Hydrogen ion specific compound 2-octadecyloxy-5-carboethoxyphenyl Ruhydrazonmenxalonitosis (Organic 5ynthesis), Ca11. Etched according to the general method described in Vol. 3 * pp 140-141. EXAMPLE 1 Preparation of L-4-dicutadecyloxy-3-nitrobenzoate.

The following mixture was run for 72 hours in a 1 liter 3-pot flask equipped with a mechanical stirrer. do: Ethyl-4-hydroxy-3-nitrobenzoate of 25J9(0,]2maj) to Lancaster Synthesis (Laoeat-De5yxtka-#O), Wisdkam, NH Catalog◆6451 96th 1-bromooctadecane Aldrich CA of 41.9 (0,12 oJ) ld de 1ck) Chemical Co., Milk Oki-1W1. Catalog φ19,949-4 16.4g anhydrous potassium carbonate SOO- anhydrous acetone After the reflux period, the reaction mixture was cooled to room temperature (i.e., about 20-27°C) and filtered. Remove the salts and remove the F solution by Brinkman (B*eki-E de(%ktaa%) Evaporate using a rotary evaporator. Dissolve the residual smoke in 500117 toluene and add Next, 4 times with 400 d of 5-dihydrogen sodium hydroxide water bath solution.

The mixture is further incubated once with a 400M saturated sodium chloride aqueous solution. Toluene coral anhydrous Dry with sodium sulfate and evaporate to dryness. The oil obtained by this process will crystallize if left for a while. become

The solid was recrystallized with hexane and decolorized with carbon.

Yield: ethyl-4-octadecyloxy-3-nitrobenzoate (yield, 9.4 8-56 pieces, 36 g (yield: 66 g).

The C nitro compound is reduced to the t-amino compound by catalytic hydrogenation. Nitration of the above Compound 10I supported on carbon': Lzeta 1 (20 Add to 01Lt of hexane. The mixture was placed in a Parr hydrogenator and heated to 62 -Heat the reaction mixture to 40°C under a hydrogen pressure of While hot (e.g. about 50°C), add Celite (Ca1ita) (p filter auxiliary medium) to [Usually filtered through Cainku± (Ma%vil1m)], and the solution was treated with hydrogen chloride gas to obtain amine hydrochloride in almost quantitative yield (m-p-141 Precipitate at -145.

Brown ethyl-4-octadecyloxy-3-7 minobenzoate hydrochloride Changed to menxalonitrile by an improved method of et al. (Brow%at　aJ) (U.S. Pat. No. 3,743,588), the above salt @ salt 8L5 fl (0, Dissolve 018 sacrificial S) in IL dimethylformamide (DMF) while warming. , to a temperature of approximately 0@. To this cold solution, add 300ml of concentrated salt and then add 500d of D Add 1,32% sodium nitrite dissolved in MFtLC to the reaction mixture for 1 hour. Next, add 1.15j of pre-warmed malononitrile and mix. Magnetically stir the product for approximately 10 minutes in a strongly basic solution (for example, AH is 9 or more). Add enough triethylamine to give . Warm the mixture to room temperature and stir for 18 hours. A precipitate forms when the stirring reaction mixture is made acidic (pH approximately 2.3) with concentrated hydrochloric acid. . The precipitate is filtered off, washed with water, and recrystallized with methanol. What I got was about 7 ．． 2-octadecyloxy-5-carboethoxyphenyl of 0Ji' (76) Hydrazo/menxalonitrile (m, p, 76, 77. Others shown in Table 1) Derivatives of were prepared in a similar manner.

Implementation example 2. PVC membrane solution rN, spread and ion selection 2-ni of 1.5Mt Trophenyl octyl ether (Flska) Chemical Company, Hawapupa Hasppasgg, NY, catalog "73732) and 0.10g 1 hydrogen ionophore (as prepared by the method described in Example 1) 0117 in tetrahydrofuran.

This solution has a very high molecular weight of 0.61 and a density of 1.385 fi/ce. Powdered polyvinyl chloride [Aldrich (Azd. 1ek) Chemical Company, Milwa Oakey %Wf, catalog +18261-3) is added. Until PYC dissolves Shake the mixture (for example, in a swirl).

The PVC solution was poured onto a stress-relieved polypropylene plate with a thickness of KI' and Conditions are maintained to allow evaporation of hydrofuran. For example, tetrahydrofuran Evaporate twice at room temperature under a humidifier so that it is removed as it evaporates. can be set. The obtained product is a plastic material containing ion-specific compounds. It is a membrane. '7 Cut the disc from the membrane using a cork borer (inner diameter 0.56%) and remove the disc. , Orion Research Co., Ltd. (0rios)' [hard body] e5earch 7sc, ), Cambridge Fuji, HA, catalog “950015 ). The internal reference electrode is a silver/silver chloride reference electrode, and the internal light filling solution is pH 7,4 phosphate buffered salt solution [Sigma (SzGyA) Chemical Co., Ltd., Seytle Lewis, MO, Katasegu+1000-3).

The pH-odor performance of the electrode was evaluated in all measurements using a 250-mm beaker. While performing magnetic stirring inside the chamber, a double contact Ay/AtC4 electrode [Corning (C er*1ny)s Catalog"476067)Y for comparison purposes. , the combination pH/reference electrode is immersed in the test solution, and the change in mV in the membrane is measured with a glass electrode. pHyi conversion was recorded in relation to the changes in pH determined.

This is evidenced by the fact that the membrane of the present invention does not tear or tear when stretched; As such, it showed good mechanical properties. Furthermore, it also shows a good minute VR performance, 6 to 6 A gradient of 55-58 Vf) was obtained over a pH range of 8. However, Performance decreased slightly for Ryoru in the range of .

For monovalent ions, the theoretical slope is ±59.1% V at 25°C. In addition, The response time, that is, the time required to reach equilibrium, is less than 1 minute, and the electrodes do not require any testing. No preparation was required.

The properties of the membrane described above are determined by the fact that the hydrogen ion selective component is the 3-chloro-2-ol The same person except that it is tadecyloxyphenylhuman 2-symmenxalonitrile. The properties were compared with those of the membrane prepared by the v4 method. Compared to the above membranes, chlorinated menxalonite The membrane prepared from RIL showed inferior performance, i.e., the slope M was lower in the same pH range. It was only below 40 aF, indicating a decrease in sensitivity.

The performance of various hydrogen ionophores is summarized in Table 1. relative to the ionophore position. Three derivatives with an 18-carbon chain that is lipophilic at the root are 8 It can be seen from this Makurai that all four of them have excellent performance. What can you infer from this? This means that the lipophilic weight adjacent to the hydrogen ion exchange site probably responds to hydrogen ions. This means that it will promote @The oily chain has a lipophilic region in the active position. may form a region, thereby increasing the selectivity for hydrogen ions.

A sensor as shown in FIG. 6 is used for urea measurement. Urea-selective sensor A second membrane containing the enzyme urease is placed inside the ion-selective electrode of the sensor. Created by incorporating. Therefore, pH membrane or ammonium sulfur ( MffI+) It can be. The phrease-containing membrane is exposed to the voltage shown in Figure 4f. Exists at poles ·, &, and C. These two M are the third 8I (for example, the membrane in Figure 6). 52).

Place the test sample (e.g. blood, blood@) and calibration sample on the electrodes as shown in Figure 4. Add.

A and B are porous materials (with a core that is made conductive by adding a wetting agent). It is. g, i, and C are ammonium io7 selective electrodes. 2 types of schools Add the positive sample to 6 and b with warease, the patient's sample will be eK7XJ.

# also has warease.

The reaction catalyzed by urease occurs with b and eK2. This is it The patient sample can be compared with the two calibration samples, thus calibrating the test card. is taken into account.

In the presence of warease, the following reaction occurs: MHz CONEI + ff t O−111÷2NH, +CO.

wn, +H, oNH,”+oH- CO! +H, 0te==: Lost BCO,''' +H1 As a result, ammonium selectivity The membrane is used to detect changes in ammonium ion (NH,-) concentration. or the pH-selective membrane can detect the increased pH caused by the formation of OR-. It can be used to

Warease-containing membrane 1; prepared as follows = 5 μ/d of warease (300% Knit/■New England x7 team center (New E% grand Esgyms Ces! sr) Boston, MA) Bath solution with pff7.4 phosphate In buffered salt solution (Sigvpha Chemical Co., catalogue 1ooo-3) Other buffers found to be effective are: 0. IMN a 10nt in CL mol/L Natriwaum Hepes (Sed<ssHspsm) (pH 7,4) and and 0. l Q mwol/L NaHzPO* in I M NaCL, 1 0 mvhollL) respace (triabaas) (pH 7,4) There is a 12 micro 7 bore size nonitrocellulose membrane [Schleicher Clear Shue] 5ahlaicλ-r & chsall, Key/, NH, 034 A 47 m diameter disc of 21 grade AE100 was placed in 700 pl of enzyme solution at room temperature. Soak for 5 minutes. Remove the membrane from the solution and scrape off the excess liquid with stirring! ! Missing page, film on a hydrophobic surface (e.g., a sheet of polypropylene) for 30 minutes at room temperature. lLiE. The membrane is stored at 4°C in a sealed container.A disk is cut out from the membrane and used as an electrode. a, h, and C (see Figure 4f), place the sample solution (25 microliters) ) can be put in C and the calibration @ solution can be put in 6 and b. 9 in a, mi, and c Rease acts on the calibration bath solution and urea in the sample, liberating ammonia and COl. Ru. Measure the increase in pH or ammonia.

For example, 40 mmol/L NaHJO4, 40 trihthol/L) Respace, and pH 7.5 mild water containing 100 gRRal/L NaCL. Contains *5 61/L urine and] Omvnal/L urine that has been diluted with glI A mild urea solution was prepared. Put 5v+m*l/L urine solution into G and C, add urea When a buffer solution containing no B was put into B, a difference of 25 tnV was observed after about 1 minute. Ta. In the case of 10 mmol/L urea solution, a difference of 50 txV was observed. Ta. No difference in electromotive force was observed in the absence of urea.

Similar behavior was observed when the pH membrane was replaced with an ammonium-selective nonacti/membrane. It was done.

Regarding Figure 4, cells 6, b, and C each contain immobilized glucose oxidizers. contains membranes with enzymes and catalases.

If glucose oxidase CGOD) and catalase are present, the following reaction will occur Gelcose + H, O + 0 *- Silyl Gluconite (H+) Foal O1H, O Goose - 2? 22=-Nyo1-==Ninini 2 Mimi Shi (O wealth 10 H I O catalase) The purpose is to recirculate the oxygen consumed in glucose oxidation, thereby The goal is to expand the linear area where the current is measured. Glucose oxidase/Katara The nitrocellulose membrane was prepared as follows: The nitrocellulose membrane described earlier was 51 to 9/ILt/lucose oxidase (300 units) in phosphorus@Kaki buffered salt solution. ツ)　/my.

Boehringer Mannhein (Be-5rt*gar Ma**kai*)) and and 1 da/- catalase (40,000-=+- nits/da sigma (Sig course 6) , immersed in 700 microliters of a solution according to catalog "(1"-100). Ta. The membrane was dried as in Example 3. # Elementary disk 6. I put it in b and c.

It has been found that the concentration of glucose is proportional to the potential difference created between - and C.

Other noglucose measuring methods use hexokinase/ATP as the enzyme system. No. Regarding the 4# figure, cell, b% and C are immobilized hexokinase and ATP, respectively. , and membranes with magnesium salts such as chlorides, acetates, or sulfates. Ru.

In the presence of hexokinase, ATP% and magnesium ion, the following reaction will occur Gel;-S+ATP≦glucose-6-phosphate 10ADP1H+ The hexokinase/ATP membrane was prepared as follows: Nitrocellulose The base film was diluted with lOs glycerol, 0.25 chitriton (7'r<gos) -100 and 0.005M) buffer consisting of reethanolamine (jlff? , 8) 109/L Magnesicum Acetate, 50"97RIATP, and 101F 1.0 i of a solution consisting of /117 hekin kinase. Glucose in sample It has been found that the concentration of carbon is proportional to the potential difference generated between carbon and carbon.

With respect to Figure 4j, the membrane containing the enzyme (in this case lipase) is separated from A/@, i, and C, where a similar reaction occurs when lipase is present. 10 grams of triglycide, 09/’:’4! ’　Glycerol + fatty acid (?) As in the previous example, the nitrocellulose membrane was soaked in 5 μ/− lipase buffer Suma Chemical Company.

The membrane was made v4 by immersing it in catalog L43841. Contains triglypeptide The decrease in pH that occurs upon addition of the sample was detected with a pH electrode.

Implementation example 6. Similar to the previous example made of v4 # elementary electrode for uric acid measurement, And regarding Figure 4.

Warikase (503-knit/co, Cedar y (SigmaG) Chemical Company, Kata log φU1878:l and catalase [1 da/d40. ooo unit/tasig Nitrocellulose membrane is immersed in a solution of Cataro/"C'-100" manufactured by Mach Chemical Co., Ltd. A specific electrode for uric acid is manufactured by v4. Cell a with enzyme membrane.

Place it in b, and c.

When cricase and catalase are present, the following reaction occurs: Urine a+0. +H, O' Liquor" 75 Intoin +co, +H, O.

g, o, h　7: Go = KO, +H, 0 The immediately generated COx causes a decrease in pH. In this case, theophylline is measured using the KG electrode and the cuff electrode. This is due to differences in the immunochemical reactivity of the proteins. Caffeine has 1 methyl group It differs from theophylline only in the following. Theophylline and caffeine are cracked each conjugate is incorporated into the PVc membrane. Ru. The theophylline membrane forms one half cell, while the force7ine membrane forms the other half cell. ing.

The caffeine membrane serves as a reference electrode, and each! Il! [is alone converted to potassium ion However, each membrane is incorporated into a concentration cell and contains potassium ions. When the same solution is placed in each half cell, the electromotive force generated between the two half cells is zero millimeter. Very close to Volt, the theophylline and caffeine conjugate is electrochemically Same thing! eJ, but immunochemical reactivity to theophylline antibodies. Great in terms of points! ! There is a difference. This difference in immunochemical reactivity and the same iontophoresis Electrochemical reactivity is the basis for differential potentiometry of competitive binding to theophylline. It becomes. This method has a certain ionic strength and also a certain potassium ion activity. [as shown in the research of Rgtksitt] The main advantage is that serum samples can be used without dilution or dialysis. It's true. The sensor arrangement can be as shown in FIG. 41.

The sensor consists of theophylline klawan ether conjugate in cells a and d and the cell Formation of caffeine-crown ether conjugate in b and c, porous connection 81 A and CVc. Add C and dK to the sample containing theophylline. , Meanwhile, add the calibration column liquid to 6 and b. The competitive binding phase involves The presence of theophylline antibodies with low mutual reactivity is required. Antibodies are sensors It may be added to the sample and calibration solution before analysis.

Alternatively, it may be included in the sensor cell A so that competitive binding occurs within the sensor. . Competition for antibody sites is caused by theophylline immobilized in the membrane and theophylline in the sample. Starts with: Ab + Ay (sample) + AI” 7j’ b A t (sample) + A&A j-1 (membrane) (membrane) Theophylline antibody binds to theophylline that is added to the membrane (a and C). When this happens, a change in potential occurs, the magnitude of which is inversely proportional to the concentration of theophily in the sample. . Therefore, as the concentration of theophylline increases in the sample, it becomes more likely to bind to the membrane. Antibodies will decrease and therefore the electromotive force will become smaller anyway. Typical measurements involve antibody binding. The change in potential difference due to the voltage difference is typically on the order of a few millivolts. Caffeine “reference” membrane In other words, when using the differential research method using theophylline membrane in combination with Another advantage of this is that it is common mode, meaning that the initial starting potential is close to zero millivolts. This means that the signal to be measured is on the order of a few millivolts. common mode If the potential is low, the sensitivity when measuring the signal voltage will be high, and therefore the background If the voltage on the main mode or common mode is lower than the signal voltage, use a more accurate Measurement becomes possible. As mentioned in Jing, theophylline half cell and kaffeifu half cell The potential difference between is almost zero when treated with the same potassium ion containing sample. It is. However, if the half-cell is replaced with an SCE or silver-silver chloride reference electrode, , the potential difference in the same potassium iodine solution increases by more than 50 v*V.

The method for synthesizing the conjugate and the method for producing the membrane are described below: θ G O @ @ The first step in the synthesis involves blocking the 7-position of 1-methylxanthine. (Hs), sink (s (%yA) and vine”’7 (UI Jmas) method (J. Am. Chem. Jot., 45:1711-1713 (1980) ) was followed. 1t Konica K 75 700yLt, ft water n A/ Fvc 3.2 P C] 8.9 mqnal) 98th-1-methylxanthyl (Aldrich Chemical Co., catalogue 2 & 098-4) and warm the mixture until dissolution occurs.

It was then cooled to room temperature. Add 2.0 JF (18,9 Add anhydrous sodium carbonate from m-02).

Next, 97 cyclones of 3-Ol (19.3 mmol) dissolved in 50 g of DMF Lomethylbiparate [All-rtek Chemical Co., Catalog φ14,118-6) was added dropwise over 1 hour, and the reaction mixture was stirred overnight at room temperature. did.

The reaction mixture was evaporated to dryness and the residue was treated with 50 m of methylene chloride. Unbath items The material was separated from P and washed with 1N HCl to obtain 1.24 g of 1-methylxanthine.

The methylene chloride solution was evaporated to dryness to obtain a mixture of -protected and diprotected xanthines. . -protected 1-methyl-7-[(pivaloyloxy)methylxanthine with ethyl acetate It was isolated from the diprotected kipuntin. By this method, 1.5J (D-protection H*) (m, p, 204-206 were obtained.-protected The ethyl acetate P solution from which the Cool the reaction mixture at reflux for 18 hours with 011t of 2N sodium hydroxide. The mixture was acidified with concentrated hydrochloric acid and extracted with chloroform. Water layer′? : Evaporate o, H 1-methylxanthine was obtained.

The second step of the synthesis is v4M of 4'-amino-benzo-15-crown-5 hydrochloride. be. 1:1 glacial acetic acid/chloroform 800 d, 30,9 (6,11 bait regret/l) benzo-15-crown-5 [Paris (Pa de 1nk) Chemical Co., Ole] While stirring the solution of A concentrated solution of 15id dissolved in glacial acetic acid was added over a period of 1 hour. The reaction mixture was stirred for 1 hour and then evaporated to dryness on a rotary evaporator. 50 residue The mixture was treated with an aqueous solution of 0.5% hydrogen carbonate and 5001Lt of IJ. Partitioned with chloroform.

The chloroform layer was washed with saturated aqueous sodium chloride solution and dried with anhydrous sodium sulfate. I set it. Evaporation of the chloroform yielded 30 g (86%) of 4'-nitrobenzo-15. - Crown - Obtained 5 crowns. The product was recrystallized from ethanol and its x5i+sa% -υ was dissolved in ditdFf7 (250117+7) and heated to 50°C under a bar (P1!ar ) Catalytic reduction over Raney (Ra%*y) nickel during hydrogenation 4.

The reaction mixture was passed through, the P solution was evaporated to dryness, and the residue was dissolved in 20011j ethyl acetate. The dissolved solution was then treated with hydrogen chloride gas to form 4'-amino-benzo-15- Kurakun-5 salt was precipitated. Yield: 9.0g (58g) The third stage of synthesis is ¥1 of 4'-benzo-15-curacun-5-(11-bromo97decanoamide) Made by @. 1 dissolved in 100d CM, CL, in a 250 conical flask. 0 g (37.7 mmol) of 11-bromo undecane [Aldrich (A ldriC &) Chemical Co., catalog φB3.280.4) has a 5.5- I took thylamine. The mixture was cooled to 0°C and 9.67p of N,N-bis[2- Oxy-3-oxazolidinyl]phosphordiamidic chloride [Bobcle (B OPCL) Chemical Dynamic Co., Ltd., added catalog "12-1370-001" Ta. The mixture was stirred for 0.5 hours and heated to 12.06 F (3?, 7 mmo D of 4'-aminope/cy 15-curacone@acid was added to 5.5% of catfish triethylamine. It was added with To this reaction mixture, 50317 was further dissolved in CH, CL. 5, 517 triethylamine was added dropwise over 1 hour 0 reaction mixture g! J is 18 Allowed to warm to room temperature for an hour. Next, water (50 d) was added and the mixture was acidified with concentrated hydrochloric acid. did. c g, C/, layers sequentially with saturated sodium chloride 9M solution of 200117 3 and 2001j 5% carbonated water Nanatrium 9M solution three times. CM, C11 The layer was dried over anhydrous sodium sulfate and evaporated to dryness. The residue was diluted with hexane/g 8.8 g (44 g) of 41-benzo-15-cra9-5- (11-bromowandecanoamide) (Q, 7.101-10 0 analysis results obtained 3) Calculation of fruit knee CHH4oBrlVO@ m:c: 56.60 Iff: 7. 60 sB de: 1 5.06, N: 2.62. Actual CH Direct: C: 56. 41. H Near, 46 B B De: 14.89 1 N: 2.60° In the 4th stage , Klawan ether was coupled to the δ1-methylxa/tin. 125 50H1f) I) MP in a conical flask of 1.0 g (3,57 1-Methyl-7-[(piparoyloxy)methyl]xanthyl/ from 1) was added.

The mixture was warmed until dissolved (e.g.

(with stirring to about 80°C), and the solution was cooled to room temperature to 0,7611 (7,1 mvs oj) of anhydrous sodium carbonate, VC and 1,90 JF (3,57 mv 0 reaction mixture with crown ether amide of soj) was stirred under nitrogen for 3 days. Remove the DMF in a zero-rotary evaporator, add 1QQd of water, and reduce the mixture to 501R1. Extracted with chloroform three times. Combined with chloroform extract, 100al! of It was washed once with a saturated aqueous sodium chloride solution and dried over anhydrous sulfurized sodium chloride. Chloroho The rum was evaporated to give a light brown oil.

The fifth step is to remove the protecting groups on an aqueous basis.

Add 75-0,2 J/Ha OH to the above oil in a 250 d round bottom flask. and 25M methyl alcohol were added.

The mixture was refluxed for 1 hour and cooled to room temperature. Cool the solution in an ice/acetone bath. Acidified with concentrated hydrochloric acid. Re-crystallization of the precipitate from ethyl alcohol-water, 1. 2 g (55%) yellowish-white crystalline solid (m, p, 166-170°) The obtained substance was confirmed to be homogeneous by high performance liquid chromatography analysis. Calculation of analysis result knee C,,H4J,O,: C': 60.47tffnear, 37HN: 11.37. Actual measurement [:C':60.25. ff Near, 23. M:11 ．． 09. The benzo-12-clawan-4 derivative was prepared lII in a similar manner.

*a-protected 1-methylxanthine of Fein-Penzo 15-Cla9)-5 conjugate 1 according to the fourth step of the reaction mechanism, except that 1,7-dimethylxanthine was substituted for Caffeine conjugate v4 was prepared.

1,0 g (5,56 mw) in 150 ml of DM in a 125- *1,7-dimethylxanthine 7 of I) was added. Do not stir until the mixture is dissolved. I warmed it up. The solution was cooled to room temperature and 0.5'l of anhydrous tetracarbonate was added. Then, add 2-95# (5,56 vmmol) of the Tarawa 7 ether amide described above to 1 The reaction mixture was stirred for 3 days, the DMF was removed in a zero-rotary evaporator, and 100 g of water was added. , the mixture was extracted three times with 59d chloroform. 100g of extracts combined Wash once with saturated sodium chloride water bath.

It was dried with anhydrous sodium sulfate. Chloroform"Ikg was released to give a brown oil, The product was crystallized with ethyl acetate to obtain 1.0129% of the product. product was decolorized from ethyl acetate/hex 7 with bleached carbon and subjected to high-performance liquid chromatography. It was found that the results were homogeneous (%, 9.130-133). Calculation of result: -C-H,, N, O, [:C:61.03. ff:? 12 irises N:1 1.12. Actual measurement: c:so, 7s wealth ff near, 53. N: 10.97°benzo The -12-curacun-4 derivative was prepared in a similar manner.

Preparation of drugs for theophylline measurement Theophylline components are o, oosg. Theophylline-crackone ether conjugate, 0.0211 tetra(arclov) bis(2-ethylhexyl) sebaque, 0.5 m ), 0.3.9' high molecular weight PVC and 5d THF. The membrane mentioned before It can be formed by a solution casting method or a dip coating method, as described above.

caffeine membrane Components of Kaffei Membrane (Caffeine-Klawan ether conjugate of 11) , 0.029 potassium tetra(p-chlorophenyl)folate, 0.5jlj Bis(2-ethylhexyl) sebacate, 0.3.9 high molecular weight PVC and 5- It was made from THF. The film is formed by solution casting or dip coating as described in 1wJ. can be set.

Orion (Qrf%) 95 each with one theophylline and caffeine membrane It was housed in an electrode body. The internal photoreceptor solution is made of phosphate buffered saline at pH 4. Therefore, the reference electrode was silver/silver chloride. Attach the two electrodes to the rubber orange 5.0d It was immersed in a phosphate buffered saline solution with a pH of 1.4.

The solution was magnetically stirred. The electromotive force measured between the two electrodes was 0.23 mV . To test the response to potassium ions, 100 st of 0.2 vph al　ECt' pipette into the stirring buffer. S) IJ tube chart V A clear peak (transient response shown as 03w) was observed in the output of the Korg. . The potential difference returned to baseline ilK within 30-60 seconds. against potassium 9m The transient response is probably due to local aa of potassium ions near the membrane surface; This is probably because it disappears as the solution is mixed.

To examine the response to protein, 3-59/dL of albumin and 3.0 9/dt globulin] 100st standard protein solution [Sigma (S) 9m4) Chemical Co., St. Louis, MO, Catalog ◆540-10) as a solution added to. Standard protein solution had no effect on the electrode.

However, 100sA theophylline antibody [Immunote'ri (lmm5so! gck), Alstore (AI Eaters), MA, Catalog 65 1 or Research Plus (Rag-GeideckFJsa), Hay:l 7 (J Fay*5ss), NJ, fil 12 guna 0l-9603-09) Y addition When this happens, the potential difference between the two electrodes is only 0.85 mF for about 20-30 minutes. moved.

This type of sensor can be used for competitive conjugation measurements. bound in the sample The remaining antibodies are measured and serve as an indirect measure of the antigen present in the sample. trial The more antibodies bound by the antigen in the sample, the smaller the electrical signal obtained. . This is because the antibodies that are used to bind to the antigen fixed in the 77-cer membrane are This is because there will be less. That is, the concentration of antigen is inversely related to the magnitude of the signal.

Creatinine is an important indicator of renal function, and its measurement in serum is routinely performed. It is a blood test. Currently, there are two methods for measuring creatinine in clinical laboratories. Ru.

A widely used method is to combine creatinine and picrate in an alkaline solution. This is the Jaffa reaction, which is based on the formation of a red complex. This method number The presence of certain metabolites and drugs will give erroneous results. The second method is # It uses the raw materials. Although the enzymatic method is very dynamic, it is difficult to The cost and time required to do so limits its routine use in clinical laboratories.

Very recently, a creatine ion-selective electrode was described [E.P.D. Diamondis (E, P, Dits - order + * dim) and T.P. Jiiana +7 (T, P.

Htsdjia 9%a Anal, Latt, 13, 1317-1332 ( (1980)), an equimolar aqueous solution of creatinine and tetraphenylhexyl sodium When the liquids are mixed and then hydrochloric acid solution is added, creatinine 9m tetra7 enylhou Taleatinicum ion exchanger vI4 is prepared by forming an elementary complex salt. Applicable The membrane was extracted with 2-nitrotoluene and the complex salt solution was transferred to an Olio tube equipped with a Teflon membrane. (0rios) 92 [Orion Research (0rios Raaea deah), Cambridge, MA) was used as a liquid ion exchanger in the electrode. This type of ion In a universal electrode, the ion exchange material is in liquid form in a receiver and passed through a membrane. Since it constantly leaches into the sample solution, it must be supplemented periodically.'! ! ! 1! i is 0.01 thol/L creatininium chloride for 24 hours before use conditioned by soaking in creatinininium at pH 3 to 2 The response has a slope of 57 mV at 20°C in the range of 10-” N10-1 gas/g/L. It was linear. 10-'~10-'meJ/L (In the D range, the slope is 37 It went down to Shin V.

The GX of the present invention utilizes a plastic membrane on which the ion exchange substance is immobilized. . 2-nitrotoluene was used as a plasticizer in pvcB. When incorporating 7-enylfluorophore, I O-”vkol/L~I O-”saJ / A slope of 44 V was observed between L creatinine chloride, and I O- '~] 0-"mol/L O rangettjA i'!! 14 mV K decreased . The clinically important range for creatinine is lO-' to 10-1 mL/L.

Others such as 2-nitro-2-cymene and 2-nitropheroctyl ether If a nitrated plasticizer is used, the sensitivity will decrease in the range of 10-4 to 10-”mol/L. It didn't get better.

Significantly improved response in the range of 10-4 to 10-” me l /L is generally formula: (In the formula, X-CL, E-de, F, CF,) 1i! tetraphenylfluoride Obtained when using salt. In one aspect.

0.02,917) Creatininium tetra(p-chloropher) boron, 1 ．． 5+j 2-nitrophenyl octyl ether, 0.31i very high molecular weight A membrane consisting of PVC, and THF of 111 ia was prepared. I mentioned the membrane before. Sea urchins were prepared by solution casting or dip coating. The layer is 10°1al A slope of 55 inertia V between /L and 101 Tsuru ml/L creatinicum chloride, Also, a slope of 40 F is produced in the range of 10-4 to 10 Li/L, which is not suitable for any test. No preparation was required. ! The substituted tetraphenyl boron salt showed unsubstituted tetraphenyl Table 2 summarizes the performance of various 5L-converted tet-2-phenylhoku salts.

Unexpected differences in the substituted tetraphenyl group compared to the unsubstituted tetra phenyl group The increased sensitivity was due to increased lipophilicity and the substituent formed by the draphenylboron salt. This can be attributed to the difference in the quality of the ion-exchanged complex. In fact, creatinine The solution containing 2B must be lowered to a pH below 3 by the addition of 52 Yes, this can be done by diluting the sample with an acid solution.

However, in the case of serum samples, significant dilution may result in severe dilution. By decreasing the concentration of =7 Measurement sensitivity may be adversely affected.

In the case of non-invention, we took a different approach. p of the sample containing creatinine H was lowered by treating the sample with a carrier containing anhydrous glycine hydrochloride. . This is nitrocellulose [Schleicher & Schull (Sc51aiek) ar　asd　Sck＊ll)　12 micron grade HE-100　 Immersing the support in a glycine hydrochloride aqueous solution (0,]1.091L/1/L) It was done by Nitrocellulose membrane V Achinini 9 Muchitra (p-/Rolov A sandwich was formed across the surface of the phenylboron PVCH. Fourth figure Regarding cells a, b, and C, each cell contains creatinicum pvcyth and glycine salt. Contains a nitrocellulose membrane containing acid salts. Serum sample in C.

Add reference solution and calibration solution to 6 and b respectively. Glycine hydrochloride is creatine Converts nin into creatinine hydrochloride, which is marked by a membrane.

The electromotive force generated between a and b can be used to calibrate the sensor. a The slope from and b) and the electromotive force generated between b and ef) further increase the creatine acid in the sample. It can be used to measure concentration.

V-4-step Taleatininium hydrochloride [Si] Sjyma Chemical Co., St. Louis, No. Catalog φC-625 73 and the corresponding sodium salt of substituted phenylhora are mixed together. Creatininium tetraphenyl boron salt was prepared. Separate the precipitated salt and add water fc') and dried. Substituted tetraphenyl derivative sodium 9m salt is casso Let (Caaaorgtta), ? Tarafati (J/cja-shi first order) Contains steps: X-F, C1, CF.

Table 2 Creatininiwam term lS2 of creatininium tetraphenylhochlorine salt Response slope to fence (inertia V) Annoyance (kei V) H5010 F 45 26 CL 55 36 CFB 57 40 All wF readings found in Table 2 are for a dual contact silver-silver chloride reference electrode. It is. The talleatinium solution used was 15 (lad/6 ml of chloride) and and 6 sM tj; 0.05M glycine-glycine hydrochloride buffer containing Kitamensucum It was prepared using a liquid (pff3.0).

Made of v4 A membrane selective for sodium ions was prepared using the following proportions of components: prepared: Cl2t methylated monensin, 0.049 tetra(,-chlorophene) nil) sodium borate, 2. 〇-2-nitrophyle octyl ether, 1 29 high molecular weight PVC112-tetrahydrofuran (THF) and 5-4- Methyl-2-pentanone.

The membrane can be formed by solution casting or dip coating as described above.

Place the electrode in the electrode housing described above and conduct the test using an aqueous solution containing sodium ions. went. The sensor produced a slope of 57 to 60 m* per lO unit at 25°C. .

Add 20-m of iodomethane to 250-m of 1,4-dioxane in a 500-m of round-bottomed flask. 5. With Tan. Of Sodium Monensin [Sigtsha Chemica Le Co., St. Louis, NO. Catalog ◆j/2513] was added. Mixture 18 It was gently refluxed for an hour and then evaporated to dryness on a rotary evaporator. 200 residue The water was dissolved in methylene chloride, and then dissolved in a 500i saturated aqueous sodium chloride solution. twice with 5% sodium bicarbonate aqueous solution of 200111t, and twice with 200- Washed once with saturated aqueous sodium chloride solution.

The methylene chloride solution was dried over anhydrous sodium sulfate and evaporated to dryness. residual The material was warmed in a 50'C water bath and treated with 50' hexane. hexane solution The mixture was cooled in a freezer for 1-2 hours and then filtered to remove precipitated salts. Heki Evaporate the sun solution to dryness, 2.5-4. A light brown oil of Of was obtained. (the oil lotus) It was used to prepare and conduct an IJ-um selective membrane.

Implementation example 10. Preparation of ion-selective electrode for potassium measurement A membrane selective for potassium ions was prepared using the following ratios of components: :0.2t palinomycin, 0.04t potassium tetraphenylborate, 2 ．． 0wt 2-nitrophenyl octyl ether, 1.2t(/:) high molecular weight PVC, 10 wt THF and 5d 4-methyl-2-pentanone.

It can be formed by a solution casting method or a dip coating method as described above. .

Implementation example 11. Preparation of ion-selective electrode for chloride measurement A membrane selective for chloride ions was prepared using the following ratios of components: 1 ．． oti's Alifor) (Aliq-sat,) 336 C Aldrich (A Tris(2-butoxyethyl)phosph of ldrich) Chemical Co., 2, Owt. [Aldrieh Chemical Co., Milwaukee, WI, CA] 13,059-1), 1.2t of extremely high molecular weight PVC [Aldrich Caldrich) Chemical Co., Milwaukee, Wl. Catalog 18, 26 1-31.12m! of THF and 5 of 4-methyl-2-pentanone, As described above, it can be formed by a solution casting method or a dip coating method.

industrial utility The present invention uses samples, particularly blood, serum, plasma, urine, saliva, and brain, to collect biological samples such as cerebrospinal fluid. The ionic content or concentration of other components of a liquid has industrial utility. present invention The ionic activity of biological samples as well as glucose, urea, triglypeptide, and Concentrations of other sample components such as reatinine, uric acid, lipase, other enzymes and drugs It is particularly useful for rapid measurements of No need to prepare for testing ion-selective membranes, fast It is suitable for use in clinical or research situations as it provides accurate results. moreover , the present invention can detect similar Can be used for measurement.

equivalent Those skilled in the art will be able to determine, using no more than routine experimentation, the specific ingredients specifically described herein. and many equivalents of the substance, which would be able to recognize or fi Una etc. (no change in content) Chopsticks 9.0Q======Od admission Clean; no change in C content) Engraving (no changes to the content) Engraving (Contents: No changes) a, b+ (procedural correction of 8dandan kuri ananium H odorushi grimin yofuenuma-1 hypthema 1 inclusion) Book□ January/2nd, 1985 1.Display of the incident PCT/US86101372 2. Name of the invention Ionophore and ion-selective membrane 3 containing it, corrector Relationship to the incident: Applicant address Name: Willis, John Kenpei 4. 0 agents Address: Room 206, Shin-Otemachi Building, 2-2-1 Otemachi, Chiyoda-ku, Tokyo 7. Contents of correction As per ff1M (note that there are no changes to the contents of the documents in (2) and (3))

Claims (1)

[Claims] 1. General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R1, R2 and R3 are independently a. halogen; b. an alkyl group having 4 to 18 carbon atoms; c. halogen-substituted alkyl group; d. alkoxy group; e. halogen-substituted alkoxy group; f. Formula -CO2R4, where R4 is alkyl having 1 to 18 carbon atoms; Ru. ); and g. a keto group of the formula -COR3, where R3 is alkyl having 1 to 18 carbon atoms; and k. selected from the group consisting of hydrogen atoms. ]. 2. General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R1R2 and R3 are independently a. halogen; b. an alkyl group having 4 to 18 carbon atoms; c. halogen-substituted alkyl group; d. alkoxy group; e. halogen-substituted alkoxy group; f. Formula -CO2R4, where R4 is alkyl having 1 to 18 carbon atoms; Ru. ); and g. a keto group of the formula -COR3, where R3 is alkyl having 1 to 18 carbon atoms; and h. selected from the group consisting of hydrogen atoms. ] ionophore. 3.2-Trifluoromethyl-4-octadecyloxy-phenylhydrazone oxalonitrile; 2-occudecyloxy-5-trifluoromethylphenylene hydrazone mesooxalonitrile; 2-octatecyloxy-5-carbethoxyphenylhydrazone mesooxalonitrile; 2-octatecyloxy-4-phenyl The ionophore of claim 1 selected from the group consisting of fluorophenylhydrazone mesooxalonitrile and derivatives thereof. 4. General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R1, R2 and R3 are independently a. halogen; b. an alkyl group having 4 to 18 carbon atoms; c. halogen-substituted alkyl group; d. alkoxy group; e. halogen-substituted alkoxy group; f. Formula -CO2R4, where R4 is alkyl having 1 to 18 carbon atoms; Ru. ); g. of the formula -CO2R3, where R3 is alkyl having 1 to 18 carbon atoms; Ru. ); and k. selected from the group consisting of hydrogen atoms. ] for potentiometric measurement of the ionic activity of a sample, which is characterized by containing an ionophore. Me sensor. 5.2-Trifluoromethyl-4-octatecyloxy-phenylhydrazone oxalonitrile; 2-octatecyloxy-5-trifluoromethylphenylene Ruhydrazone mesoosalonitrile; 2-octatecyloxy-5-carbethoxy Phenylhydrazone mesooxalonitrile; 2-octatecyloxy-4-fur The sensor according to claim 4, characterized in that it contains an ionophore selected from the group consisting of olophenylhydrazone mesooxalonitrile and derivatives thereof. Sir. 6. a. General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R1R2 and R3 are independently a. halogen; b. an alkyl group having 4 to 18 carbon atoms; c. halogen-substituted alkyl group; d. alkoxy group; e. halogen-substituted alkoxy group; f. Formula -CO2R4, where R4 is alkyl having 1 to 18 carbon atoms; Ru. ); g. a keto group of the formula -COR3, where R3 is alkyl having 1 to 18 carbon atoms; and k. selected from the group consisting of hydrogen atoms. A membrane consisting of an ion-selective compound: 7. a. The ion-selective compound makes up about 1-10% of the membrane by weight, and the 2-trifluoride Lomethyl-4-octadecyloxyphenylhydrazone mesooxalonitrile; 2-octadecyloxy-5-trifluoromethylphenylhydrazone mesooxalonitrile Salonitrile; 2-octadecyloxy-5-carbetoxyphenylhydrazone Mesooxalonitrile; 2-octadecyloxy-4-fluorophenylhydrazone selected from the group consisting of mesooxalonitrile and derivatives thereof; and b. The thermoplastic or plastic constitutes approximately 10-30% by weight of the membrane and Polyvinyl chloride; cellulose acetate; polyvinyl acetate; and silicone rubber. 7. A membrane according to claim 6, selected from the group consisting of: 8. Approximately 50 to 80% by weight of the membrane, and phthalate ester; adipic acid ester at least one selected from the group consisting of: ether type; sebacic acid ester type; aliphatic and aromatic ether type; The membrane according to claim 6, further comprising a plasticizer. 9. The plasticizer comprises about 50% to about 80% by weight of the membrane, and the 2-nitrophenyl octyl 9. The membrane of claim 8, which is a ether. 10. In the method in which an ionophore selective for hydrogen ions is used for the potential difference measurement of the hydrogen ion activity of a sample, the ionophore has the general formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [wherein R1R2 and R3 are independently a. halogen; b. an alkyl group having 4 to 18 carbon atoms; c. halogen-substituted alkyl group; d. alkoxy group; e. halogen-substituted alkoxy group; f. Formula -CO2R4, where R4 is alkyl having 1 to 18 carbon atoms; Ru. ); g. a keto group of the formula -COR3, where R3 is alkyl having 1 to 18 carbon atoms; and k. selected from the group consisting of hydrogen atoms. ] The method as described above. 11. Testing the activity of an enzyme in a sample that catalyzes a reaction that causes a change in the pH in a sample, or the activity of a component other than an enzyme that is a substrate for a reaction that causes a change in pH in a sample. It is a sensor for measuring the difference in concentration in a sample, and has a general formula: ▲Mathematical formula, chemical formula, table, etc.▼ [In the formula, R1, R2 and R3 are independently a. halogen; b. an alkyl group having 4 to 18 carbon atoms; c. halogen-substituted alkyl group; d. alkoxy group; e. halogen-substituted alkoxy group; f. Formula -CO2R4, where R4 is alkyl having 1 to 18 carbon atoms; Ru. ); g. a keto group of the formula -COR3, where R3 is alkyl having 1 to 18 carbon atoms; and k. selected from the group consisting of hydrogen atoms. ] The above-mentioned sensor is characterized by comprising an ionophore having the following properties. 12. a. General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R1, R2 and R3 are independently 1. halogen; alkyl group having 2.4 to 18 carbon atoms; 3. Halodane-substituted alkyl group; 4. Alkoxy group; 5. 6. Halogen-substituted alkoxy group; Formula -CO2R4, where R4 is alkyl having 1 to 18 carbon atoms; Ru. ); 7. 8. a keto group of the formula -COR3, where R3 is alkyl having 1 to 18 carbon atoms; and 8. selected from the group consisting of hydrogen atoms. ]; b. dissolving a plasticizer and the ionophore of (a) in a volatile organic solvent; and c. Removal of volatile organic solvents: A method for producing a hydrogen ion selective membrane comprising the steps. 13. a. placing the membrane material in a solvent; b. removing air trapped in the membrane material; removing the membrane material from the solvent; d. Membrane material and 1. General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R1R2 and R3 are independently a. halogen; b. an alkyl group having 4 to 18 carbon atoms; c. halogen-substituted alkyl group; d. alkoxy group; e. halogen-substituted alkoxy group; f. Formula -CO2R4, where R4 is alkyl having 1 to 18 carbon atoms; Ru. ); g. a keto group of the formula -CORs, where Rs is alkyl having 1 to 18 carbon atoms; and k. selected from the group consisting of hydrogen atoms. ]; 2. Thermoplastic resin or plastic; and 3. a plasticizer; and a. A method for producing a hydrogen ion selective membrane, comprising the steps of: drying the membrane material. 14. General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R is a drug; X is an oxygen atom, or a nitrogen atom bonded to a hydrogen atom or an alkyl group; and n1 and n2 are 1 ) is an integer greater than or equal to . 15. General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R is a drug; X is an oxygen atom, or a nitrogen atom bonded to a hydrogen atom or an alkyl group; and n1 and n2 are 1 an integer greater than or equal to ). 16. R is theophylline or caffein; X is bonded to a hydrogen atom; n1 is equal to 10; and n2 is equal to 1 or 2, The ionophore according to item 15. 17. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (where R is the drug; X is an oxygen atom, or a hydrogen atom or a nitrogen atom bonded to an alkyl group; and n1 and n2 are equal to 1 or A sensor for potentiometric measurement of the concentration of a drug in a sample, comprising an ionophore, characterized in that it consists of an integer greater than or equal to . 18. General formulas: ▲Mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, A compound which is a substituted tetraphenylboron salt having a group (which can be a group). 19. General formulas: ▲Mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, An ionophore which is a substituted tetraphenylboron salt having a group (can be a group). 20. a. General formulas: ▲Mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, b. a thermoplastic or plastic; and c. Plasticizer: A film consisting of. 21. Thermoplastics or plastics include polyvinyl chloride; cellulose acetate; 21. The membrane of claim 20, selected from the group consisting of vinyl phosphoacetate; and silicone rubber, and wherein the plasticizer is 2-nitrophenyl octyl ether. 22. General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R is a drug; X is an oxygen atom, or a nitrogen atom bonded to a hydrogen atom or an alkyl group; and n1 and n2 are equal to 1. A sensor for potentiometric measurement of the concentration of creatinine in a sample, characterized in that the sensor comprises an ionophore of 1.0 or greater. 23. a. A solution of known hydrogen ion activity, a first ion selective electrode and a second ionic a first ion-selective electrode and a second ion-selective electrode. A selective electrode has a general formula: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, R1, R2 and R3 are independently a. halogen; b. an alkyl group having 4 to 18 carbon atoms; c. halogen-substituted alkyl group; d. alkoxy group; e. halogen-substituted alkoxy group; f. Formula -CO2R4, where R4 is alkyl having 1 to 18 carbon atoms; Ru. ); g. a keto group of the formula -CORs, where Rs is alkyl having 1 to 18 carbon atoms; k. selected from the group consisting of hydrogen atoms. ] consists of an ion-selective membrane with ionophores, and the first and second ion-selective membranes are Selective electrodes have a porous material between them to provide ion flow between them. b. A sample of unknown hydrogen ion activity is brought into contact with a third ion-selective electrode, wherein the electrode has an ionophore of general formula (a), and the third electrode and the second electrode An ionizer having a porous chamber between the third electrode and the second electrode to provide an ion flow between the electrodes. c. The emf between the solution that is in contact with the first electrode and the solution that is in contact with the second electrode, and the emf between the solution that is in contact with the second electrode and the solution that is in contact with the third electrode are determined by potential difference. measure; d. calibrate the sensor; and e. A method for measuring the hydrogen ion activity of a sample, which consists of: determining the hydrogen ion activity of the sample from the emf generated between the solution in contact with the second electrode and the solution in contact with the third electrode. 24. The activity or activity of an enzyme in a sample that catalyzes a reaction that causes a change in the pH of the sample. is a method for potentiometrically measuring the concentration of a substance that is a substrate for a reaction that causes a change in the pH of a sample, and includes: a. A solution with a known concentration of the enzyme or substrate to be measured and a first ion selector. a selective electrode and a second ion-selective electrode, wherein the first electrode comprises a membrane having an ionophore according to claim 2 and an enzyme or substrate immobilized thereon. , and the second electrode is an ionizer according to claim 2. It consists of a membrane with nophores and an inactive protein immobilized on it. The first and second electrodes are porous to provide ion flow between them. have quality between them; b. A solution with an unknown concentration of the enzyme or substrate to be measured and a third ion selection. a selective electrode and a fourth ion-selective electrode, wherein the third ion-selective electrode is a membrane having an ionophore according to claim 2 and an enzyme or substrate immobilized thereon. The fourth ion-selective electrode is composed of a membrane having an ionophore according to claim 2 and a membrane having the same inert protein as the second electrode immobilized thereon; and the fourth electrode is that have a porous material between them to provide ion flow between them; c. First train measuring the change in potential difference between the pole and the second electrode and between the third and fourth electrode; and Te d. Calculating the activity of the enzyme or the concentration of the substrate in the sample.