JPS5933389B2 - Polarography sensor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a polarographic sensor used by being inserted into a blood vessel, and particularly to a technique for preventing a blood coagulation reaction that occurs when the sensor is inserted into a blood vessel.

Generally, the concentration of an oxidizing substance or a reducing substance contained in an aqueous solution can be measured by polarography using a measuring electrode (working electrode) of a noble metal such as platinum or gold.

Taking the case of measuring 02, which is an oxidizable (reducible) molecule, as an example, a measurement electrode (cathode) made of PtAu or Ag, etc.
0.5 to 0 for the Ag/AgCl reference electrode (anode).
．． A low voltage of 7 [V] is applied. With this measurement electrode,
A reduction reaction of 02+2h20+4e→40H occurs, and an oxidation reaction of 4Ag+4C1→4AgC1+4e occurs at the reference electrode, so a current flows between the two electrodes.

In addition, when measuring H2, which is a reducing substance, a measurement electrode made of Pt or Pt, Au, etc. with a layer of Pt attached (
(anode) to the Ag/AgCl reference electrode (cathode).
A high voltage of 1 to 0.4 [V] is applied. An oxidation reaction occurs at the measuring electrode, and a reduction reaction occurs at the reference electrode, causing a current to flow between the two electrodes.

Under appropriate conditions, the aforementioned current is proportional to the concentration (partial pressure) of 02 or H2, so the concentration of 02 or H2 can be measured based on the current value. This method can also be applied to oxidizing substances or reducing substances other than H2. However, it is necessary to appropriately select the voltage value to be applied between the two electrodes and the material of the electrodes depending on the type of the substance to be excavated. Even for substances that are difficult to directly measure using polarography, it is possible to immobilize an enzyme that acts on the substance to be measured on the reaction surface of the measurement electrode or its vicinity, and then use polarography to detect the reactant or product produced by the enzyme. In many cases, the concentration of the substance to be measured can be determined by measurement. To immobilize enzymes, the reaction surface of the electrode is coated with a porous plastic membrane, a natural polymer membrane, a thin layer of glass or ceramic, etc., and an appropriate chemical reaction is applied to the surface of the pores in these material layers. Methods are used to bind enzyme molecules or to confine enzyme molecules in these pores and cover them with a semipermeable membrane to prevent leakage. In the case of measuring glucose, glucose oxidase is immobilized as an enzyme on the surface of a measuring electrode made of a noble metal and in its vicinity, and in the measurement of the measuring electrode, glucose + 02 + H2O → gluconic acid + H2O27' ~ V voice z'z' Jr. The concentration of glucose can be determined by carrying out the reaction and measuring the concentration of 02 by the cathodic reduction method described above, or by measuring V)H2O2 by the anodic oxidation method as shown in the following formula.

H2O2+20H→・2H20+e- Regarding substances other than the above-mentioned glucose, ascorbic acid, uric acid, noradrenaline, etc. present in the blood can be reduced by fixing enzymes such as ascorbate oxidase, urigase, and monoamine oxidase to the measurement electrode. By carrying out the reaction shown in the chemical formula and measuring O2 or H2O2 as described above, it is possible to perform measurements with much higher selectivity (specificity) than direct polarography.

``- φ-1 crystal 1 - [MoRmi: 3 Although it is possible to measure an extremely large variety of substances using direct boularography using a noble metal electrode as described above or indirect boularography using an electrode on which an enzyme is immobilized,
If these electrodes are formed into an elongated shape and inserted into a blood vessel to form an intravascular polarographic sensor, the concentration of various components in the blood can be continuously measured and recorded, making it clinically useful. This will allow us to make extremely important observations from a research perspective.

However, in measurements using such an intravascular polarographic sensor, there have conventionally been the following two difficult problems. In other words, (1) medium-high molecular weight substances such as proteins, peptides, lipids, and nucleotides present in high concentrations in blood adhere to the electrode reaction surface and contaminate the electrode surface, and (V) the electrode activity decreases over time. descend. (2) Most of the substances that form the electrodes have the property of causing blood coagulation, so blood coagulation occurs on the electrode surface9, which poses a danger to animals and humans and worsens the reactivity of the electrode. let By the way, the above-mentioned problem (1) can be solved relatively easily by covering the electrode reaction surface with a semipermeable membrane. However, solving problem (2) was extremely difficult. The present invention relates to a technique for solving the above-mentioned problem (2), in which direct or enzymatic (indirect) boularography is applied to animals and humans to obtain 02N20, H2, ascorbic acid, glucose, The purpose of this method is to prevent danger to animals and humans when continuously measuring the concentration of important blood components such as uric acid-adrenergic hormones, and to improve accuracy of measurement. The following can be considered as blood coagulation prevention means for equipment that comes into contact with blood. (a) Cover the equipment with a highly anticoagulant substance such as Teflon or silicone. (b) Chemically bonding heparin, a natural anticoagulant, to the surface of the device.

(c) applying a substance containing heparin to the surface of the device;
The heparin is allowed to gradually dissolve into the blood.

When taking the above method (a), brass nuts with extremely high anticoagulant properties such as Teflon and silicone have a high degree of hydrophobicity without exception and are impermeable to water and electrolytes, so they must be relatively large. However, electrolyte non-permeable composite electrode (
It cannot be used for polarographic electrodes other than Clark type electrodes, and cannot be applied to electrolyte-permeable intravascular polarographic sensors, which can be formed extremely thin.

In addition, when taking the above-mentioned method (b), the method of chemically bonding heparin to the surface of the device is technically relatively easy, but generally a sufficient blood coagulation prevention effect cannot be obtained. This is thought to be because heparin molecules are a substance that primarily exhibits anticoagulant effects when they are in a state of abundance. Therefore, the present inventor investigated the above-mentioned means (c), and developed a polarographic sensor in which the electrodes were coated with a film in which heparin was uniformly dispersed in the form of fine particles on a hydrophilic brass stick, as will be described in detail later. The anti-coagulation effect could be maintained. The present invention will be explained in detail below using the drawings. 1A to 1D each show an embodiment of the present invention.
FIG. 2 is a longitudinal cross-sectional view of a polarographic e-sensor.

The diameters of the four types of polarographic sensors shown in Figures 1A to 1D are all 0.3 to 2.0 mm.
1.The length of each of the lengths shown in Figure 1A1, Figure 1C and Figure 1D is about 100 to 300 mTL, and the length of the length shown in Figure 1B is about 150 to 1200 meters. What is shown in FIG. 1A is a polarographic sensor having a needle-like separate measuring electrode.

The outer surface of the noble metal wire 1 is covered with an insulating layer 4 made of glass, enamel, or the like, except for the electrode reaction surface 2 and the surface near the end that serves as a connection terminal 3 to an external electric circuit. The outer surface of the insulating layer 4 other than the electrode reaction surface 2 and its vicinity is covered with a metal tube 7 made of a stainless steel tube for an upper injection needle or the like coated with an adhesive such as epoxy resin. A portion of about several millimeters at the tip of the insulating layer 4 protrudes outside the metal tube 7, and at this protruding portion, a part of the side surface of the noble metal wire 1 is exposed from the insulating layer 4 as an electrode reaction surface 2. ．．
The outermost surface of the polarographic sensor is made of a plastic coating 6 with water permeability φ in which heparin is homogeneously dispersed to prevent blood coagulation. That is, the electrode reaction surface 2
The portion of the outer surface of the insulating layer 4 that is not covered with the metal tube 7 and the outer surface of the metal tube 7 are covered with the plastic coating 6. In addition, in this embodiment, in order to prevent the Vc electrode reaction surface 2 from being contaminated and inactivated by proteins in the blood, etc., the gap between the electrode reaction surface 2 and the insulating layer 4 in the vicinity thereof and the plastic coating 6 is prevented. A protective coating 5 is provided. This contamination prevention coating 5 is semipermeable (blocks relatively large molecules such as acyl celluloses, cellulose ethers, collodion, cellophane, vinyl acetate, hydron, etc.).
It is made of hydrophilic plastic and has the property of being transparent to relatively small molecules. The plastic coating 6 in which heparin is homogeneously dispersed will be described in more detail below. That is,
These plastics include hydrocollagen, various cellulose esters (acetylcellulose, butyrylcellulose, nitrocellulose, etc.), various cellulose ethers (methylcellulose, carboxymethylcellulose, ethylcellulose, propylcellulose, etc.), vinyl acetate, etc. Plastics that are hydrophilic and have high water permeability, as well as certain polyamides, polyesters, phenol resins, vinyl chloride resins, etc., are not generally classified as water-absorbing plastics, but they are hydrophilic to some extent. Any type of plastic that has water permeability and ion permeability to the extent necessary for the electrode reaction can be used. Plastic coating 6 in which such hevarin is dispersed
To form this on the outermost surface of the polarographic sensor, first dissolve either the hydrophilic or slightly hydrophilic plastic described above in a suitable solvent to form a solution with a concentration of about 0.5 to 10%. V, 1000 to 10 while stirring vigorously.
A small amount (1/1
When 00 to 1/10 volume) is added dropwise to the above-mentioned plastic snack solution, the aqueous heparin solution is dispersed in the plastic solution, and as a result, heparin precipitates to form a fine and homogeneous dispersion. Usually, this dispersion is further treated with ultrasonic waves to increase the degree of dispersion, and then the polarographic sensor 1, on which the anti-contamination film 5 is formed near the tip, is immersed in the above-mentioned plastic solution in which heparin is homogeneously dispersed. It is then taken up into the air to evaporate the solvent in the plastic and form a thin film. Such soaking and drying operations usually take 2 to 5
This method is repeated 9 times, but it is necessary to carry out this method by appropriately changing the conditions depending on the type and concentration of the plastic stick. Although the above-mentioned plastic coating in which heparin is dispersed is effective in preventing blood coagulation even if it is formed only near the tip of the sensor, it is necessary to form it on almost the entire outermost surface of the sensor as in this example. is desirable. In this example, the electrode reaction surface 2 was provided on the side surface near the tip of the noble metal wire 1.
As shown in FIGS. 1B to 1D, which will be described later, the tip end surface of the noble metal wire 1 may be used as the electrode reaction surface. In this embodiment, the anti-blood coagulation coating 6 is applied to the outer surface of the anti-contamination coating 5, but since the latter generally also has the effect of preventing contamination, the former is excluded and the anti-blood coagulation coating 6 is applied directly onto the insulating layer 4. A film may be applied.

This also applies to the cases shown in FIGS. 1B, C, and D, which will be explained below. Furthermore, although the electrode protection metal tube 7 is used in this embodiment, a tube other than metal can also be used, and if a strong material is selected for the insulating layer 4, this tube can be omitted.

This also applies to FIGS. 1C and 1D, which will be explained below. Next, what is shown in FIG. 1B is a boularography sensor comprising a flexible separate measuring electrode.

In FIG. 1B, parts corresponding to those in FIG. 1A described above are given the same reference numerals and detailed explanations will be omitted. In Figure 1B, Figure 1A
A flexible tube 8 made of Teflon, silicon, etc. is used in place of the metal tube 7 , and the tip of the surface of the noble metal wire 1 is covered with a hard insulating layer 4 made of glass, enamel, etc. The part following the part covered with is covered with an insulating layer 9 made of flexible resin. For this reason, most of this polarographic sensor is highly flexible, so it can be inserted deep into a blood vessel (for example, into the heart). Although the electrode reaction surface 2 is provided on the tip end surface of the noble metal wire 1 in FIG. 1B, it may be provided on the side surface of the noble metal wire 1 as shown in FIG. 1A. In the case of using a flexible plastic tube 8 of a flexible anticoagulant material such as Teflon silicone in the boralography sensor 1 as in this embodiment, heparin is homogeneously applied only to the vicinity of the tip. A sufficient anti-blood coagulation effect can be obtained by coating with a film of dispersed plastic. The polarographic sensor 1 shown in FIG. 1C is a syringe-shaped composite electrode having both a measuring electrode 1 and a reference electrode 10 in an integrated sensor.

The measuring electrode is made of noble metal wire 19, as in the polarographic sensors shown in FIGS. 1A and 1B, and the reference electrode 10 is made of Ag/AgCl. The reference electrode 10 is made by thinning a portion of a metal tube 7 made of stainless steel or the like near the electrode tip, depositing silver on it, then silver plating, and then partially removing it by electrolytic method in hydrochloric acid or potassium chloride. It is formed by silver chloride. The metal tube 7 is made of this Ag/AgC
A terminal 11 to an external electrical circuit is attached to the end of this metal tube 7 by a special soldering method, since it also serves as a lead wire for the reference electrode. However, as mentioned above, the metal tube 7 can be excluded, but in this case, a silver tube or a silver chloride silver wire is used as the reference electrode, and a thin coated electric wire is used as the lead wire instead of the metal tube 7. . Also, in the case of the composite electrode shown in FIG. 1C, the method of forming the semipermeable anti-contamination coating 5 and the anti-blood coagulation coating 6 is the same as that described with reference to FIG. 1A above. FIG. 1D shows a polarographic sensor 1 which forms an electrode for indirect polarography applying an enzyme reaction, and a contamination prevention coating 5 on the electrode reaction surface 2 of the measuring electrode similar to the embodiment described with reference to FIG. 1A.
A porous plastic layer 12 in which enzymes are uniformly dispersed and fixed is further formed on top of the porous plastic layer 12, and the top thereof is further covered with a blood coagulation prevention coating 6. In this case, the enzyme is
Various types of enzymes are used to measure glucose, such as glucose oxidase, urigase to measure uric acid, and ascorbic acid oxidase to measure ascorbic acid.
．． As described above, the concentration of the active substance of the enzyme can be selectively measured by detecting and measuring 02) using a polarographic method. FIG. 2 is a diagram showing a method of applying the various intravascular polarographic sensors shown in FIGS. 1A to 1D to a blood vessel.

Fig. 2 shows a case where a separate type measurement electrode sensor (A, B, D in Fig. 1) is used. The attached Teflon insertion catheter (vanilla nail) 25 is inserted into the blood vessel 27, and the distal end with the electrode reaction surface is made to protrude from the catheter into the blood 28. The insertion catheter 25 is connected at its side to a reference electrode container 24 containing a reference electrode 22 and an electrolyte 23, so that the reference electrode 22 is electrically connected to the tip reaction surface of the measuring electrode by the electrolyte. . The reference electrode 22 is usually an Ag/AgCl reference electrode, and the electrolyte 23 is physiological saline, which is usually injected gradually into the blood by dripping. A predetermined additional voltage depending on the substance to be measured is applied between the measurement electrode and the reference electrode of the electrode system configured as described above by the power supply circuit 31, and the current flowing between the electrodes is connected to the two sensors of the current amplifier. Using the method shown in FIG. 2, a catheter 25 was placed in the left and right femoral arteries of a dog under Nembutal anesthesia, and a voltage of -0.6 V was applied to these measurement electrodes 21 with respect to the control electrode 22. The dogs were allowed to breathe air, with occasional pulses of 10 seconds or 5 minutes of 50% oxygen breathing, while the electrolytic current values of both sensors were recorded using a two-pen recorder.
The vertical axis of the figure is the oxygen partial pressure (
(Oxygen concentration) value, and according to custom, it is expressed in millimeters of mercury pressure. Note that this experiment was conducted at 38°C, so the 100mmHg(7)02 partial pressure is 0.
．． This can be converted to an 02 concentration of 137 μM Moles/l. The horizontal axis is time, which is shown in minutes, but data for 300 minutes (5 hours) is omitted. Now,
The recorded value A in this Figure 3 is the recorded value of the sensor of the present invention which has been treated with a blood coagulation prevention coating9, and the recorded value B is the recorded value of the conventional sensor which is not subjected to this treatment. In order to make pen crossing possible with the simultaneously recorded values of the recorder 1, the time of B is increased by a distance 32 corresponding to 5 minutes from that of A and recorded with the recorder 33. Concentration can be known continuously. In the case of a composite electrode as shown in FIG. 1C, the measurement electrode and the reference electrode are combined and both are in contact with blood, so the reference electrode 22, electrolyte 23, and reference electrode container 24 in the case of FIG. is necessary.

In the above explanation of the structure of the polarographic sensor in FIGS. 1A to 1D, we have explained the case where a noble metal wire is used as the measuring electrode structure. Since it is only the reaction surface, a base metal wire with a noble metal wire connected only to the tip may be used, or a base metal wire whose tip is vapor-deposited or plated with a noble metal may be used.

FIG. 3 shows a case where the intravascular polarographic sensor of the present invention in the embodiment explained based on FIG. This is an example of experimental results comparing the stability and responsiveness in blood of a sensor of the present invention subjected to 6) and a conventional sensor not subjected to 6). In this experimental example, the deviation is as described above. For both A and B, a coating coated with 7.5% cellulose and diacetate 5 times was used as a contamination prevention coating, and in the case of A, an additional 200 uni coated on top of this was used.
It was coated twice with 5 (fl) cellulose triacetate dispersed with t/ml heparin. The two measuring electrodes are initially placed so that their tips are halfway to the catheter 25 in Figure 2, and the PO2 values are measured using physiological saline for testing (a solution equilibrated with nitrogen or air at 37°C). 0mm
Hg (with a value of 152 mmHg) was passed gradually to standardize the sensitivity before recording was started.

The recorded values from a to b in FIG. 3 show the PO2 values of both sensors when in contact with this air-balanced liquid. After completing the assay in this way, when the sensor is pushed in at point b and its tip protrudes from the catheter 25 and comes into contact with blood, the recorded values are as shown by curves A and B for arterial blood and venous blood, respectively. It will now show the value. Next, at point c, the dog's breathing air is removed from the air at 50 (f) for 10 seconds.
)02, both recorded values A and B show sharp peaks. Next, when 5 minutes of 50% 02 breathing is performed at point d, in the case of curve A the PO2 value reaches a value of approximately 295 Hg, but in the case of curve B it reaches a value of approximately 285U77! It only reaches Hg. In addition, the difference in air breathing values gradually becomes noticeable between A and B, and at 50 minutes, the PO2 values become 100 mmHg and 96 mmHg, respectively. This is B
In the case of , this indicates that blood coagulation has begun to adhere and the electrode sensitivity has begun to decrease. When the measurement is continued for another 5 hours, the sensitivity of B decreases significantly and shows only about half of the initial value, but in the case of A, the sensitivity close to the initial value is still maintained. 10 seconds and 5 at points f and g
Looking at the effects of 50% 02 breaths, it can be seen that in case A, there is almost no change from the initial state, but in case B, the shape is significantly distorted and the responsiveness of the electrode has deteriorated. At time h, the electrode tip was pulled into the catheter 25 again and brought into contact with the test solution (P゛02152TLT1LHg), and the electrode sensitivity at the final point of the experiment was compared. , a decrease in sensitivity of approximately 50% was observed in sensor B. Furthermore, when visually observed at the end of the experiment, sensor B, which did not have an anti-blood coagulation coating, showed red blood clots (viscous blood clots) all over the tip. ) was attached, or this was not observed in A, which was coated with a blood coagulation prevention coating. From the above experimental results, it is clear that the anti-blood coagulation coating according to the present invention has a remarkable effect on maintaining the sensitivity and responsiveness of the intravascular polarographic sensor, and is also effective in preventing risks caused by blood coagulation. It is estimated that

[Brief explanation of the drawing]

1A to 1D are longitudinal cross-sectional views showing the polarographic e-sensor of the present invention, FIG. 2 is a diagram showing a method of measuring intravascular substances by applying the polarographic e-sensor of the present invention, and FIG. 1 is a diagram showing records of changes over time in the characteristics of the polarographic e-sensor 1 of the present invention and the conventional polarographic e-sensor 1 when both sensors are applied to the artery of a dog as oxygen measuring electrodes. In FIG. 1, 1... Precious metal wire, 2... Electrode reaction surface, 3... Connection terminal, 4... Insulating layer, 5... Contamination prevention coating, 6... Blood coagulation prevention coating. , 7... Metal tube, 8... Tube (flexible), 9... Insulating layer (flexible), 10... Reference electrode, 11... Terminal, 12...
・Blastic, in Figure 2 21...Measurement electrode, 22...Control electrode, 23...Electrolyte, 24...
Control electrode container, 25... Catheter for insertion, 26...
・Liquid leakage prevention ring, 27... Blood vessel, 28... Blood, 31... Power supply circuit, 32... Current amplifier, 33.
...Recorder, in Fig. 3, A...Measurement value of 02 partial pressure by the polarographic sensor for arterial blood of the present invention, B...Measurement of the partial pressure of 02 by the conventional polarographic sensor for arterial blood. Value, a... Time when the tips of both sensors are brought into contact with the test solution, b... Time when the tips of both sensors are protruded into the blood, c... Oxygen breathing period of 10 seconds, d・゛゜5 minute oxygen breathing period, e...recorder record value omission period, f...10 second oxygen breathing period, g...5 minute oxygen breathing period, H゜...the tips of both sensors. The point at which it is drawn into the catheter and brought into contact with the assay solution again.

Claims (1)

[Scope of Claims] 1. A metal wire whose electrode reaction surface is made of a noble metal, and a surface of the metal wire except for the surface that is the electrode reaction surface and the surface that is a connection terminal to an external electric circuit is coated. A polarographic e-sensor comprising an insulating layer, an anti-blood coagulation coating made of a water-permeable plastic in which heparin is homogeneously dispersed and provided to cover the outer surface of the insulating layer and the electrode reaction surface. 2. The polarographic sensor according to claim 1, further comprising a semipermeable and hydrophilic anti-contamination coating between the electrode reaction surface and the insulating layer in the vicinity thereof and the anti-blood coagulation coating. 3. A metal wire whose electrode reaction surface is made of a noble metal, with a surface near the electrode reaction surface of the surface of the metal wire excluding the surface that is the electrode reaction surface and the surface that is a connection terminal to an external electric circuit. a hard first insulating layer covering a predetermined portion including;
A soft material that covers a surface of the metal wire that continues from the surface covered with the first insulating layer, excluding the surface that is the electrode reaction surface and the surface that is the connection terminal to an external electric circuit. a second insulating layer, a flexible plastic tube covering the outer surface of the first insulating layer and the outer surface of the second insulating layer other than the electrode reaction surface and the vicinity thereof, and at least the electrode reaction surface and the first insulating layer. A polarographic sensor having an anti-blood coagulation coating made of a water-permeable plastic in which heparin is homogeneously dispersed and which is provided so as to cover the outer surface of an insulating layer. 4. The polarographic sensor according to claim 3, further comprising a semipermeable and hydrophilic anti-contamination coating between the insulating layer on and near the electrode reaction surface and the anti-blood coagulation coating. 5. A metal wire whose electrode reaction surface is made of a noble metal, an insulating layer covering the surface of the metal wire excluding the surface that is the electrode reaction surface and the surface that is a connection terminal to an external electric circuit, and the insulation. A blood coagulation device comprising a control electrode provided on the outer surface of the layer, and a water-permeable plastic in which heparin is homogeneously dispersed and provided so as to cover the outer surface of the insulating layer and the outer surface of the target electrode, including at least the reaction surface of the electrode. Polarographic sensor with a protective coating. 6. The polarographic sensor according to claim 5, further comprising a semipermeable and hydrophilic anti-contamination coating between the insulating layer on and near the electrode reaction surface and the anti-blood coagulation coating. 7. A metal wire whose electrode reaction surface is made of a noble metal, an insulating layer covering the surface of the metal wire excluding the surface that is the electrode reaction surface and the surface that is a connection terminal to an external electric circuit, and the electrode. A thin layer of a porous material provided to cover the reaction surface and the outer surface of the insulating layer in the vicinity thereof, in which an enzyme that acts on the substance to be measured is uniformly dispersed and fixed, and at least the outside of the thin layer of the porous material. A polarographic sensor that has a blood-coagulation prevention coating made of water-permeable plastic that covers the surface and has heparin homogeneously dispersed therein. 8. The polarographic e-sensor according to claim 7, further comprising a semipermeable and hydrophilic anti-contamination coating between the electrode reaction surface and the insulating layer in the vicinity thereof and the plastic layer to which the enzyme is immobilized.