JPS6254153A - Adp sensor - Google Patents

Abstract

PURPOSE:To permit the selective measurement of adenosine diphosphate by using a specific bicyclo di-ammonium salt as a sensitive material to constitute an adenosine diphosphate sensor of a liquid film type electrode. CONSTITUTION:The bicyclo di-ammonium salt expressed by formula L and formula II is used as the sensitive material. In the formula, R, R' denote an alkyl and substd. alkyl of >=3C, X<-> denotes a monovalent anion, Y<--> denotes a divalent anion. For example, the bromide, etc. of 1, 4-diazabicyclo (2, 2, 2) octane are used. Such compd. is impregnated to porous ceramics or polymer to form a liquid film. The electrode is so constituted that both faces of the resultant sensitive film contact respectively with an internal liquid and measuring liquid to obtain the sensor. Since the above-mentioned sensitive material is used, the sensitive material is selectively sensitive with the adenosine diphosphate film and the accurate measurement is made possible.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Applicability) The present invention relates to a sensor for measuring adenosine diphosphoric acid (hereinafter abbreviated as ADP). This sensor is -A
L) P-AE Since it has a negative impact on energy metabolism in living organisms, it is used for research on energy metabolism mechanisms. Also,
This sensor performs A D P in one energy conversion.
It is used as a testing device to quantify the amount of enzyme involved in reactions where the concentration changes.

(Conventional techniques and problems) In measuring the chemical components of minute impurities in living organisms, the method using electrodes has the characteristics of being quick and capable of continuous measurement. It is used to analyze many chemical components including ions such as , Na, Ca, 11.0#, etc.

Electrodes can be divided into glass electrodes, solid membrane electrodes, M1-type 'hawk electrodes, enzyme electrodes, etc., depending on the type of sensitive membrane used. Among these, liquid membrane electrodes use a liquid membrane solvent to transfer the sensitive substance. The liquid is fixed in a matrix such as a porous ceramic support, polyvinyl chloride, or a silicone film in order to maintain the form of the film. Since such a liquid film m field can use an organic substance W as a sensitive substance, electrodes corresponding to various substances 'a' can be manufactured. An example is "Ion N, Pole and Enzyme Electrode" (Suzuki Shu, edited by Kodansha University, 1981)
As shown in p. 16-17, palinomycin kumquat was added to Kf! Ca electrodes using di(n-octylphenyl)phosphonate gold, vitamin BIN electrodes using tetraphenyl boron salt, etc. are commercially available and prototypes are being produced.

In the living body, ADP is involved in the synthesis of nucleic acids, protein, sugar, and lipid metabolism together with A'rP (adenosinphoic acid trisulfate), and is also involved in mechanical energy such as muscle contraction and bioluminescent light. Although it is a source of energy and is an extremely important substance that mediates the storage, supply, and transport of energy for living organisms, no sensor has been published to measure the concentration of ADP, and these values are currently unknown. is determined by column chromatography, electrophoresis, etc. However, these methods require complicated operations such as pretreatment, and also have the disadvantage that measurement takes time.

(111 points to be solved by the invention) The reason why sensors for measuring AI)P have not yet been announced is that they are selectively VC sensitive to AI)P, that is,
Although the potential changes as the ADP concentration changes, no change in the potential occurs or is so small that it can be ignored even when the concentration of other substances changes. Therefore, the problem to be solved by the present invention is, do you need to buy supplies? The aim is to obtain ADP henanoser as a NFTM-responsive material.

(Means for solving the problem) Chi2-C)12 where C, B' is an argyl group or substituted alkyl group having 3 or more carbon atoms, X- is a -valent anion, and Y~ is a divalent anion. It has been found that a bicyclodiammonium salt represented by the ion (representing an ion) has good selectivity for ADP, and by using this, a sensor with good selectivity for ADP can be obtained.

In the above general formula, the alkyl group includes straight-chain alkyl groups such as propyl, hexyl, octyl, decyl, cetyl, and stearyl, branched alkyl groups such as instearyl, and cyclohexyl. Cyclic alkyl groups such as and some of these alkyl groups are chloro groups,
Those substituted with a hydrophobic substituent such as a halogen group such as a fluoro group, an alkoxy group such as a methoxy group or an ethoxy group, or an allyl group such as a phenyl group or a naphthyl group are used. Note that B and R' may be thresholds or different.

Next, as an example of X- in the above general formula, (support) -
1Br- etc. (7) Hapgen ion, CeO4-18O
Ions such as N-5No3-, and examples of Y- are 8
Examples include anions such as 04-. Among these anions, Ce- has a high salt solubility in water, so Br, NO3-1so, "- is particularly preferred as an anion.

The above bicyclodiammonium salt I used in the present invention
Taking iN,N-dicetyldiazabicyclo(21212)octane dibroside as an example, it can be synthesized as follows. That is, 1,4-diazabicyclo(2,
2,2) Age 41” / 1.0 g (8,9nu+
nol) Todicetyl dibromide 16.2 g (53
mmol) f dimethyl formaldehyde 90rr
Dissolve LeK, heat at 70°C for 2 days, cool the solution after the reaction, and repeat the recrystallization of the precipitate with dimethylformamide to obtain crystals of N,N'-distearyldiammonium dibromide. . (m, p235-23
(6°C, yield 81%) To prepare a sensor using the above bicyclodiammonium salt as a sensitive substance, a liquid film kt! ! Must be fabricated as a polar membrane. For the preparation of liquid films, see G.G., Moody ((4, T.
``Selective Ion Sensitive Electrode (8elective io
n 5ensistive electrodes
) J (Japanese version “Ion Selective Electrode” Kyoritsu Shuppan) 9
(1979) It is described in detail in 7 Kusa. That is, the liquid film is prepared by dissolving the bicyclodiammonium salt in a hydrophobic solvent and infiltrating it into a porous ceramic or polymer, or by dissolving the bicyclodiammonium salt in a porous ceramic or polymer, or by dissolving the bicyclodiammonium salt in a porous ceramic or polymer. It can be obtained by dissolving it in a polymer or a polymer with a glass transition temperature higher than room temperature and a plasticizer added thereto, but it is preferable to dissolve it in a polymer from the viewpoint of ease of handling the membrane and longevity of the membrane. The electrode film can be formed by dissolving the bicyclodiammonium salt in a polymer solution, forming the entire film from this, and using this as an electrode film, or by applying a polymer solution to the electrode surface. This is done by forming a film. Examples of the polymer compound used include silicone resin, polyvinyl chloride, polychlorostyrene, polyvinylidene chloride, etc. Among these, polyvinyl chloride is preferable from the viewpoint of stability of its potential. When a polymer having a glass transition temperature Ii higher than room temperature, such as polyvinyl chloride, is used as a support, it is necessary to add a plasticizer to increase the mobility of ions.

As such plasticizers, any plasticizer that is generally not used for molding can be used. Examples of these plasticizers include dioctyl phthalate, dioctyl adipate, tricresyl phosphate, and the like. As described above, the method for obtaining an At)P-sensitive film using these polymer compounds is to dissolve the bicyclodiammonium compound, the polymer compound, and, if necessary, a plasticizer in a suitable solvent [4r], and add this to glass. It is produced not only by casting on a plate, etc. and evaporating the solvent, or by heating the solution on a 100% TTL surface, but also by using prepolymers, monomers, etc. instead of polymers and polymerizing them after molding. It is also possible to do so. The above bicyclodiammonium salt is preferably added in an amount of 1.01 to 2% by weight based on the total isomorphic content.

The t'tti used in the present invention is of the type that has internal electrodes and an internal liquid with a constant concentration in an insulating container, and both sides of the ADP-sensitive membrane are in contact with the internal liquid and the measurement liquid, respectively. , the sensitive film was applied directly onto conductors such as poles, metal plates, and metal wires! There are extremes. The At)P sensor of the present invention can also be obtained by removing the metal gate sensitive film of the MOS-FET and coating the gate part of the MOS-FET pH sensor with the sensitive film so as to be in direct contact with the measuring liquid. I can do it. The method for manufacturing this FETpfi sensor is described in, for example, Japanese Patent Laid-Open No. 53-96890, which has the advantage that a very small sensor can be manufactured.

The sensitive membrane used in the present invention has the drawbacks that the solubility of the sensitive substance is low and the conductivity of the membrane is low compared to other ordinary liquid film type electrode membranes. Sensors are particularly suitable.

This is because FET sensors essentially do not require electrical conductivity of the gate sensitive film, and it is possible to form a thin sensitive film directly on the gate surface.

(Function) The bicyclodiammonium salt used in the present invention
It is used as a sensitive substance because the two ammonium groups in the compound selectively coordinate to the anion part of the phosphate group of ADP, creating a hydrophobic compound. This is due to the generation of an interfacial potential depending on the Al)F concentration at the interface between the membrane and the ADP solution. Therefore, this C112-C112 must be) 1,1 (' is for shifting the water and membrane of the complex with ADP toward the membrane, and has 3 carbon atoms.
Any of the above alkyl groups may be substituted with various groups. However, if the number of carbon atoms in B and R' is 2 or less, the complex with ADP becomes hydrophilic, so such compounds are likely to elute from the membrane into water, causing drift during measurement and storage, and sensitivity. This causes a decrease in the temperature. Furthermore, if the number of carbon atoms in these alkyl groups or substituted alkyl groups becomes too large, it becomes difficult to increase the concentration of the ligand, so it is preferable that the number of carbon atoms is 30 or less.

(Example) Example 1 1.4-diazabicyclo(2,2,2)octane 1.0
g (8,9 mmol) and stearyl iodite 20
g (53 mmol) Vc dimethylformamide (1
3M, F) 50ml of gold was added and heated at 70°C for 2 days. Excess stearyl a-dite and monoammonium salt were removed, and recrystallization was repeated with the remaining solid IDMF.
An iodide salt of a diammonium compound was obtained. Iodine ions were replaced with bromide ions using hydrochloric acid to obtain a bromide salt.

This diammonium! M1.0mg and polyvinyl chloride 0
．． 2 g of dioctyl phthalic acid as a plasticizer and 0.8 g of gold tetrahydrofuran were dissolved by heating at 65°C.

The active solution thus obtained was cast onto a glass plate, and the solvent was evaporated in dry air to form a transparent solution with a thickness of ±0.2 mm.
A film was prepared. A solution of PVO in tetrahydrofuran was pasted on the end of this PVC pipe with a diameter of ICm, and dried in a desiccator for 24 hours. Using this membrane, the relationship between the electromotive force at both ends of the battery and the ADP concentration in the measurement solution was measured as described below.

The difference between the electromotive force of this sensor and the logarithm of ADPω degrees is AI)
A good linear relationship was established between the P concentration 10''M~10-3~1, and the slope was 22yFLk/log(A D
)') So, I got h.

Example 2 A PET pal sensor manufactured by the method shown in JP-A No. 54-66194 was inserted into a nylon catheter with a diameter of 1 mm, leaving the gate part.
PVC used in c, tetrahydrofuran solution of dioctyl phthalate and bicyclodiammonium salt i k'
The ET gate portion was dip coated to produce a sensor. The PVCff4 film thickness of this sensor was about 30μ.

This sensor was connected at a drain current of 30 μA and a drain-source voltage of 5 V.
Figure 1 shows the results of measuring the relationship between source constant pressure. A.T.
In the absence of P, the relationship between the logarithm of ADP concentration and output is ADP
f) It shows good linearity over a very wide range from 3°10 mNi to 0.001rnM, and its slope is 30
rnV,/log(A D P ), which agrees with the Nernst equation. In addition, when ATPlmM coexists, the pH
5, the sensitivity curve deviates from the M theory curve, but p14 (i
When adjusted to -7, linearity is exhibited in the range of A D P O,2rnM or more, and the output is saturated at concentrations below that.

From this curve, the selection coefficient of ADP to ATP is 0.
It is calculated as 049. This means that this sensor is AT
It is 20 times more sensitive to ADP than P (meaning it has a sensitivity).The selectivity coefficient for other substances is AMPo, 047, Na a2804 o, o 20, N
aC1 is O, Na2t (PO40,004), indicating that this sensor is specifically sensitive to ADP.

Example 3 Salts substituted with Br-'t-anions of each type as shown in Table 1 used in Example 1 were prepared and their response characteristics to AI)P were measured. The results are shown in Table 1. AD for any anion.
It is possible to measure P6 degrees, but in addition to the above-mentioned Br-, NO, 2804-1Cp- showed suppressed results.

Ce04 Q △ Some noise 8ON △
〃NO300 Good 804 0 0
t.

Ce−○ ○ 〃＋】 ○
...Has a sensitivity of 20mV/(logADP) or more *20...Has linearity at 0.001 to 1mM △・
...Saturation in the low concentration region (effect of the invention) ADP plays an important role in the energy metabolism of the living body, especially in the energy conversion of muscle contraction, and measuring the total amount in the living body is a research on energy conversion N8) structure. Importantly, the sensor obtained in the present invention exhibits a Nernst response in a wide range of ADPg from 0.001 tnM to 10mM, and also has ATP.

The relative sensitivity to AMP is 20 times and 21 times, respectively, indicating good selectivity. For measurement, it is sufficient to simply immerse the electrode directly in the pH-adjusted sample, and by using the sensor of the present invention, it becomes possible to easily and quickly selectively measure the ADP concentration. Furthermore, in the upper P energy conversion, ADPe4
Ai)P enzyme 1k involved in reactions that change the degree
It can be determined from the rate of increase and decrease of the enzyme layer, and the ADP sensor has the potential to be used as a testing device for quantifying these enzyme layers.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship (at 25° C.) between the ADP concentration (mu) and the source potential (mV) of the sensor of the present invention. In the figure, ○...AL) Aqueous solution of P only (pa
l 7 ), Δ-.-A T P 1 mM coexisting aqueous solution (pH 5), . . AT 1' l mAI coexisting aqueous solution (Pi(7)).

Claims (1)

[Claims] General formula ▲ Numerical formula, chemical formula, table, etc. ▼ or ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R and R' are an alkyl group or substituted alkyl group having 3 or more carbon atoms, A liquid film type adenosine diphosphate sensor using a bicyclodiammonium salt represented by (X^- represents a monovalent anion and Y^-^- represents a divalent anion) as a sensitive substance.