JPH01250046A - Reducible compound compatible with water and method of measuring material to be analyzed by using said compound - Google Patents

Abstract

PURPOSE:To rapidly and quantitatively measure the material to be analyzed in liquid of physiological pH by using the compd. which has the structure expressed by general formula I and contains the part compatible with water by releasing species R<5> when the compd. is reduced at <=9pH. CONSTITUTION:This reducible compd. has the structure expressed by general formula I. R<1>, R<2> and R<3> denote substituents excepts hydrogen, at least one among which is an electron-accepting group. X is a group II contg. an oxygen atom., sulfur atom. or nitrogen atom. Time denotes the group which cleaves and releases R<5> with an N-X single bond as a trigger when said bond is cloven. R<5> is the movable and detectable species and the species R<5> is released when this reducible compd. is reduced at <=9pH. When R<5> is substd. with H in formula I, the species itself has E.1/2 of at least +100mV when measured in water.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] Kinoe 8A relates to clinical chemistry. In particular, the present invention relates to water-compatible reducible compounds that can be reduced to produce detectable species, and analytical compositions and elements containing the compounds. The invention further relates to a method for determining an analyte, for example in a biological fluid.

[Conventional technology]

Chemical analysis of liquids such as water, milk and biological fluids is often desirable or necessary for health maintenance and diagnosis. A wide variety of compositions and elements are known to facilitate such analysis. Such compositions and elements include:
It generally includes a reagent composition for measuring the substance to be analyzed (herein referred to as analyte). The analyte is
It may be a living organism or a non-living chemical. The reagent composition produces a detectable change (eg, pigment formation) when it interacts with the analyte.

Recently, much research has been done to develop compositions and elements useful for rapid and highly quantitative diagnostic or clinical analysis of biological fluids, such as whole blood, serum, plasma or urine.

For example, for rapid and effective diagnosis and treatment of infectious diseases, it is desirable to be able to detect the bacteria causing the disease as quickly as possible. Urinary tract infections are the most common bacterial infections, often second only to those of the respiratory tract. In fact, in many hospitals, urinary tract infections are the most common form of nosocomial infection, often resulting from the use of catheters and various surgical procedures. Most urinary tract infections result from ascending infection by microorganisms derived from the urethra and range in severity from undetected infection to severe systemic symptoms. Such infections are usually associated with a bacterial count of more than 0.000 bacteria per liter of urine, a condition referred to as marked bacteriuria. Under normal conditions, urine is sterile, but up to 1000 microorganisms per/- can be present in a properly collected and transported sample due to contamination from external genitalia.

Significant advances in the detection of microorganisms and other analytes capable of reducing reducible compounds have been made in European Patent Application Publication No. J//
, rri No. 2 publication.

The analytical method described in that publication utilizes a reducible compound that releases a detectable species in the presence of the analyte.

Mafc Japanese Patent Publication No. 2003-110002 also discloses an analytical method using a similar reducing compound.

[Problem to be solved by the invention]

Although the reducible compounds described in European Patent Application No. 2II, R1 offer very advantageous analytical methods, they generally have limited solubility in aqueous solutions. Therefore, water-solubilizing surfactants must be used to prepare compositions of reducing compounds.

The use of surfactants in such compositions has significant drawbacks. Detergents have a tendency to lyse certain cells, such as white blood cells, making their detection or identification very difficult. Therefore, there is a need for a method that avoids the use of surfactants for rapid and sensitive analysis using reducible compounds.

JP-A-61-2rrt was developed for this purpose.
This is the method disclosed in No. tJ. However, although this method is an excellent method, it is severely limited in this field where it is difficult to introduce a substituent for imparting water compatibility and it is necessary to supply the substituent easily and in large quantities. Therefore, there is a strong need for a reducible compound that is easy to synthesize, is compatible with water, and has a suitable E- group.

[Means to solve the problem]

The above target point has the general formula / structure and has a pH of 2 or less.
contains at least 7 moieties that can be reduced to release a mobile and detectable species, making it compatible with water, and R5 is replaced with H in This was achieved using a water-compatible reducible compound having an Ei of at least +10 OmV.

In the formula, R1, B2 and 3 are substituents other than hydrogen, and at least one of them represents an electron-accepting group.

At least one is an electron-accepting group.

几1,! :B2, R3 and R3, R3 and R4, and R4 and FLl may be bonded to each other to form a ring.

Time is a group that cleaves and releases R triggered by the cleavage of the single bond between N-X, and t is o-
or / to cause thorns.

R5 kills mobile and detectable species.

In the formula, solid lines represent bonds, and dashed lines represent at least seven bonds.

The object of the invention was further achieved by a composition comprising the water-compatible reducing compound described above, buffered below pEl.

It is further an object of the present invention to: A. contact a liquid sample suspected of containing an analyte with said water-compatible reducible compound; and B. release as a result of the presence of the analyte. This was accomplished by a method for measuring an analyte, which includes the step of detecting a detectable species.

In the present invention, more useful reducible compounds are those represented by the following general formula.

In the formula, R represents an atomic group necessary to bond to the nitrogen atom (N) and X to form a 3- to t-membered monocycle or fused heterocycle.

Other meanings are the same as those stated in the general formula/.

Examples of the groups FL1, B2, 几3, and 几4 in the general formula/or are as follows.

Nitro group, cyan group, carboxy group, sulfo group, halogen atom (e.g., fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), alkyl group, aralkyl group (alkyl group, aralkyl group that may be substituted. For example, Methyl group, trifluoromethyl group, benzyl group, chloromethyl group, dimethylaminomethyl group, ethoxycarbonylmethyl group, aminomethyl group, acetylaminomethyl group, ethyl group, -1(≠-toticaylaminophenyl)ethyl group , carboxyethyl group, allyl i, J I J l 3-1lichloroprobyl group, rope α-pyl group, 1so-propyl group, n-butyl group, is□-butyl group, 5ec-gutyl group, t-butyl group group, rho is a methyl group, 5ec- is a methyl group, t-pentyl group, cyclopentyl group, n-hexyl group, 5ec-
-hexyl group, t-hexyl group, cyclohexyl group, low octyl group, 5ec-octyl group, t-octyl group, etc.), alkenyl group (alkenyl group that may be substituted with 1i1).

Examples, t, vinyl group, cochlorovinyl group, /-
methylvinyl group, -cyanovinyl group, cyclohexen-/-yl group, etc.), alkynyl/'fig (optionally substituted alkynyl group).

Examples, t, ethynyl group, /-propynyl group, -monoethoxylponylethenyl group-1, etc.), aryl group (aryl group that may be substituted. For example, phenyl group, naphthyl group, 3-hydroxyphenyl group, -”Rollophenyl group, ≠-acetylaminophenyl group, couhexadecanesulfonylaminophenyl group, -1methanesulfonyl-goonitrophenyl 4% j-nitrophenyl group, μ-
Methoxyphenyl group, l-acetylaminophenyl group,
≠-methanesulfonylphenyl i1.2゜hiro-dimethylphenyl group f! ,to), ? J Barocyclic group (optionally substituted polycyclic group. For example, /-
imidazolyl group, -furyl group, copyridyl group, j-
ditro-pyridyl group, 3-pyridyl M, Jlj-dicyano-biridyl group, yo-tetrazolyl group,! -phenyl-7-tetrazolyl group, Cohentziazolyl group, Cohenzimidazolyl group, -monobenzoxazolyl group, -monooxazoline-yl group, morpholino group, etc.), acyl group (optionally substituted acyl Groups, such as acetyl group, propionyl group, butyroyl group, 1so-butyroyl group, co,-dimethylpropionyl group, benzoyl group, 3.μmm dichloroCI2indiyl, 3-acetylamino-Hiro-methoxybenzoyl group, ≠-methyl benzoyl group, etc.), sulfonyl group (optionally substituted sulfonyl group).

For example, methanesulfonyl group, ethanesulfonyl group, chloromethanesulfonyl group, furo/ξsulfonyl group, methanesulfonyl group, low octanesulfonyl group, benzenesulfonyl group, μ-toluenesulfonyl group, etc.), carbamoyl group (substituted Moyoi cal; moyl group.

For example, carbamoyl group, methylcarbamoyl group, dimethylcarbamoyl group, bis-(-monomethoxyethyl)carbamoyl group, diethylcarbamoyl group, cyclohexylcarbamoyl group, di-0-octylcarbamoyl group,
3-dodecyloxypropylcarbamoyl group, hexadecylcarbamoyl group, 3-octanesulfonylaminophenylcarbamoyl group), sulfamoyl group (sulfamoyl group that may be substituted. For example, sulfamoyl group, methylsulfamoyl group, dimethylsulfamoyl group) group, bis-(-monomethoxyethyl)sulfamoyl group, diethylsulfamoyl group,
C-n-7-tylsulfamoyl group, methyl-n-octylsulfamoyl group (A4% n-hexadecylmethylsulfamoyl group, 3-ethogycypropylmethylsulfamoyl group, etc.).

At least one of R1 to R4 represents an electron-accepting group.

The electron-accepting group may be any group that accepts electrons, but is preferably one represented by the following formula (EAG).

Formula (EAG) V represents an atomic group forming a 3-r-membered ring with Zo and z2, and n is an integer from 3 to t. z -z -z5-1v, knee Z3-
Z4-Z5-Z6-1 V7: -Z3-Z, -Z5-Z6-Z7-1v8 knee Z
3-Z, -Z, -Z6-Z7-Z8-.

Sub Sub represents 10-1-S-1 or 18O2-, and Sub represents a simple bond (π bond), a hydrogen atom, or a substituent described below. Each Sub may be the same or different, and each Sub may be combined with each other to form 3~?
A negatively saturated or unsaturated carbocyclic or heterocyclic ring may be formed.

In general formula (A), the substituent constant σp
8u so that the total sum is +0.02 or more, more preferably 100.3 or more, most preferably +o,tiz or more.
Select b.

Examples when 8ub is a substituent are listed below. (The number of carbon atoms is preferably O-20 in each case.) Substituted or unsubstituted alkyl groups (e.g. methyl group, ethyl group, 5ec-butyl group, t-octyl group, benzyl group, cyclohexyl group, chloromethyl group, dimethylaminomethyl group) , trifluoromethyl group, J, J, !-trichloropropyl group, methoxycarbonylmethyl group, etc.)
, substituted or unsubstituted alkenyl groups (e.g. vinyl group, cochlorovinyl group, /-methylvinyl group, etc.), substituted or unsubstituted alkynyl groups (e.g. ethynyl group,
/-Flovinyl group, etc.), cyan group, nitro group, halogen atom (fluorine, chlorine, bromine, iodine), substituted or not! Heterocyclic residues on the sleeve (-1-pyridyl group, /-imidazolyl group, penzothiazol-1-yl group, morpholino group, penzoxazol-yl group, etc.), sulfo group,
Carbamoyl group, substituted or unsubstituted aryloxycarbonyl or alkoxycarbonyl group (for example, methoxycarbonyl group, ethoxycarbonyl group, tetradecyloxycarbonyl group, comethoxyethoxycarbonyl group, phenoxycarbonyl group, μm cyanophenylcarbonyl group, -monochlorophenoxycarbonyl group), substituted or unsubstituted carbamoyl group (e.g. carbamoyl group, methylcarbamoyl group, diethylcarbamoyl group, methylhexadecylcarbamoyl Ms, i+
Hiro, ≦-) IJ dichloroenylcarbamoyl group, N-
Ethyl-N-phenylcarbamoyl Mfx), hydroxyl group, substituted or unsubstituted azo group (e.g. phenyla, p-methoxyphenylazo group, -cyano-methanesulfonylphenylazo group, etc.), substituted or unsubstituted azo group Substituted aryloxy or alkoxy groups (e.g. methoxy, ethoxy, benzyloxy,
(phenogia group, Hiro-methoxyphenoxy group, 3-acetylaminophenoxy group, 3-methoxycarbonylpropyloxy group, -monotrimethylammonioethoxy group, etc.), sulfino group, sulfeno group, mercapto group, substituted or unsubstituted acyl group (For example, acetyl group, trifluoroacetyl group, n-butyroyl group, t-butyroyl group, benzoyl group, λ-caldoxybenzoyl group, 3-
(nitrobenzoyl group, formyl group, etc.), substituted or unsubstituted aryl or alkylthio groups (e.g. methylthio group, ethylthio group, t-octylthio group, hexadecylthio group, phenylthio group, co, ≠, t-trichlorophenylthio group, comethoxy group) 1-1-octylphenylthio group, -1acetylaminophenylthio group, etc.)
, a substituted or unsubstituted aryl group (e.g. phenyl group, naphthyl group, 3-sulfophenyl group, ≠-methoxyphenyl a, s-ra r:y roylaminopheno group, etc.),
Substituted or unsubstituted sulfonyl groups (e.g. methylsulfonyl group, methylsulfonyl group, n-octylsulfonyl a, n-hexadecylsulfonyl/'M, 5e
c-octylsulfonyl group, p-toluenesulfonyl!
, $-chlorophenylsulfonyl group, 3-nitrophenylsulfonyl group, etc.), substituted or unsubstituted sulfinyl 2!1i- (e.g., methylsulfinyl group, phenylsulfinyl group, Buddha-nitrophenylsulfinyl group, etc.), substituted or Unsubstituted amino groups (e.g., methylamino group, diethylamino group, amine group, methyloctadecylamino group, phenylamino group, ethylphenylamino group, acetylamino group, trifluoroacetylamino group, N-hexadecylacetylamino group, N-methylbenzoylamine 7 groups, methoxycarbonylamino group, phenoxycarf-nylmethylamino group, N-methoxyacetylamine 7 groups, amidinoamino group, phenylaminocarbonylamino group, ≠-cyanophenylamino 7carbonylamino group, N -xthylethoxycarbonylamino group, N-(cocyanoethyl)-p-toluenesulfonylamino group, trimethylammonio group), substituted or unsubstituted sulfamoyl group (e.g. dimethylsulfamoyl group, sulfamoyl group, hexadecyl Sulfamoyl group, λ-cyanoethylhexadylsulfamoyl group, phenylsulfamoyl group, N-(J,≠-dimethylphenyl)-N-octylsulfamoyl group, dibutylsulfamoyl group, bis-(co methoxyl f
substituted or unsubstituted acyloxy groups (e.g., acetoxy, benzoyloxy, chloroacetoxy, etc.), substituted or unsubstituted sulfonyloxy groups (e.g., methylsulfonyloxy M, p-1- luenesulfonyloxy group, p-chlorophenylsulfonyloxy group, etc.).

A more specific example of an electron-accepting group is a group 11Jt substituted with at least one electron-withdrawing group.
For example, ≠-nitrophenyl group, λ-nito-Hiro-N-methyl-N-octadecylsulfamoylphenyl group 1.2
-N,N-dimethylsulfamoyl-hiro-nitrophenyl group, λ-Schiff/-S-octadecylsulfonylphenyl! 1.2゜gudinitrophenyl group, -, g, tricyanophenyl group, -12trohiro-N-methyl-N
- Octadeflu power rubamoylphenyl! , -2-=drawooctylthiophenyl group, ! , 4t-dimethanesulfonylphenyl%, J, J--dinitrophenyl i,
-2-Chloro-Hiro-Nidrow 7-Meflphenyl group, Konitrow 3. j-difi+Luhiro-tetradecylsulfonylphenyl group, co, ≠-dinitrosu7tyl group, λ-
Ethylcarbamoyl-nitrophenyl group, -14t
-bis-dodecylsulfonyl-z-tovfluoromethylphenyl group, co, 3. ≠, j, J-pentafluorophenyl group, coacetyl-Hiro-nitrophenyl group %Jl≠
-diacetylphenyl group, -trifluoromethylphenyl group, etc.), substituted or unsubstituted heterocycles (e.g., -1pyridyl group, -1pyrazyl group, -2tro-pyridyl group, j- N-methylrubamoyl-biridyl group, ≠-pyridyl group, 3.!-dicyano-2-pyridyl group, !-methanesulfonyl-biridyl group, !-cyano-biridyl group, Hiro-nitrotheophe -yl group, !-nitro/1,2-dimethylimidazol-≠-yl i, j, j-diaceter-hyricyl group, /-dodecyl-yo-carbamoylpyridinium-coyl group, etc.), substituted or Unsubstituted quinones (
For example, /, goupenzoquinone-yl group, J, j-
, t-trimethyl-/.

Hiro-penzoquinone-coyl group, 3-methyl-/.

≠-naphthoquinone-coyl group, 3. Bu-dimethyl-hexadylthio-/, Hiro-benzoquinone-λ-yl group,! -methyl/, -pensoquinone-μmyl group, etc.) or, in addition to the vinyllobes listed above, a nitroalkyl group (e.g., -12tro-furopyl group)
, a nitroalkenyl group (e.g., conitroethenyl group), a monovalent group of an α-diketo compound (e.g., -1-ogisopronoyl group), nitroalkanes, nitroalkenes, and the like.

R6 is bonded to a nitrogen atom and an oxygen atom as described above, and is 3-
．． represents the Atsuko group necessary to form an r-membered heterocycle,
Below are some examples of this heterocycle.

Substituents may be introduced into these rings.

In this invention! A reducible compound represented by the following general formula 3 is preferred.

In the formula, 几 and 几 are each a simple bond, a hydrogen atom, or a group capable of substituting this, and may be bonded to each other to form a saturated or unsaturated carbocyclic or heterocyclic ring.

Y is a monovalent linking fund, and EAG is an electron-accepting base. Other meanings represent the same meanings as stated in the general formula /.

Specifically, substitutable groups for R7 and R8 can be selected from the same range as explained for R1 to R4. R
, R may be combined with each other to form a saturated or unsaturated carbocycle or heterocycle.

Particularly useful water-compatible reducible compounds are those having a structure represented by the general formula -≠.

In formula (1), Y represents a monovalent linking group, preferably -C- or -5O2-.

Preferred examples of R7 include a hydrogen atom, a substituted or unsubstituted alkyl group (methyl group, ethyl group, 1-butyl group, ophtateryl group, phenethyl group, carboxymethyl group, etc.)
, substituted or unsubstituted aryl group (phenyl group, 3-
Nitrophenyl group, methoxyphenyl group, μm acetylaminophenyl group, Hiro-methanesulfonylphenyl group, μ-sulfophenyl group, ≠-carboxyphenyl group, etc.), substituted or unsubstituted heterocyclic groups (λ-pyridyl group, , cofuryl group, 3-pyridyl group, etc.), and preferred examples include hydrogen atoms, substituted or unsubstituted alkyl groups (methyl group, hydroxymethyl group, C
H2-(Time)t-FL3 group, etc.), substituted or unsubstituted aryl group (phenylated or unsubstituted heterocyclic group (
≠-pyridyl group, etc.). Furthermore, particularly preferred examples of the case where 几 and 几 form a ring to form a condensed ring include the following.

In the general formula ≠, R5 gives off a detectable species when released from the reducing compound. When R5 is substituted with H, it has an E- of at least +10 OmV, and at least 7 of R7, 几8, 几9, B10 or R11 have at least a water-compatible moiety. This includes one item.

In the general formula, R9, BIO and R11 will be explained in detail.

R9, RI O1 is an alkoxy group (including those having a fig substituent. For example, a methoxy group, -1-methoxyethoxy group, phenoxy group, Hiroshi-n-hexadecylcarbamoylphenoxy group, ethoxy group, n- xyloxy group, n-hexadecyloxy group, methoxypropyl group), acyl group (including whole substituents; for example, acetyl group, n-dodecanoyl group, benzoyl group,
co-ethoxycarbonylbenzoyl group, -2-1dimethylpropanoyl group, etc.), alkoxy or aryloxycarbonyl group (including those with substituents; for example, methoxycarbonyl group, ethoxycarbonyl group, n-
octyloxycarbonyl group, n-hexadecyloxycarbonyl group, phenoxycarbonyl group, etc.), sulfonyl group (including those with substituents. For example, methylsulfonyl group, aryalmethylsulfonyl group, ethylsulfonyl group, n-1 -f-silsulfonyl group, n-tetradecylsulfonyl group, phenylsulfonyl group, ≠-methylphenylsulfonyl group, t-dodecylsulfonyl group, etc.)
, carbamoyl group (including gold having a substituent. For example, carbamoyl group, dimethylcarbamoyl group, diethylcarbamoyl group, n--f thylcarbamoyl i, J-
(co,4!-di-t-pentylphenoxy)furobylcarbamoyl i, N-methyl-N-n-octylcarbamoyl group, (3-hexadecylsulfamoyl)phenylcarbamoyl group, N-methyl-N-n-octadecyl Carbamoyl group, n-hexadecylcarbamoyl group, J
-n-dodecyloxypropylcarbamoyl group, etc.)
, sulfamoyl group (including whole substituents; for example, methylsulfamoyl group, dimethylsulfamoyl group, diethylsulfamoyl group, dibutylsulfamoyl group, N-methyl-Nn-hexylsulfamoyl group) Moyl group % N-'ti-N-n-octylsulfamoyl group,
N-methyl-N-n-hexadecylsulfamoyl group,
N-methyA/ -N-rl-octadecylsulfamoyl group, n-dodecylsulfamoyl group, N-phenyl-N-hexadecylcarbamoyl group, N-mef-ruN
-3-methoxypropylsulfamoyl group, bis(comethoxyethyl)sulfamoyl group, nato), nitro group, cyano group, halogen atom, calr6oxy group or trifluoromethyl group, sulfo group, C02e group, 5O
A group selected from 3e groups, 几 and B 10
At least one of is selected from a nitro group, a cyano group, a sulfonyl group, or a trifluoromethyl group. At least one of R and R is preferably a nitro group or a sulfonyl group. Particularly preferred is one in which at least one of R and R is a nitro group.

At this time, R9, one group other than the nitro group of RIO and/
Alternatively, R is preferably a sulfonyl group, a sulfamoyl group, an alkoxycarbonyl group, a carbamoyl group, an acyl group, a trifluoromethyl group, or a sia, *' group.

- It is preferable that R9 is a nitro group, R10 is a sulfamoyl group, carbamoyl group, alkoxycarboxyl group or trifluoromethyl group, and Bll is a hydrogen atom.

In general 2/~≠, Time represents a group that releases all of 5 through the subsequent reaction, triggered by cleavage of the N-X bond. t is Q or / fully clothed.

Various groups represented by Tirne are known, for example, in Japanese Patent Application Publication No. 2003-120002, published in Japanese Patent Application Publication No. 2003-120003 (!), pages 6 to 6, 4/4.
Examples include the groups described in European Patent Publication No. 20,74 ILJ (/0) to pages (J, 2).

A water-compatible reducible compound useful in the practice of the present invention broadly refers to a mobile compound that is capable of being reduced and mobile at physiological pH (i.e., below Defined as a water-compatible organic compound containing a detectable species. The term "mobile" refers to (1) a chromogenic moiety that has a first spectral absorption band when bound to a reducible compound and a second spectral absorption band when released; (2) a fluorogenic moiety having a first spectral excitation and emission band when bound to the reducible compound and a second spectral excitation and emission band when released; a chemically or biologically useful moiety that is inert, blocked, or inaccessible when bound, but active, unblocked, or accessible when released; or (3) a chemically or biologically useful moiety that is active or accessible when bound to a reducible compound, but inactive or inaccessible when released. be done.

Thus, a detectable species is chemically transformed when bound to a reducible compound; for example, for (1) above, the spectrum/ζ node of the reducible compound is The band is “moved” from the band it had when it was released. Generally, but not necessarily, the band shifts substantially further to shorter wavelengths when the species is part of a reducible compound.

In all cases, the bands do not overlap to any significant degree.

The change from one spectral band to another may be due to mere release of moieties from the reducible compound, or such release may involve interaction of the released moieties with ions or mordants on the gold or the analysis. This can occur due to changes in the environment (for example, changes in pH). With these changes in the environment, one must stay below the 9H bar.

Also, as mentioned above, a mobile, detectable species is inert or blocked or inaccessible when bound to a water-compatible reducible compound, but at physiological pH. It may be a chemically or biologically useful moiety that, once released, becomes biologically or chemically active or accessible for subsequent interaction. The released active species is detectable per se or 7
A detectable species can be produced by one or more subsequent chemical, physical or biological reactions. The method of the invention can be applied to a variety of chemical or biological purposes, preferably at physiological pH, by means of releasing such moieties, such as electron transfer agents, enzymes, enzymes, etc. upon reduction of reducible compounds. Provide a substrate, enzyme inhibitor, cofactor, catalyst or reactant.

Furthermore, the mobile, detectable species is active or accessible for seven or more subsequent chemical, physical, or biological reactions when bound to a reducible compound, but does not ■ It may be a chemically or biologically useful group that, when released, becomes inert or inaccessible to such reactions.

The reducible compounds described herein are compatible with water [from non-polar organic solvents to polar organic solvents (e.g. Eva, alcohol, acetonitrile, N,N-dimethylformamide or dimethyl sulfoxide), water or a small amount of It is defined as being more easily soluble in an aqueous solution containing four or more types of polar organic solvents.

Which solvents are polar or non-polar can be readily determined by those skilled in the art. This water compatibility is conferred by seven or more water-compatible substituents on the compound. Such a substituent is broadly defined as a group having a hydrophobic parameter (Pi) of -co, O. Such parameters are, for example, Martin, Mar.
cel Dekker.

Inc., New York, 1971) is a standard value for a given group.

These moieties are either readily ionizable in water (e.g. carboxy or sulfo groups) or non-ionizable in water (e.g. iodoxy or glucosyl groups).
A preferred water-compatible substituent is a carboxy group. Other useful substituents are hydroxy groups, quaternary ammonium groups,
Includes sulfonamide groups, etc.

In the present invention, the measurement of R5, the mobile and detectable species defined by the general formula l-≠, is carried out using differential pulse polarography or cyclic voltametry.
(e.g., Sawyer and John Wiley).
& 5ons, 2: L-York, /27 years, see]. E-value measured in water: 0/oomv ~+ti
Details regarding the determination of the Pi value, preferably 00 mV, are given further below in Table 1. Desired E-
fl, achieved by means well known to those skilled in the art. This can be achieved, for example, by appropriate electron-withdrawing groups on the 釠, R, and 锡 ports of general formula 3, or by strained fused rings attached to the nucleus, or a combination of both.

The water-compatible reducible compound used in the present invention is 7
The mode of this reduction and the subsequent cleavage of a mobile and detectable species has already been described in the field of photography by European Patent Publications Coco. It is described in detail in F≠No.6.

When compared to reducible compounds used in the photographic field,
What distinguishes the compounds of the present invention is that they have high E-values, which facilitates reduction and subsequent release of mobile and detectable species at physiological pH (i.e., below pH).

An example of a mobile detectable species in the present invention is a detectable species having a first spectral band with the rest of the molecule, but which, when desorbed from a compound of the invention, has a second spectral band. I can list things. This species is released in an amount that can be directly proportional to the amount of reducing agent present. The specific composition of FA() can vary significantly depending on the type of detectable species desired and the particular detection means used.

Mobile, detectable species include substances that can be detected directly by suitable means, as well as substances that can react with other substances, such as analytes, enzymes or other reagents, to produce a detectable species. It's fine. Such species include chromogens (e.g. dyes or pigments) that can be detected colorimetrically and fluorogenic substances (e.g. fluorescent dyes or probes) that can be detected fluorometrically. It includes those detectable by means.

Additionally, the detectable species may be a phosphorescent species, a chemiluminescent species, or any other detectable species known to those skilled in the art.

Particularly useful mobile, detectable species are chromogenic and fluorogenic substances that have a first spectral band before release and a second spectral band when measured after release. Examples of useful chromogens are azo dyes, azomethine dyes, nitrophenol dyes, indophenol dyes, indoaniline dyes and triarylmethane dyes, and others known in the art, with azo dyes being preferred.

Examples of useful fluorogenic substances include coumarins, μm oxo-≠I(-benzo-(d,e)anthracene and its derivatives, phenalenones and benzophenalenones,
Fluorescein and rotamine fluorescein dyes, as well as others known in the art. Phenalenone dyes are particularly useful.

Useful phosphorescent species include phosphors such as 2/, z/-dibromofluorescein and a/, Z/-short fluorescein. A useful chemiluminescent species is luciferin.

Specific examples of the reducible compound used in the present invention are listed below.

All of the water-compatible reducible compounds of the present invention can be prepared by methods known from European Patent Publication No. 10, 7 No. 0, etc., or by
They can also be prepared using techniques and starting materials readily known to the skilled synthetic chemist.

In particular, Japanese Patent Application No. 2813 describes a detailed method for synthesizing synthetic intermediates for introducing a movable and detectable species R into the compounds of the present invention. is extremely useful.

Synthesis Example t Synthesis of the compound in the batter (chlor compound) The raw material chlor compound (m, p, JjJo) was prepared by patent application Sho/,
2-jjl! It was synthesized using the method described in No.

The chloride J, lr ft-DMFJ was dissolved in O-, and sodium hydride≠00■ (to% dispersion oil) was added thereto and stirred at Z'C for 70 minutes.

Compound A/, 7P was dissolved in 10 ml of DMF in VC, 100 ml of sodium hydride (to%, dispersion oil) was added, and the mixture was stirred for 10 minutes at room temperature. After mixing both and stirring at 4 to C for 3 hours, the solvent was distilled off, tetrahydrofuran was added and the mixture was heated and filtered, and the mother liquor was crystallized to obtain 7.4 (r/).

m, p, J70'c or higher.

2 Synthesis of Compound 3 (Chlor Form) The starting material chlor form (m, p, /ter/ri4to) was synthesized using the method described in Japanese Patent Application No. 6-2813.

Compound B/, Ri! ? was dissolved in DMFJO seeds, t-butoxypotassium/,/ff was added thereto, and the mixture was stirred at room temperature for 30 minutes. There are 3 chloroforms in it. r? DMF solution 1 of
Gradually add 0txlf and stir at μo'C for μ hours after the dropwise addition.

After the reaction was complete, the solvent was completely distilled off, brine was added, and the mixture was extracted repeatedly with ethyl acetate. The solvent was distilled off, and the solid material obtained was recrystallized from ethyl acetate to obtain Compound 3 (η, -2).

m, p,, 2A00°3 Synthesis of compound RI (Yual form) The raw material chlor form (m, p, /riO~/ri100 decomposition) is a known compound.

Dye moiety C is also a well-known compound in the photographic industry (for a similar reaction of this dye, %Kaishoj/-1j40
It is described in μ7 etc. ).

Compound Cj was dissolved in 4tr't-DMF/Ots, and sodium hydride≠00mg (40% dispersion oil) was added thereto and stirred at room temperature for 70 minutes.

On the other hand, chloride 3. /f was dissolved in DMFjOd and hydrogenated) IJum4too■ (40% dispersion oil) was added thereto and stirred at room temperature for 70 minutes. Mix both at room temperature 5
, the reaction mixture was stirred for 3 hours at o'C. The solvent was distilled off, tetrahydrofuran was added, the mixture was filtered while hot, and the solution F was allowed to stand, yielding the desired compound (7.32). m, p,
J70'c or higher.

The water-compatible reducible compounds described herein can be in aqueous compositions containing suitable buffers and optionally water-miscible solvents.

The compositions can be manufactured in the general manner described below, and specific details of such manufacture are described in the Examples below. A reducible compound is added at a concentration according to its molecular weight.
- In the shell there is a buffer / per at / ~ ioo■, preferably! ~totrI! Dissolve in buffer. or,
The reducible compound can be dissolved in a water-miscible solvent and then mixed with a suitable buffer.

Buffers generally maintain the assay at (below) physiological pH. The concentration of buffering agent in the aqueous composition can vary widely, but is generally between 0.0/-0.1 molar. Typical buffering agents include phosphate, borate and
) includes those reported in

The water-compatible reducible compounds described herein include aqueous and non-aqueous liquids, such as biological fluids, manufacturing process liquids,
It is useful in compositions for analytical measurements (ie, qualitative or quantitative measurements) of wastewater or food materials. Analytes in strains can be determined by a reaction or series of reactions that reduce the compound and release a detectable species. The analyte is a living cell (e.g. bacteria, white blood cells, yeast or fungi),
Enzymes such as oxidoreductases such as lactate dehydrogenase, pyruvate dehydrogenase or glucose-6-phosphneetotehydrokenase, oxidases such as glucose oxidase, lactate oxidase or α-glycerophosphate oxidase, transferases such as alanine aminotransferase or aspartate amine Transferases, hydrolases, e.g., t-rehase, calphocysterase, and others (7) NADH, FADH, or oxidase-based assays), biological or chemical reducing agents other than living cells that reduce reducible compounds ( for example ascorbate, cysteine, glutathione or thioredoxin),
Metabolizable substances (e.g. glucose, lactic acid, triglycerides or cholesterol), immunoreactants (e.g. antigens,
antibodies or haptens).

Enzyme redox reaction and reaction with 7-rabin adenine dinucleotide (FAD-FADH) i substrate and nicotinamide adenine dinucleotide (NAD-NADH)
The composition can be used to monitor reactions with substrates and (NADP-NADPH) t-substrates. In such cases, a reducible compound that produces a detectable amount of water can be used in place of NADH, FADH or NADPH.

The invention is particularly useful for detecting or quantifying viable cells in biological samples. Although biological samples suspected of containing viable cells (e.g. food, tissue, ground water, chilled water, pharmaceutical products or sewage), bacteria, yeast or fungi can be analyzed by the present invention, aqueous liquids, e.g. It is particularly useful for bacterial detection in human and animal fluids (eg urine, brain fluid, blood and fecal excreta) and suspensions of human or animal tissue. The practice of the invention can be carried out in urine (diluted or undiluted).
Particularly important in detecting urinary tract infections.

When measuring viable cells using reducible compounds, it is preferred that the viable cells interact with an electron transfer agent (hereinafter referred to as ETA) for rapid dye release during such measurements. The presence of ETA results in effective dye release for analytical determination of non-viable analytes. ETA is a mobile compound that acts as an intermediary between the substance to be measured (eg, living cells) and the reducible compound. Certain ETAs work particularly well when measuring certain organisms (see examples//below).

Generally, ETA compounds useful in the practice of the present invention are prepared using differential pulsed polarography techniques in an aqueous buffer (p.
H7), measured against a standard hydrogen electrode, -3
It has an E in the range of TohirooornV. In general, ETA
The potential of should be more positive than the potential of the substance to be measured (ie, the analyte) and less than the potential of the reducible compound. That is, the ETA should be more easily reduced than the analyte and less easily reduced than the reducible compound. Generally, the ETA compound is present at a concentration that depends on the analyte concentration, preferably /X
It is present in concentrations from 10-7 molar to 10 molar.

ETA compounds useful in the practice of this invention include phenazine methosulfate, phenazine ethosulfate and similar compounds known to those skilled in the art.

Combinations of different ETA compounds can be used if desired.

Furthermore, preferred ETA compounds useful in the practice of the present invention that give rise to the advantage of low background are those described in European Patent Application Publication No. 0,730, 2003. Generally, these compounds are substituted benzo- and naphthoquinones. The quinones of this strain are, for example, core 3
-dimethyl-1-hydroxymethyl-/, 4t-benzoΦnon, -2! -dimethoxy-/, cubenzoquinone,
Ko, 3°7-)! J Methyl-1. Goobenzoquinone, tetramethyl-7,4LL-<nzoquinone, -23-dimethoxy-! -Methyl-7. ≠-benzoquinone, co-hydroxymethyl-/9μm naphthoquinone and co-(,2-
hydroxymethyl)-/, 44-naphthoquinone.

Detection of viable cells, and especially microorganisms, is often carried out with the presence of nutrients for these cells, but the presence is not essential. Any nutrient medium containing a useful carbon and optional nitrogen source can be used. Suitable nutrient media with suitable ingredients and [)H are well known in the literature. Particularly useful nutrients are glucose or tryptose, alone or in combination.

The invention is applicable to solution or dry analysis. In solution analysis, a solution containing the reducible compound, and preferably ETA, is prepared and brought into contact with and mixed with a liquid test sample suspected of containing the viable cells or analyte to be measured. It is also possible to mix with the ETA'lr test sample before mixing with the reducible compound.

General K: Place the reducible compound in a suitable container (e.g. test tube,
Mix with the test sample in a container (dish or cuvette). The resulting solution is mixed gently and heated for a relatively short period of time (i.e.,
(30 minutes or less), keep warm.

The detectable species is then detected, for example, at a wavelength in the spectral absorption band of the chromogenic species, or at a wavelength in the emission band of the fluorogenic species (which band occurs before the reducible compound releases 81 Evaluate the test sample by measuring (different bands). Such an evaluation can be performed using a suitable detection device.

Prior to analysis, pretreatment steps to remove interfering substances or concentrate cells can also be performed, if necessary.

Solution analysis can also be carried out by contacting a porous absorbent material, such as a paper strip, containing the test sample with a solution of the reducible compound. The analyte in the test sample can move from the porous material into solution and initiate the analytical reactions necessary for the measurement. In solution analysis, the amount of water-compatible reducing compounds present is at least o, o
oi millimolar concentration, preferably o, oi ~/, O millimolar concentration. Other reagents may be present in amounts readily determined by those skilled in the art.

Further, the method of the present invention can be carried out by a dry analysis method using a dry analysis element. Such elements may include absorbent carrier materials, i.e. thin sheets or strips of self-supporting absorbent or water-absorbing material, containing the reducible compound or the dried residue of a dispersion containing it; It may be. Such elements include test strips,
It is known as JT element, datzo stick or diagnostic agent.

When the reducible compounds described herein are used in dry analytical elements, they can be incorporated into or coated onto suitable absorbent carrier materials by absorption or impregnation. can.

Also, compounds can be mixed into the element during analysis.

Useful carriers'X are those which are insoluble and maintain their structural integrity when contacted with water or physiological fluids such as urine or serum.

Useful carrier materials can be made from paper, porous particulate structures, cellulose, porous polymer films, glass F11 fibers, woven and non-woven fabrics (synthetic and non-synthetic), and the like. Materials and procedures useful for making all such elements are described, for example, in U.S. Pat.
, 1r02.1112, same No. 3. Ri/yo, t≠7,
Same 3rd. ri/7. ≠jJ No. 3. Jt, No. 317, No. ≠, -μr, No. R, No. ≠, 26! , 31
μ issue and 1st issue! , 270,220.

Dry analysis can be carried out particularly advantageously using an analytical element comprising a support having at least 7 porous diffusion zones thereon as absorbent carrier material. The reducible compounds can be present in a diffuse zone or in different zones (eg reagent zone, recording zone or hydrophilic zone). The diffusion zone can be made from any suitable fibrous or non-fibrous material or mixtures of one or both.

The diffusion zone can be fabricated using a polymeric composition using fibrous materials mixed with suitable binders or woven into a cloth, such as those described in U.S. Pat. [e.g. brush polymers] or U.S. Patent No. 3. Tatar,/! ♂ issue, same issue μ, ko! ? , 00/ issue and the same issue ≠, ≠ 30゜ ≠ 3 in the specification of the issue and JP-A No. 7 (/
It can be manufactured from particulate material (with or without binding adhesive) as described in Publication No. PI Co.-10/760. The diffusion zone is isotropically porous,
That is, it is desirable that the porosity be the same in all directions of the zone, such as caused by continuous spaces or pores between particles, fibers, polymer strands, etc.

Although the dry analytical elements of the present invention may be a single free-standing porous diffusion zone containing the reducible compound and other reagents desired for a particular application, such zone may be formed on a suitable support. Preferably, it is supported on Such supports may be of any suitable dimensional stability, preferably non-porous,
0~yo.

A transparent core radiation-transmitting film or sheet material that transmits electromagnetic radiation at wavelengths of nm. The support selected for a particular element should be compatible with the intended detection method (reflection, fluorescence or transmission spectroscopy) and inert to the chemical reagents and liquid samples used in the analysis. . Useful support materials include polystyrene, polyesters, polycarbonates and cellulose esters.

The element may have more than seven zones, such as reagent zones, recording zones or priming zones. The zones are generally in liquid contact with each other (meaning that liquids, reagents, and reactive components will pass between the overlapping regions of adjacent zones). Preferably, the bands are in separately stacked layers, but there may be more than one band in a single layer. In addition to the above-mentioned patents, e.g.
172.

Suitable element structures and components are also described in US Pat.

In elements of the invention, the amount of water-compatible reducible compound can vary widely, but is generally at least o, oi?
/ms preferably o, ot~o.

It is present in an amount of 5i of 2 t/m. Preferred, but not essential, reagents are generally present in the following coatings: ETA: generally at least 0.00/l/m2, preferably from 0.0/f/m2.

Nutrient 2 Generally at least 0.0! ? /m2, preferably o, i~cof/W12C (used only when detecting viable cells), buffer (pH ≦): generally at least 0.127 m
2, preferably 0.3 to -9/m2. Bands 7 and above are used for analysis of various other desired but non-essential components (including activators, binders (generally hydrophilic) or antioxidants known in the literature) and specific analytes. It may contain any necessary reagents.

In one embodiment of the invention, the element for the detection of microorganisms (eg yeast, fungi or bacteria) in an aqueous liquid comprises an electron transfer agent and a reducible compound as described above.

These elements desirably further include nutrients for viable cells and a buffer to maintain physiological [)H during analysis (eg when contacted with/~200 μl sample of test liquid). Such an element can be detected by physically contacting the sample and the element in a suitable manner and detecting the amount of detectable a released from reducing compounds as a result of the presence of bacteria, e.g. by detection at a suitable wavelength. It can be used to detect bacteria in a sample (eg, one that has been pretreated to remove interfering substances or concentrate cells).

In another embodiment of the invention, the element is used to measure non-viable biological or chemical analytes in an aqueous liquid.

If the analyte cannot directly reduce the reducible compounds described herein to release a detectable glaze, the analysis may further include interacting with the analyte to reduce the reducible compound. An interactive composition containing >/S of reagents that produce a product is used.

This interactive composition can be incorporated into the element or added at the time of analysis. Examples of such analytes are described above. The amount of detectable species detected can be correlated to the analyte present in the liquid sample.

Elements of the invention may further include other reducing agents, such as ascorbate (ascorbic acid and an equivalent alkali metal salt),
It is useful for measuring cysteine, glutathione, thioredoxin, etc.

According to the present invention, various elements can be manufactured that differ depending on the analysis method. The elements can be formed in the form of articles including long tapes, sheets, slides or chips of any desired width.

The analysis of the present invention can be done manually or automatically. Generally, when using a dry element, the element is taken from a supply roll, chip package or other source, and a sample of the liquid to be tested (e.g. 1-200 μl) is collected such that the sample mixes with the reagents in the element. The analyte or viable cells are measured by contacting the analyte or living cells. Such contacting may be accomplished by any suitable method, for example by dipping the element into the sample, or preferably by applying one or more drops of the sample using suitable dispensing means. This is accomplished by manually or mechanically spotting the sample onto the element.

After the sample has been applied, the element is subjected to conditioning, such as incubation, heating, etc., as desired to further enhance or facilitate the acquisition of any test results.

Detection of analyte or viable cells 1 is achieved when a water-compatible reducible compound is reduced and releases tv4 molecules that can be detected by an appropriate method. As mentioned above, detectable species can be detected using standard colorimetric or fluorometric equipment. Preferably, it is a colorimetric or fluorescent dye that can be detected using a detection procedure. If the detectable species is other than a chromogen or fluorophore, such as a chemiluminescent or phosphorescent group, suitable chemiluminescent or phosphorescent detection equipment can be used.

Spectroscopic measurements can be made at the maximum wavelength of the detectable species or at wavelengths other than the maximum wavelength.

Escherichia coli (A.T.
CC-5 octopus) and Staphylococcus aureus (8 taphylococcus aureus,
ATCC-yooctopus 3) cells in BHI medium, respectively.
Grown overnight at 70C without shaking and transplanted daily.

〇*l cells were harvested by centrifugation and resuspended in 10 d of 0.0 molar 0 HEPE8 buffer (pH 7, r). The absorbance measured at lp20nm was adjusted to 0.2 and 7.0, respectively. The absorption of o,r33(jJQnm) was found to be equal to 1x10 cells/-. Candida albicans
s, ATCC/Hiroo! 3) Cells = 28 AB grown overnight in uj'c with agitation, harvested by centrifugation, washed, and resuspended in HBPE8 buffer. The cell suspension was adjusted to an optical density of /,0.

Example/: Preparation of an aqueous composition A composition of the invention was prepared as follows: A compound of the invention was made acidic by adding 0.7 g of concentrated sulfuric acid;
N-dimethylformamide (DMF)-! The resulting solution was dissolved in cojtl's 0005 molar potassium phosphate buffer or o, or molar HEPES buffer (pH
7, r).

Comparison of hardware This example shows the analysis of the present invention using a composition that does not contain a surfactant and the analysis of the present invention using a composition that contains a surfactant. For comparison with the analysis, two water-compatible compounds of the invention (6 l and io above) are used.

E. coli cells were grown in IBHI medium overnight at 37°C without shaking. μO − cells were collected by centrifugation. The resulting pellet was soaked in potassium phosphate buffer (pH 7,
, μ=0. OJ), 2Jtd and the resulting suspension was centrifuged. Wash the let and add buffer-ra
A known amount of the suspension was diluted with the same buffer to obtain an absorbance of A20nm''??0./.

A. The composition of the present invention was prepared from the following solutions: 7-enazinemethosulfate (FMS) or co-hydroxymethyl, t-dimethyl-/.

Goobenzoquinone (HDMBQ) (methanol spray, l
×io mol), compound of the invention/or 10 (N,
in N-dimethylformamide (DMF)/, 7X/(
1) molar) and glucose solution (70% in water).

Cuvette (volume t4'm/) K cell suspension 0
．． /1alc final concentration txio cells/d), phosphate buffer J, Otd, glucose solution jOμl (final concentration r, Ix10 molar concentration) and human PMS or HDM
BQJzμ! (final concentration 7.7×10 5 molar). Melt and seal the cuvette to prevent evaporation.
Equilibrate with heat at °C. Compound of the present invention/or 7 of 10
The reaction was started by the addition of Ml (final concentration 7.1 x 10 molar). A control solution was prepared without cells.

B. Prior art compositions using TRITON %(2) Compounds of the Invention/or/o (7./X10-2 molar concentration in DMF), (3 N!0 μE solution - 1700 μl of Triton X
-100 Shishimen active agent +j1! Ll phosphate buffer, (4
) glucose solution (io% in water) and (5) 6 x 70 cells/Ml in phosphate buffer.

Add solution 3 of /Irrtd (maximum H.
Compound concentration F17, & x io 'mol @ degree)
, 6 μl of solution ≠ (final glucose concentration r, rxto-
3 molar concentration) and/ko,! μl of solution! (final cell concentration x 10 cells/d). The cuvette was covered and heat equilibrated at 37°C.

Next K, 3 μl solution/(final ETA concentration 7.7 x 10-
The reaction was started by addition of 5 molar). A control solution ftm was prepared without cells.

The reaction is followed by monitoring the appearance of the dye at 2 nm. The results shown in Section 16 below are the average of 3 or 3 measurements and demonstrate the increased cellular response from the compositions of the present invention.

Table ■/FMS! -1r
/, J〃1 control)〃 ≠2 0
．． 1// ) 11) MBQ! /
0.1 (control) -ta
Q・/io FMS to
i, i〃(control) // j3
/, Ott HD M B Q
I≠ /, 3 (control) tt
The t20,j analysis was performed on a milliliter HAj' [well filtration plate (
Milliliter HAf4 Filtrat
ion Plate).

excitation! Dynatech Microfluor Read modified to read at 10nrn and color 1r20nm.
er) was used to measure the fluorescence.

A control composition was prepared as follows.

Solution (control compound/J■/d acidified DMF) co! Oh!
, Triton X-100fc surfactant solution 200μ1%H
EPES buffer (pH 7, r) λ! d1 trimethyl-7
, goobenzoquinone (TMBQ) (i, zmy7g4
methanol)! O0μ11 and glucose solution (7 in water
0% solution) j00μ10 The control compound is the compound of Table 1 except that it has a cyano group instead of a carboxy group≠
It was the same.

The composition of the present invention was prepared as follows.

Compound 4t (/7'9/da sexified DMF), 2JOt
tl! %HEPES buffer-jμg, TMBQ(/,
200 μl of methanol) and glucose solution (
70% solution in water>200g.

Add Staphylococcus aureus cell suspension (final concentration/×l0) to the wells.
-7 cells/1m)/jOμE and plate 37
Warm to °C and control and test compositions/! The analysis was performed in duplicate by adding Qμl. A blank solution without cells was prepared. Then at zero time and 3
Fluorescence was measured after 30 minutes of incubation at 7°C. The data are shown in Table ■ below. These data demonstrate that the present invention increases dye release in the absence of surfactants.

Table ■Comparison /j 2770 exam
/30) Jrit@2 Comparison This example shows the enhancement of cellular responses in compositions of the invention using one water-compatible compound of the invention and three electron transfer agents.

The compound of the present invention used was 3,μ,! , Otsu, and/
-It was.

The ETA used was ETAI, i.e., tetramethyl-
/, 4t-benzoquinone, BTA, 2, i.e. -,
? , j-) Rimeteru/, Hiro-penzoquinone, ETAj
, that is, Tip 3 - Dimethoxy! -methyl-7, ≠-
It was benzoquinone.

Solutions were prepared from each water-compatible compound and each ETA in a microtiter plate.

The control solution (containing Triton x-ioo surfactant) contains: HEPE8 buffer (pH 7, j
r) P, Jau, compound (final concentration 7.4x10 molar in acidified DMF) O1/μl, Triton X-100 surfactant 0. - μl, 10% glucose solution O, μA'x ETA (final concentration J in methanol
,I! x 10 molar) Q, co μl and E. coli cells (final concentration 10 cells/go HEPES buffer). A test solution was prepared from the same ingredients except that the Triton X-100 surfactant was not present. To measure background, a blank solution containing all components except cells was prepared.

Fluorescence was then measured at time zero and after 30 minutes of incubation at 37°C. The data are shown in Section I below.

Example B: Solution analysis of leukocytes This example demonstrates the use of the invention to measure leukocytes in solution analysis. For convenience, this is compared with a similar analysis carried out using the composition of the aforementioned European Patent Application Publication No. 1/191.

A composition of the invention was prepared as described in Example 7.

Control A composition was prepared in the same manner as the control composition of Example 7, and Control B used a water-compatible compound of the invention! Same as Control A except that. Controls A and B
contained a surfactant.

Blood samples (r, jd) were collected in standard blood collection tubes containing acidic citrate dextrose/jrdf. tube 71
Fixed hit/,! Add 6% dextran solution to each in an amount of ~d and mix the tube contents by inversion;
It was left to stand for 3 hours. The plasma layer from the tube was transferred to a sterile centrifuge tube and 093 molar potassium phosphate buffer (pH 7, t) containing 71 r, yy of Naα was added. Then the tube and its contents f10 minutes / 000 rpm
The supernatant was decanted. The resulting cell pellet was treated with ammonium chloride per cell lysis solution Cal,
3t, sodium carbonate/sodium and 0.3t sodium ethylenediaminetetraacetate. Resuspend in 10 wl of OJf) and then leave the tube for 1 minute to clear the solution.

The tube contents were centrifuged again for 70 minutes at 11000 rpm and the supernatant was decanted. The resulting pellet was again centrifuged at 111ii L% in phosphate-buffered saline, the supernatant was decanted, and the pellet was resuspended in phosphate-buffered saline at fO,j,1.

Commercially available Coulter Callan p-(C
using a counter) device,
White blood cells were counted using standard procedures after adjustment. Corrected to match numbers. Cells were frozen overnight before evaluation and then diluted 13-fold in phosphate-buffered saline before use.

A diluted leukocyte suspension (2,/×10 cells/l at 7!θμI) was added to a microliter plate well, containing the compound composition of the invention (/!Oμg), trimethylbenzoquinone ETA (/.!my/− methanol Solution-2!μl
) and glucose (70% W/V solution -!μl) were added. Plate 'f: , left at 370 C, and the released dye was fluoresced using a commercially available fluorometer equipped with a xenon arc lamp source (emission 420 nrn, excitation j 4tOnm).
Evaluation was made by measuring. Each test was repeated 1-3 times.

The analytical results shown in Table V below show that leukocytes contain compounds that are compatible with water in compositions devoid of surfactants! Indicates that it can be detected only by using .

Table 7 Control A -1 Hiro Control B -j Example! - Measurement This example shows the measurement of a type of bacteria, namely Escherichia coli and Staphylococcus aureus, and a yeast, namely Candida albicans, using a dry element containing the compound t of the invention.

Whatman 3mm glue C1? A strip of spiky paper was immersed in the following solutions: methanol 7al, solution (compounds of the invention t, o
, in N,N-dimethylformamide containing i% sulfuric acid.
0.21r molar concentration)/d, ETA solution (2,J-dimethoxy-!-methyl-7゜hiro-benzoquinone, 0.01
molar methanol)/− and glucose solution (i in water
o External bath solution/wLl.

The strips were then dried in the dark at -J-'C for 7 hours.

Cut disks (approximately //3 inches in diameter) from the dried strip using a standard paper punch and place the disks in Corning Cell Well 2.
ng Cell Wells, trademark).

Control (containing buffer only) and test cell suspensions (E. coli, ~r×io8 cells/d, Staphylococcus aureus, P-1×1
0 cells/d and Candida albicans, ~jX1
A sample of 0 IIE cells/ml) was dropped onto a sporatra paper strip at 10) tl and measured at zero and 37° using a Dynatic Micro70-L reader.
After incubation at C for 30 minutes, fluorescence (excitation J'≠Onm, emission t (Jnm)) was measured.

The results are shown below as changes in relative fluorescence after fJO minutes. - List the average value of the readings. It is clear that the invention can be used to measure microorganisms using dry elements.

Table ■ Control, zr.

E. coli core 0/Staphylococcus aureus cotri Candida albicans
, 20J! This example illustrates the use of a reducible compound of the invention (compound l of the invention) for determining several microorganisms. All cells were grown as described above at 37 °C without shaking, except for M. juteus).
and Bacillus subtilis (Bacillus ussubtilis) is 3
Pseudomonas bacterium (Pseudomonas
aeruginosa) were grown without shaking.

The example above! Analyzes were performed as described in and t. Different types of ETA%FMS and HDMBQ (both described in Example! and Part II above) were used. The results obtained are shown in Table 2 below.

〔Effect of the invention〕

The present invention is useful for the rapid and highly quantitative determination of analytes, such as enzymes, metabolites or viable cells (such as bacteria, yeast, fungi or white blood cells) in liquids at physiological pH (i.e. below pH). Provides a means for using reducible compounds. These reducible compounds can be dissolved in the analyte composition or solution analysis composition without the use of surfactants. Therefore, the problems encountered with the use of surfactants (adverse effects on cells +W) are avoided.

Additionally, it has been found that the absence of surfactants improves the sensitivity of the assay. The present invention further provides a means to release chemically or biologically useful groups that can be converted into detectable species.

These advantages have been achieved by incorporating groups into the reducible compound that make it compatible with water.

Patent applicant: Fuji Photo Film Co., Ltd. 1986//
month day

Claims (1)

[Claims] 1. A moiety having the structure of general formula 1, capable of releasing a mobile and detectable species R^5 when reduced at a pH of 9 or less, and making it compatible with water. Contains at least one R^
A water-compatible reducible compound which, when 5 is substituted with H, has an E(1)/(2) of at least +100 mV when measured in water. ▲There are mathematical formulas, chemical formulas, tables, etc.▼General formula■ In the formula, R^1, R^2, and R^3 are substituents other than hydrogen, and at least one of them represents an electron-accepting group. X represents a group containing an oxygen atom, a sulfur atom, or a nitrogen atom (there are mathematical formulas, chemical formulas, tables, etc.) (here, R^4 represents a group other than a hydrogen atom or a simple bond). X is ▲
There are mathematical formulas, chemical formulas, tables, etc. If ▼, then R^1
At least one of ~R^4 represents an electron-accepting group. R^1 and R^2, R^2 and R^3, R^3 and R^4, R
^4 and R^1 may each be bonded to each other to form a ring. Time represents a group that cleaves and releases R^5 triggered by the cleavage of the single bond between N-X, and t is 0.
Or represents 1. R^5 represents a mobile and detectable species. In the formula, a solid line represents a bond, and a broken line represents at least one bond. 2. Claim 1: A liquid sample believed to contain an analyte
A method for determining an analyte comprising contacting with the described reducible compound and then detecting a detectable species R^5 released as a result of the presence of the analyte.