KR100232446B1 - Test strip containing merocyanine protein error indicators for the detection of protein - Google Patents

Abstract

The present invention provides merocyanine protein error indicator compounds. Merocyanine compounds are a novel class of protein error indicators and provide useful analytical means for detecting proteins in a sample.

Description

Test strip containing merocyanine protein error indicators for the detection of protein

The present invention generally relates to protein detection, and more particularly to a novel class of protein error indicators.

Protein detection is important in the diagnosis of disease, medical research and industry. There are several ways to detect proteins in a sample. All protein detection methods, except the use of protein error indicators, are often very complex equipment and multi-step methods that require hours to complete.

One common method used to measure proteins in a sample is the Biuret method. According to this method, the sample is first acidified to precipitate any protein present in the sample. The precipitated protein is then redissolved in a suitable alkaline medium and treated with a solution containing copper ions. Peptide bonds of proteins and copper ions react to form colored chelates. The absorbance of the treated solution is then measured using a spectrophotometer. From this data, the corrected spectroscopic absorption curve is used to calculate the amount of protein in the sample. This method generally takes 1 to 3 hours to complete.

A variation of the burette method is the Lowry method. According to this method, after re-dissolving the precipitated protein, phospho tungstomolybdic acid reagent is added to this solution under alkaline conditions to oxidize all the phenolic compounds in the solution. As long as virtually all proteins contain some phenolic compound (eg tyrosine), this method can measure the protein in the sample. The absorbance of the treated solution is then measured with a spectrophotometer. The absorbance measured using the calibrated spectroscopic curve is used to determine the amount of protein in the sample. However, many buffers and other compounds interfere with this test because they contain amine groups such as TRIS, glycine and amide buffers.

Another method of measuring the presence of proteins in a sample is to measure turbidity after acidifying the sample. According to this method, the turbidity of a sample is measured using a spectrophotometer after adding a protein precipitant, generally an acidulant, to the sample. The presence of the protein in the sample is determined by comparing the calculated turbidity of the sample with a standard curve measured with a spectrophotometer. Common precipitants used in this process include sulfosalicylic acid, trichloroacetic acid and tannic acid.

Methodologies using protein error indicators are widely used to measure the presence of proteins in a sample. Using protein error indicators is inexpensive, quick, simple and convenient. Phenolsulfonphthalein compounds such as bromophenol blue, bromocresol green and coomassie blue are perhaps the most widely used protein error indicators. Often methodologies using protein error indicators include reagent strips impregnated with protein error indicators. According to this method, the reagent test strip is contacted with a small amount of sample. If the protein is present in the sample, the strip simply changes color to indicate the presence of the protein. The color observed can vary depending on the protein concentration in the sample. These various color changes are used to quantify the protein in the sample. Reagent strips of this type require minimal training to be used correctly. These reagent test strips provide an accurate, convenient and fast vehicle for protein instant determination. Such test strips are widely used by those skilled in the industry, research and clinical trials.

More specifically, the protein error indicator is a pH indicator comprising an ionizable group having a pKa value that is replaced by the presence of a protein. In the case of phenolsulfonphthalein, the ionizable group is a phenolic hydroxyl. Release of protons from phenolic hydroxyls results in an observable color change, which indicates the protein in the sample being tested. Protein error indicators that are generally considered useful for the analytical determination of proteins in a sample are described in US Pat. No. 4,013,416.

The present invention provides merocyanine protein error indicator. Merocyanine protein error indicators are a new class of protein error indicators. Until the present invention, merocyanine dyes are not known as protein error indicators. Merocyanine protein error indicators are useful for the detection of proteins. The merocyanine protein error indicator of the present invention can be represented by Formula 1:

In Formula 1 above,

Q is -Cl, -Br or -I,

m is an integer from 1 to 6,

R is S, Se, O or C (C _n H _{2n + 1} ) ₂ , where n is an integer from 1 to 6,

T -S0 ₃ Or -H.

In one embodiment of the invention, m is 3 or 4, R is C (CH ₃ ) ₂ and T is -SO ₃ to be.

Another important aspect of the present invention is to provide an analytical reagent strip comprising merocyanine protein error indicators for protein detection in liquid samples.

Another aspect of the invention relates to a method for detecting protein in a liquid sample. The method includes the step of soaking the reagent strip for analysis with a liquid sample. This test strip consists of an absorbent carrier impregnated with at least one of the aforementioned merocyanine protein error indicators. The test strip is then observed to detect color change. The color change indicates the protein in the liquid sample.

1 to 6 are schematic diagrams of a method for synthesizing merocyanine protein error indicator.

FIG. 7 shows 0.3 mM SPDIB (1- (ω-sulfopropyl) -2- (4'-hydroxy-3 ', 5'-dioodostyryl) -3,3-dimethylindolenium beta at pH 2.5 Dose response curves for albumin of analytical test strips impregnated with phosphorus) are shown.

The present invention relates to the discovery of a new class of compounds useful as protein error indicators. According to one aspect of the present invention, the merocyanine compounds of the present invention have been found to react with proteins to exhibit observable color changes in the merocyanine compounds. According to another aspect of the invention, it has been found that analytical reagent strips for protein detection in liquids can be obtained by impregnating absorbent paper test strips with the novel protein error indicators of the invention. According to another aspect of the present invention, the novel protein error indicator of the present invention has been found to be useful for aqueous liquid analysis. This is particularly advantageous compared to the fact that protein error indicators of the prior art, for example bromophenol blue, require organic solvents that can react adversely with the proteins in the sample to be tested. The protein error indicator of the present invention has a pKa value of at least 3.5. This is advantageous because various buffers can be used to prepare a liquid or dip-stick reagent for immersion comprising the indicator of the present invention.

The novel protein error indicators described below are orange and gradually develop a deep pink color upon contact with a sample containing protein. The concentration of the color reflects the concentration of the protein in the sample. The resulting pink color is distinct from the orange color of the negative test. The protein error indicator of the present invention positively detects a protein range of about 15 to about 500 mg / dl in the sample.

With reference to the observable color change from orange to pink, the test strips prepared according to the present invention produce different and distinct colors in the presence of proteins, which are distinct from the orange of the negative test, thereby detecting the protein in biological fluids. It is a diagnostic tool for. This is distinguished from other test strips which slightly change from one color tone to another in the presence of albumin (eg from yellow to yellow-green). The orange color for the negative test and the pink color in the positive test appear to be a significant development that is distinct from the previous methods and prior indicators used to detect proteins in liquid samples. More specifically, the present invention provides clinicians reliable methods for the detection of proteins in samples. The change from orange to pink simplifies the interpretation of the results. This reduces the likelihood of misjudgment and thus saves the user money.

The merocyanine protein error indicator of the present invention is a compound of Formula 1:

Formula 1

In Formula 1 above,

Q is -Cl, -Br or -I,

m is an integer from 1 to 6,

R is S, O, Se or C (C _n H _{2n + 1} ) ₂ , where n is an integer from 1 to 6, and

T -SO ₃ Or -H.

More preferably, Q is -Br or -I, m is an integer from 2 to 4, R is C (CH ₃ ) ₂ and T is -SO ₃ to be. Most preferably, Q is -I and m is 3.

It is to be noted that the present invention describes a first use as a protein error indicator of the merocyanine class of coloring agents and therefore encompasses a variety of substituted derivatives. It is apparent that the aromatic ring in the above general formula may have various substituent groups without departing from the scope of the present invention. Such substituent groups may be limited only by the ability of one of ordinary skill in the art to prepare stable compounds having the protein error indicator properties of the present invention, including unsubstituted or substituted alkyl, unsubstituted or substituted Aryl, alkoxy, aryloxy, halo (eg fluoro, chloro, bromo), nitro and substituted amino groups (eg dialkylamino).

In the context of the present invention, 'alkyl' is a straight chain and branched chain form of an unsubstituted hydrocarbon residue of the general formula —C _n H _{2n + 1} , preferably methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl and the like and 'lower alkyl' aliphatic forms and substituted forms thereof.

In addition, in the context of the present invention, 'aryl' includes organic residues derived from aromatic hydrocarbon rings or ring systems by removing hydrogen atoms and includes unsubstituted hydrocarbon ring residues such as phenyl and naphthyl and substituted forms thereof. do. Aryl moieties for purposes of the present invention include those containing one or more identical or different functional groups or substituents that may be selected by one of ordinary skill in the art to provide the merocyanine protein error indicators of the present invention.

More specifically, when 'aryl' and 'alkyl' are substituted, such substitutions include the above groups or substituents which are monosubstituted or polysubstituted by functional groups that do not substantially impair the useful properties of the compounds of the invention. do. Such functional groups include chemical groups which can be introduced synthetically and which provide stable and useful merocyanine protein error indicator compounds of the present invention. Non-limiting examples of such functional groups are halo (eg fluoro, chloro, bromo), substituted amino (eg dialkylamino), nitro, alkoxy, aryloxy, alkyl and aryl.

Examples of merocyanine protein error indicators of the present invention include 1- (ω-sulfopropyl) -2- (4'hydroxy-3 ', 5'-dibromostyryl) -3,3-dimethylindolenium Betaine; 1- (ω-sulfobutyl) -2- (4'-hydroxy-3 ', 5'-dioodostyryl) -benzothiazolium betaine; 1- (ω-sulfoethyl) -2- (4'-hydroxy-3 ', 5'-dioodostyryl) -3,3-dimethylindolenium betaine; 1- (ω-sulfopropyl) -2- (4'-hydroxy-3 ', 5'-dioodostyryl) -3,3-dimethylindolenium betaine; 1- (ω-sulfobutyl) -2- (4'-hydroxy-3 ', 5'-dioodostyryl) -3,3-dimethylindolenium betaine and 1- (n-butyl)- 2- (4'-hydroxy-3 ', 5'-diiodostyryl) -3,3-dimethylindolenium iodide. Details for the preparation of the merocyanine protein error indicators listed above are shown in the following examples.

1-6 generally show the synthesis of various merocyanine protein indicator compounds of the present invention. Such chemical methods are simple and generally involve coupling an aromatic hydroxyaldehyde and a heterocyclic quaternary salt under basic reaction conditions. The merocyanine protein error indicator of the present invention is water soluble. This is advantageous because these indicators can be added directly to aqueous systems for detecting proteins, such as, for example, urine, blood, serum, aqueous gels (electrophoretic gels), aqueous solutions. The general method used to prepare the merocyanine protein error indicator is shown in FIGS. 1 to 6 and is discussed in detail in the Examples below.

One aspect of the invention relates to an assay test strip for protein detection in a liquid sample or gel, comprising an absorbent carrier impregnated with one of the aforementioned merocyanine protein error indicator compounds. The absorbent carrier of the test strip is preferably filter paper. Other materials useful as absorbent carriers include felt, porous ceramic strips, and woven or mat shaped glass fibers described in US Pat. No. 3,846,247. Also suggested are wood, cloth, sponge materials and the clay materials described in US Pat. No. 3,552,928. Alternatively, the absorbent carrier may be a nonporous material such as various polymer films, glass, and the like. All these absorbent carrier materials are suitable for use in the present invention as others. However, it has been found that filter paper is particularly suitable.

The absorbent strip is preferably impregnated in the buffer. Any buffer system that can be adjusted to a pH of about 1.5 to about 4.5 is useful in practical applications of the present invention. Preferably, the buffer system is adjusted to pH about 2.0 to about 4.0, most preferably to pH about 3.5. According to the invention, the analytical test strip is contacted with a liquid sample or gel sample. Then observe the color change of the strip. The color change indicates the protein in the sample.

The following examples are intended to illustrate preferred embodiments and usefulness of the present invention and do not limit the invention unless otherwise stated within the claims appended hereto.

Example 1

1- (ω-sulfopropyl) -2- (4'-hydroxy-3 ', 5'-dibromostyryl) -3,3-dimethylindolenium betaine

3,5-Dibromo-4-hydroxybenzaldehyde (2.0 g, 7.14 mmol) in EtOH (30 ml) (manufactured by Lancaster Synthesis, Ltd., Wyndham, NH), 1 -(ω-sulfopropyl) -2,3,3-trimethylindoleninium betaine (2.0 g, 7.11 mmol), see Belg. 726,639; CA 73: P82538a] and a solution of piperidine (0.4 ml) are maintained under an inert gas atmosphere. The solution is refluxed for 50 minutes and cooled in an ice bath. The reaction mixture is evaporated in vacuo to anhydride and dissolved in a minimum amount of methanol (MeOH). The solution is then chromatographed on silica gel (600 g) using MeOH / CHCl ₃ (1: 4 v / v) development. Fractions containing the main purple product bands are combined and acidified with excess HCl in 2-propanol (i-PrOH) to produce a purple to golden color change. The solution is evaporated to anhydrous in vacuo. The residue is dissolved in hot EtOH (about 25 ml) and cooled to crystallize. The separated solids were collected by filtration, washed with ice-cold EtOH / hexanes (3: 1 v / v) and vacuum dried to analytically pure compound 1- (ω-sulfopropyl) -2- (4'-hydroxy-3 ', 5'-dibromostyryl) -3,3-dimethylindolenium betaine (0.96 g, 25%) is obtained as golden crystals. The compound has no constant melting point and darkens in color at temperatures above 200 ° C. The above-mentioned method for preparing the compound is generally shown in FIG. 1. Spectroscopic analysis data identifying the compound are as follows.

C ₂₁ H ₂₁ Br ₂ NO ₄ S Elemental Analysis for ½EtOH

Calc .: C, 46.65; H, 4. 27; N, 2.47

Found: C, 46.48; H, 4.50; N, 2.33.

Example 2

1- (ω-sulfobutyl) -2- (4'-hydroxy-3 ', 5'-dioodostyryl) -benzothiazolium betaine

3,5-Diiodo-4-hydroxybenzaldehyde (3.74 g, 10 mmol) in EtOH (30 ml) (Lancaster Synthesis, Wyndham, Hampshire, USA), 3- (ω-sulfobutyl)- 2-methylbenzothiazolium betaine (3.71 g, 13 mmol) [Brit. 742, 112; CA 50: P11149c] and piperidine (0.8 ml) are maintained under an inert gas atmosphere. The solution is refluxed for 1 hour and then cooled to ambient temperature. The reaction mixture is added with a sufficient amount of 1.93 M hydrochloric acid in i-PrOH to cause a color change from purple to yellow, thereby separating the solids from the solution. The solids are collected by filtration, washed with EtOH and dried. The solid was then dissolved in warm (55 ° C.) EtOH / MeOH / H ₂ O (3: 2: 1 v / v / v) (300 ml) containing 2M aqueous sodium hydroxide (5.2 ml) and celite [US Filtered through Jones-Manville Corp., Denver, Colorado, and then precipitated by addition of 3M aqueous hydrochloric acid ((6 ml). After cooling in an ice bath, the solids were collected by filtration, EtOH Washed with and then dried in vacuo, then the solid was boiled in acetic acid (HOAc) (600 ml), filtered and then dried under vacuum at 115 ° C. to give analytical pure compound 1- (ω-sulfobutyl) -2- (4'-Hydroxy-3 ', 5'-diiodostyryl) -benzothiazolium betaine (5.10 g, 79%) is obtained as a yellow powder.The above methods for preparing the compounds are generally described. It is shown in Figure 2. The spectroscopic data identifying the compound is as follows.

Elemental Analysis for C ₁₉ H1 ₇ I ₂ NS ₂ O ₄

Calc .: C, 35.58; H, 2.67; N, 2.18

Found: C, 35.52; H, 2.75; N, 2.06.

Example 3

1- (ω-sulfoethyl) -2- (4'-hydroxy-3 ', 5'-dioodostyryl) -3,3-dimethylindoleninium betaine

3,5-Diiodo-4-hydroxybenzaldehyde (3.73 g, 10 mmol), 1- (ω-sulfoethyl) -2,3,3- in EtOH / MeOH (2: 1 v / v) (60 ml) Trimethyl-indoleninium bromide (6.61 g; 19 mmol), see US Pat. Nos. 2,503,776 and CA 44; P5738i] and piperidine (2.0 ml) are maintained under an inert gas atmosphere. The solution is refluxed for 4 hours, cooled to ambient temperature and then evaporated in vacuo to anhydride to leave a brown residue. The brown residue is dissolved in MeOH (2-3 ml). The solution is treated with triethylamine (NEt ₃ ) (2 ml) and chromatographed on silica gel using MeOH / CHCl ₃ (1: 4 v / v) development. Fractions containing purple product bands are collected and evaporated to anhydride under vacuum. The crude product is dissolved in EtOH (10 ml) and acidified with a sufficient amount of 1.93 M HCl in i-PrOH to cause a color change from purple to yellow. The solution is evaporated to anhydrous. The residue is dissolved in EtOH / hexane (3: 1 v / v) and frozen until the solution crystallizes. The separated crystalline solid is collected by filtration. These solids are washed with ice-cold EtOH followed by EtOH / hexanes. Vacuum drying the remaining solid to yield compound 1- (ω-sulfoethyl) -2- (4'-hydroxy-3 ', 5'-dioodostyryl) -3,3-dimethylindolenium betaine (0.80 g; 12.8%). Recrystallization from EtOH / HOAc affords analytically pure compound as a dark reddish brown powder. The aforementioned method for preparing the compound is generally shown in FIG. 3. Spectroscopic analysis data identifying the compound are as follows.

C ₂₀ H ₁₉ I ₂ NO ₄ S Elemental Analysis of ½EtH ₂ O

Calc .: C, 39.02; H, 3. 43; N, 2.16

Found: C, 39.25; H, 3.47; N, 2.25.

Example 4

1- (ω-sulfopropyl) -2- (4'-hydroxy-3 ', 5'-dioodostyryl) -3,3-dimethylindoleninium betaine (SPDIB)

3,5-Diiodo-4-hydroxybenzaldehyde (3.73 g, 10 mmol), 1- (ω-sulfopropyl) -2,3,3-trimethylindoleninium betaine (3.65 g) in EtOH (about 50 ml) , 13 mmol) and piperidine (0.8 ml) are maintained under an inert gas atmosphere. The solution is refluxed for 2.75 hours and cooled in an ice bath. The solution is acidified with 1.93 M HCl in i-PrOH (5.0 ml). The dark tar is separated, filtered off and ground with boiled HOAc. The mixed abrasives are evaporated in vacuo to anhydride, dissolved in HOAc (20 ml) and then crystallized. The separated solid was collected by filtration, washed with HOAc, and then dried in vacuo to give compound 1- (ω-sulfopropyl) -2- (4'-hydroxy-3 ', 5'-dioodostyryl)- 3,3-dimethylindolenium betaine (4.37 g, 68%) is obtained as an orange powder. The compound is recrystallized from HOAc to give an analytically pure compound. The aforementioned method for preparing the compound is generally shown in FIG. 4. Spectroscopic analysis data identifying the compound are as follows.

C ₂₁ H ₂₁ I ₂ NO ₄ S Elemental Analysis for ½H ₂ O:

Calc .: C, 39.02; H, 3. 43; N, 2.17

Found: C, 39.01; H, 3. 46; N, 1.94.

Example 5

1- (ω-sulfobutyl) -2- (4'-hydroxy-3 ', 5'-dioodostyryl) -3,3-dimethylindolenium betaine

3,5-Diiodo-4-hydroxybenzaldehyde (1.87 g, 5 mmol), 1- (ω-sulfobutyl) -2,3,3-trimethylindolenium betaine (2.36 g, in EtOH (35 ml) 8 mmol) [see: RB Mujumdar et al., Cytometry 10, 11-9 (1989)] and piperidine (0.4 ml) are maintained under an inert gas atmosphere. The solution is refluxed for 2.5 hours and then cooled to ambient temperature. The reaction mixture is acidified with excess 1.93 M HCl in i-PrOH and evaporated to dryness in vacuo to leave a residue. The residue is dissolved in EtOH (10 ml). Leaving in the refrigerator separates solids from the mixture. The solid was collected by filtration, washed with ice-cold EtOH / hexane (3: 1 v / v) and then dried in vacuo to give an orange solid (3.36 g). The crude product is dissolved in boiling EtOH (about 30 ml) and precipitates quickly. Additional boiling EtOH is added (approx. 220 ml), but the solids are not redissolved. Cool in ice, collect the solids, filter, wash with EtOH, and dry in vacuo to give analytically pure compound 1- (ω-sulfobutyl) -2- (4'-hydroxy-3 ', 5'-dioyo Dostyryl) -3,3-dimethylindolenium betaine (1.54 g, 47%) is obtained as an orange powder. The aforementioned method for preparing the compound is generally shown in FIG. 5. Spectroscopic analysis data identifying the compound are as follows.

Elemental Analysis for C ₂₂ H ₂₃ I ₂ NO ₄ S

Calc .: C, 40.57; H, 3.56; N, 2.15

Found: C, 40.59; H, 3.50; N, 1.99.

Example 6

1- (n-butyl) -2- (4'-hydroxy-3 ', 5'-dioodostyryl) -3,3-dimethylindolenium iodide

3,5-Diiodo-4-hydroxybenzaldehyde (3.73 g, 10 mmol), 1- (n-butyl) -2,3,3-trimethylindoleninium iodide (4.46 g, in EtOH (40 ml) 13 mmol) [see: DP Maisuradze et al., Soobschch. Akad. Nauk Gruz, SSR 50, 77-82 (1968); CA 69: 106526r) and a mixture of piperidine (0.8 ml) are maintained under an inert gas atmosphere. The solution is refluxed for 1 hour and cooled to ambient temperature. The solution is evaporated to dryness in vacuo to leave a residue. The residue is dissolved in EtOH (10 ml) and treated with 1.93 M HCl in i-PrOH (3.0 ml). The solution is then evaporated back to dryness in vacuo to leave a residue. The residue is dissolved in EtOH (4 ml). When the solution is frozen, crystals form naturally. The separated crystalline solid was collected by filtration, washed with ice-cold EtOH, and then dried in vacuo to give 1- (n-butyl) -2- (4'-hydroxy-3 ', 5'-diiodostyryl ) -3,3-dimethylindolenium iodide (4.90 g, 80.7%) is obtained. The crude compound is dissolved in hot EtOH (60 ml), filtered through filter paper and concentrated to about 30 ml in vacuo. Allow the solution to crystallize. The separated crystalline solid was collected as above, washed and dried to give analytically pure compound 1- (n-butyl) -2- (4'-hydroxy-3 ', 5'-dioodostyryl) -3 , 3-dimethylindolenium iodide (3.90 g, 56%) is obtained as a light orange powder. The above method for preparing the compound is generally shown in FIG. 6. Spectroscopic analysis data identifying the compound are as follows.

C ₂₂ H ₂₄ I ₃ NO Elemental Analysis for EtOH

Calc .: C, 38.68; H, 4.06; N, 1.89

Found: C, 38.55; H, 3.96; N, 1.91.

Example 7

Analytical Performance for Human Serum Albumin

The utility of the merocyanine protein error indicator of the present invention in liquid assays for determining the concentration of proteins in liquid test samples is described in the table below. At a pH at least 0.5 units below the pKa of the compound, a solution of that compound in 200-250 mM Na + or K + citrate buffer is prepared and its absorbance is measured at the wavelengths listed below. The solution is then treated with a sufficient amount of human serum albumin so that the albumin concentration is 100 mg / dl and the absorbance is measured again. The reported absorbance increase (ΔABS) is proportional to the amount of albumin present, which indicates the relative sensitivity of the dye for protein measurement.

Example 8

Preparation of Reagent Strips

One of the methods of preparing the analyte protein reagent strips described herein is described below. The method described is a continuous method for mass production of protein reagent test sheets.

According to this method, the thin absorbent paper strip is moved along the line at a suitable speed of about 4 ft / min. One preferred paper is E & D 237 (Ahlstrom Filtration, Inc., Mount Holley Springs, PA, U.S.A.). The paper is soaked in a pH 2.5 buffer bath containing the merocyanine protein error indicator SPDIB dissolved in ethanol. According to one preferred method, the bath contains from about 0.03 to about 0.08 mM SPDIB, 0.5 M potassium citrate buffer (pH 2.5) and 20% ethanol. The test strip is then passed at a rate of about 4 ft / min through a drier at 1 inch air pressure and 60 ° C. temperature conditions. The test strip is then cut and packaged.

Example 9

Dose Reaction of Reagent Strips Containing SPDIB

Buffered liquid samples (50 mM potassium citrate pH 2.5) that were found to be albumin free by immunoassay were tested using Pentex ^R human serum albumin [HSA; Spikes to various clinically significant levels using Miles Inc., El Cart, Indiana, USA. Protein measurements are performed using an analytical test strip containing 0.3 mM SPDIB using a Clinitek 200 device (manufactured by Miles Inc.).

The results are quantitatively expressed in Δ K / S between albumin concentrations, as shown in FIG. 7. K / S is calculated by Equation 1:

In Equation 1 above,

R is the reflectance fraction from the test apparatus,

K is a constant,

S is the light scattering coefficient of the particular reflective medium.

Equation 1 above is a well-known Kubelka-Munk equation [Gustav Kortum, 'Reflectance Spectroscopy', pp. 106-11, Springer Verlas, New York (1969). K / S is measured at 25 seconds.

The reagent strips described above are analyzed for their response to different protein concentrations. The results of this survey are summarized in FIG. As shown in Figure 7, the strip of the present invention is sensitive to a wide range of protein concentrations. In addition, significant analysis between protein concentrations can be seen in the present invention.

Of course, the present invention may have various modifications and other forms, but specific embodiments thereof are shown as examples and described in detail herein. However, this is not intended to be limited to the specific forms disclosed herein, but on the contrary is intended to cover all modifications, equivalents, and modifications falling within the spirit and scope of the invention as defined by the appended claims. Must understand.

Claims (3)

An assay test strip for detecting proteins in a liquid sample, comprising an absorbent carrier impregnated with a merocyanine protein error indicator compound of Formula 1. Formula 1 In Formula 1 above, m is an integer from 1 to 6, Q is -Br, -I or Cl, R is S, Se, O or C (C _n H _{2n + 1} ) ₂ , where n is an integer from 1 to 6, T -SO ₃ Or -H. The compound of claim 1, wherein m is an integer of 3 or 4, Q is -Br or -I, and R is C (C _n H _{2n + 1} ) ₂ , wherein n is an integer of 1 to 3. , T -SO ₃ Analytical test strips. The assay test strip of claim 1, wherein m is an integer of 3 and R is C (CH ₃ ) ₂ .