ACCELERATING A NINHYDRIN REACTION

Field

The present disclosure relates to accelerating a ninhydrin reaction within an indicator solution. In particular, the present disclosure relates to accelerating a ninhydrin reaction within an indicator solution under ambient temperature conditions.

Background

Ninhydrin (1,2,3-triketo-hydrindane hydrate) has been recognized as a reagent for detecting amino acids, amines and amino sugars. When reacting with amines, a deep blue or purple product called Ruhemann's Purple forms. The reaction from ninhydrin to the conjugate Ruhemann's Purple is shown below:

Ninhydrin Ruhemann's Purple

Ninhydrin has practical applications. For example, in forensics ninhydrin can be used to develop latent fingerprints, which contain amino acid, on porous surfaces and will produce the color change to identify the print. However, a variety of factors can affect the reaction time for achieving a color change. For some applications, it may be desirable to accelerate the reaction time for achieving a color change of the ninhydrin.

Summary

The present disclosure relates to accelerating a ninhydrin reaction within an indicator solution. In particular, the present disclosure relates to accelerating a ninhydrin reaction within an indicator solution under ambient temperature conditions. "Ambient temperature conditions" as used herein means generally temperature conditions without

applying an additional heat source. In one embodiment, "ambient temperature conditions" means a temperature between 10° C (50° F) and 35° C (95° F).

In one embodiment, an indicator solution is disclosed for detecting the presence of a protein or amine. The indicator solution comprises ninhydrin and an accelerant selected from the group consisting of at least 50% wt. isopropyl alcohol and a lithium salt. The indicator solution produces a color change in the presence of a protein or amine in less than 5 minutes under an ambient temperature condition.

Detailed Description Ninhydrin gives a visually discernable color change in the presence of amines and proteins. For cleaning applications, such as in a bathroom, biological contaminants may exist or in a kitchen bacteria, such as E. coli or Salmonella may be present. The user initially has no indication as to the actual presence of such contaminants. Further, following cleaning, the user has no indication as to the actual cleanliness of the surfaces cleaned. The user may have just spread the soil or may have captured it. For providing a clean environment, the user needs an indication of the cleanliness of a surface.

In kitchen cleaning in particular, amines and proteins are present in meats such as ground beef and chicken, which if contaminated may contain E. coli or Salmonella, respectively. Therefore, if ninhydrin is exposed to a kitchen surface and a color change becomes apparent, the indication may be that a meat residue is present and contaminations such as E. coli or Salmonella may be present. Therefore, an indicating solution containing ninhydrin may be used to indicate the cleanliness of a surface.

As identified above, many variables will affect the reaction time for getting the visible color change of the ninhydrin. First, the types of amino acids and the concentration of the amino acid determine the reaction time. Second, temperature and humidity influence the development of a color change of the ninhydrin. Third, the concentration of the ninhydrin affects reaction time. Typically, the reaction is slow and heating is often required for faster results. Also, with all of these variables, the development time and the intensity of the color change may differ widely from test to test.

For many applications, cleaning in particular, a fast response of the color change is desirable. A visual color change should be apparent in less than 5 minutes, in another embodiment, less than 2 minutes, and in another embodiment, less than 60 seconds. The ability to control the concentration of the amine or protein is limited. In addition, introducing a heat source to increase the temperatures above room temperature is undesirable and impractical for use, especially during cleaning.

An indicator solution for detecting the presence of a protein or amine is disclosed. The indicator solution comprises ninhydrin and an accelerant present so that the indicator solution produces a color change in the presence of a protein or amine in less than 5 minutes under an ambient temperature condition. Although such an indicator solution has application relating to cleaning, such an application is not limiting. The indicator solution with an accelerated reaction time may be used in other applications where detection of amines and/or proteins is needed, such as forensics.

In one embodiment, the accelerant is isopropyl alcohol comprising at least 50% wt. of the indicator solution. The ninhydrin is soluble in isopropyl alcohol. The solubility of the ninhydrin in higher chain alcohols decreases significantly. Further, shorter chain alcohols do not have the same amount of accelerating ability as the isopropyl alcohol has on the ninhydrin reaction time. The isopropyl alcohol solution achieved surprising and advantageous result regarding accelerating the reaction time for achieving a color change of the ninhydrin in the presence of a protein.

Without being limited to any one particular theory, it is believed that the isopropyl alcohol provides superior denaturing of the protein that allows the amine groups access to react with the ninhydrin. In addition, the ninhydrin is relatively soluble in the isopropyl alcohol. In another embodiment, the accelerant is a lithium salt. Suitable lithium salts include lithium acetate (LiAc), lithium chloride (LiCl), lithium hydroxide (LiOH), and lithium carbonate (Li ₂ COs). In one embodiment, the lithium salt and the ninhydrin are in solution with water due to the solubility of the lithium salt and ninhydrin. Other solvents may be used so long as the lithium salt and the ninhydrin are relatively soluble in the solvent. In one embodiment, the lithium salt concentration of the indicator solution is at

least 0.1M. In one embodiment, the lithium salt concentration of the indicator solution is at least IM.

Without being limited to any one particular theory, it is believed that due to its strong electrical coordination capacities, the lithium ion helps better align the ninhydrin molecules during conjugation reaction to achieve the Ruhemann's Purple product.

Typically, the ninhydrin is present in the indicating solution from 0.5% to 10% wt. In another embodiment, the ninhydrin is present in the indicating solution from 1% to 5% wt. It is understood that the ninhydrin is present in the indicating solution in sufficient concentration to produce a reaction while still being dissolved in the indication solution.

In addition to the accelerant, the indicating solution may include a variety of other components such as, but not limited to, anti-oxidants, dyes, pigments, surfactants, soaps, detergents, fragrance, and anti-microbial agents, disinfectants. In embodiments where the indicating solution is used on surfaces that come into contact with people or pets, the components of the solution should be safe and nontoxic. Disinfectants may be particularly suitable for the indicating solution intended for cleaning purposes. Common surface disinfectants comprise biocides such as alcohols, biguanides, cationic surfactants, and halogen or halogen containing compounds. Suitable alcohols include ethanol and IPA in 70% water [IPA/H ₂ O (70/30), EtOH/H ₂ O (70/30)]. Suitable biguanide (chlorhexidine) include polyhexamethylene biguanide, p-chlorophyenyl biguanide, and 4-chlorobenzhydryl biguanide. Commercially available biguanides are Nolvasan® available from Wyeth of Fort Dodge, IA and ChlorhexiDerm® Disinfectant available from DVM Pharmaceuticals of USA. Examples of cationic surfactant (Quaternary Ammonium Compounds, Quats) include Parvosol® available from Hess & Clark of Randolph, WI, Roccal-D® Plus available from Pfizer of New York, NY,

Unicide™ 256 available from Brulin & Coompany Inc. of Indianapolis, IN, and benzalkonium chloride. Typical halogen or halogen containing compounds are either chlorine or iodine based. The disinfectant, and any other additives, chosen should not interfere with the ability of the ninhydrin to produce a color change. The indicating solution may be preloaded into a substrate such as a cleaning cloth, foam, or sponge. Such a cleaning cloth may be formed from a woven, knitted, or

nonwoven material. Suitable nonwoven materials include lofty fiber webs of natural or synthetic fibers. The preloaded substrate would be exposed to the surface to be cleaned. Then, the user will inspect the surface of the substrate to see if a visual color change results. If a visual color change results, then the user knows that a protein is present on the surface. Then, the user can disinfect the surface. In one embodiment, the preloaded substrate includes a disinfectant so the indicating and disinfecting occurs in one step. Then, a second preloaded wipe can be exposed to the surface to be cleaned. The user will inspect the surface of the substrate to see if a visual color change results. If a visual color change is not apparent, then the user knows that the protein has been eliminated. If a visual color change results, then the user repeats the steps above.

Alternatively, the indicating solution may be in a container, such as a spray container, and delivered directly to a surface. In this embodiment, the indicating solution may react on contact with the surface giving the user the indication that a protein is present on the surface. Then, the user will disinfect the surface. In one embodiment, the disinfectant is included with the indicating solution. In this embodiment, the user will remove the indicating solution from the surface with some kind of wiping substrate. The indicating solution may react once wiped onto the wiping substrate. Following wiping, the user may spray the surface again. If no color change occurs, then the user knows the protein has been eliminated. If the color change appears, then the user repeats the steps above.

Although specific embodiments of this invention have been shown and described herein, it is understood that these embodiments are merely illustrative of the many possible specific arrangements that can be devised in application of the principles of the invention. Numerous and varied other arrangements can be devised in accordance with these principles by those of ordinary skill in the art without departing from the spirit and scope of the invention. Thus, the scope of the present invention should not be limited to the structures described in this application, but only by the structures described by the language of the claims and the equivalents of those structures.

Example 1: Ninhydrin with Isopropyl Alcohol

Preparation of Stimulus:

A meat juice solution was prepared. Approximately 10 gram of cubed fresh pork chop meat was extracted with 20 mL of water for 16 hours and the mixture was filtered. The total protein in the meat juice was measured according to Pierce Coomassie Plus Protein Assay (product # 23238) available from Pierce Biotechnology Inc., Rockford, IL and ranges from approximately 11 mg/mL to 23 mg/mL.

Product List

Ninhydrin available from Aldrich Chemical Co. of Milwaukee, WI. Isopropanol (IPA) available from EM Science of Gibbstown, NJ.

Reaction Measurement The prepared indicating solutions containing 1% wt. ninhydrin were exposed to the prepared meat juice at ambient conditions. In particular, the temperature was approximately between 15.5° C (60° F) and 24° C (75° F). The ratio of indicating solution to meat juice solution is noted in Table 1. A visual inspection was conducted to determine when a noticeable visual color change occurred. The reaction time was measured and is noted in minutes:seconds.

Table 1: Ninhydrin/Isopropanol Solutions

The data in Table 1 indicates that an increase of isopropyl alcohol content decreases the time to a color change in the presence of protein as compared to a sample without isopropyl alcohol (sample 4). Also, the above data in Table 1 indicates an increase of

isopropyl alcohol concentration in the ninhydrin solutions will decrease the time to a color change in the presence of protein.

Example 2: Ninhydrin with Lithium Acetate

Product List

Ninhydrin available from Aldrich Chemical Co. of Milwaukee, WI. Lithium acetate available from Aldrich Chemical Co. of Milwaukee, WI. Human serum albumin available from Baxter Healthcare Corp. of Glendale, CA. PBS (phosphate buffer saline), 0.1M, pH 7.4.

Reaction Measurement

The kinetics of the response of ninhydrin to protein in the presence of lithium acetate was monitored by UV-Vis spectrometry. The characteristic absorption peak of "Ruhemann's Purple," the protein-ninhydrin conjugate, was determined to be 578nm.

One milliliter of the water solution of various concentrations of lithium acetate and 1 ml of 10% human serum albumin in 0.1 M PBS (pH 7.4) were added into a UV cuvette, and the background was recorded. The absorption at 578 nm was measured every 3 seconds after 1 ml of 2% ninhydrin water solution was added. The reaction time was measured as the absorption reaches 2.5 AU and is noted in minutes:seconds.

Table 2: Ninhydrin/Lithium Acetate Solutions

The data in Table 2 indicates that an increase of lithium acetate concentration decreases the time to a color change in the presence of protein as compared to a sample without

lithium (sample 4). Also, the data in Table 2 indicates an increase of lithium concentration in the ninhydrin solutions will decrease the time to a color change in the presence of protein.

Example 3: Ninhydrin with Lithium Acetate

Product List

Ninhydrin available from Aldrich Chemical Co. of Milwaukee, WI. Lithium acetate available from Aldrich Chemical Co. of Milwaukee, WI.

Preparation of Stimulus:

A meat juice solution was prepared. Approximately 10 gram of fresh pork chop meat was extracted with 20 mL of water for 16 hours and the mixture was filtered. The total protein in the meat juice was measured according to Pierce assay and ranges from approximately 11 mg/mL to 23 mg/mL.

Reaction Measurement

The prepared indicating solutions (250μL) containing 2% wt. ninhydrin were exposed to 25 μL of lithium acetate solutions (0, 1.0M, and 4.0M) and 25 μL prepared meat juice at ambient conditions. In particular, the temperature was approximately between 15.5° C (60° F) and 24° C (75° F). A visual inspection was conducted to determine when a noticeable visual color change occurred. The reaction time was measured and is noted in minutes:seconds.

Table 3: Ninhydrin/Lithium Acetate

The data in Table 3 indicates that an increase of lithium acetate concentration decreases the time to a color change in the presence of protein as compared to a sample without lithium (sample 3).

Example 4: Ninhydrin/Various Lithium Ions

Product List

Ninhydrin available from Aldrich Chemical Co. of Milwaukee, WI. Lithium acetate, lithium carbonate, lithium chloride, lithium hydroxide all available from Aldrich Chemical Co. of Milwaukee, WI.

Human serum albumin available from Baxter Healthcare Corp. of Glendale, CA. PBS (phosphate buffer saline), 0.1M, pH 7.4.

Reaction Measurement

Tables 4 and 5 list the time when a noticeable purple color starts to appear after 100 μL of the prepared lithium solution is added to the mixture of 100 μL of 10% human serum albumin in 0.1 M PBS and 100 μL of 2% ninhydrin in DI water at ambient temperature conditions. In particular, the temperature was approximately between 15.5° C (60° F) and 24° C (75° F). The reaction time was measured and is noted in minutes : seconds .

Table 4: Ninhydrin/Various Lithium Ions

The data in Table 4 indicates that an addition of lithium ions decreases the time to a color change in the presence of protein as compared to a sample without lithium ion (sample 5). In addition, different lithium counter ions resulted in different reaction times.

Table 5: Ninhydrin/Various Lithium Ions

The data in Table 5 indicates that an addition of lithium ions decreases the time to a color change in the presence of protein as compared to a sample without lithium ion (sample 5). Different lithium counter ions resulted in different reaction times. Also, the data in Table 4, in comparison to the data in Table 5, indicates an increase of lithium concentration in ninhydrin solutions will decrease the time to a color change in the presence of protein.

Although specific embodiments of this invention have been shown and described herein, it is understood that these embodiments are merely illustrative of the many

possible specific arrangements that can be devised in application of the principles of the invention. Numerous and varied other arrangements can be devised in accordance with these principles by those of ordinary skill in the art without departing from the spirit and scope of the invention. Thus, the scope of the present invention should not be limited to the structures described in this application, but only by the structures described by the language of the claims and the equivalents of those structures.