Living cells use adenosine triphosphate (ATP) as an energy source for cell function. As the amount of ATP present indicates cell viability and cell numbers, ATP measurement is performed to evaluate the degree of bacterial contamination in hospitals and food processing environments. The technique is also used to test cell status in biological, medical and pharmaceutical research.

 

The most common method of measuring ATP is bioluminescence assay, which features high sensitivity and selectivity. However, a luminometer and other dedicated instruments are needed to detect the weak light generated from bioluminescence reactions. For situations where only the approximate number of cells needs to be known or simple information on the presence or absence of bacterial contamination is required, a simple and inexpensive method based on visual determination without the need for dedicated equipment is desirable. A technique for quantitative analysis using commonly available and versatile devices such as colorimeters rather than luminometers is also required.

 

We developed a colorimetric method for ATP to meet these demands. The assay results exhibit yellow-orange/purple-blue colors when the ATP concentration of a sample is lower/higher than the predetermined level, respectively. This makes it easy to determine at a glance whether the ATP concentration in a sample meets the relevant criteria as shown below. For situations where quantitative results are required, a laboratory-installed colorimeter can be used.

The principles of the present colorimetric assay method are as follows: First, enzymatic cycling using pyruvate kinase and adenylate kinase along with ATP contained in a sample is performed for a given period of time (Step 1). The number of cycles can be arbitrarily fixed by setting the reaction time to produce pyruvate at higher concentrations than the initial ATP. Even ultra-traces of ATP can be detected with the naked eye under enzymatic cycling. Second, hydrogen peroxide is generated from pyruvate using pyruvate oxidase (Step 2). Third, ferrous ion is oxidized by hydrogen peroxide under acidic conditions (pH: approx. 2). Ferric ion forms complexes with xylenol orange (XO) even though ferrous ion does not bind with XO because of low stability constants under these conditions (Step 3).

The complexes are in three forms: Fe(xo), Fe(xo) 2 and Fe2(xo). They exhibit orange, red and purple-blue, respectively, and the combination of these colors produces an assay result color depending on ferric ion concentration as shown in the figure below. If the ferric concentration is higher than 20 μM, purple is produced. Thus, for example, if the assay conditions are fixed so that 100 pM of ATP should produce 20 μM of ferric ions, a purple assay result indicates that the sample contains ATP at a higher concentration than 100 pM, while yellow indicates a concentration lower than this level.
The present assay reaction produces four dyes (including unreacted XO (yellow)) and three complexes to provide a wide spectrum of color changes ranging from yellow to deep orange to purple. This is a particular characteristic of the present assay as compared to common colorimetric assay, which provides only monochromic changes with a single dye.

In this study, the colorimetric assay method described here was applied to bacteria count screening, and the presence of five million bacteria cells per ml (equivalent to 500 pM ATP) was determined. Collaboration with another university is currently under way on the development of a rapid colorimetric assay system for ATP in the blood to assess the severity of conditions in patients based on the present methodology. *1 pM: a trillionth of a mol per liter.

 

This research was supported by the Japan Science and Technology Agency (JST).

 

Related information

1) Akihiko Ishida, Yasuko Yamada, Tamio Kamidate, Colorimetric method for enzymatic screening assay of ATP using Fe (III)-xylenol orange complex formation, Anal. Bioanal. Chem., 392, 987 – 994 (2008). http:// www.ncbi.nlm.nih.gov/pubmed/18726586
November 13, 2009.

2) Hokkaido University, Assay method for adenosine triphosphate in samples, Japan Patent 4940432, March 9, 2012.

 

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