Validation of Caffeine Dehydrogenase from Pseudomonas sp. Strain

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Validation of Caffeine Dehydrogenase from Pseudomonas sp. Strain CBB1 as a Suitable Enzyme for a Rapid Caffeine Detection and Potential Diagnostic Test. Sujit K. Mohanty, Chi Li Yu, Sridhar Gopishetty, and Mani Subramanian J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf501598c • Publication Date (Web): 14 Jul 2014 Downloaded from http://pubs.acs.org on July 15, 2014

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Journal of Agricultural and Food Chemistry

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Validation of Caffeine Dehydrogenase from

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Pseudomonas sp. Strain CBB1 as a Suitable Enzyme

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for a Rapid Caffeine Detection and Potential

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Diagnostic Test.

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Sujit K. Mohanty‡, Chi Li Yu‡†, Sridhar Gopishetty§, and Mani Subramanian‡,§,*

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‡

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Center, Iowa City, IA 52242, U.S.A.

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§

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100, Coralville, IA-52241, U.S.A.

Department of Chemical and Biochemical Engineering, University of Iowa, 4133 Seamans

The Center for Biocatalyst and Bioprocessing, University of Iowa, 2501 Crosspark Rd. Suite C-

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Corresponding Author

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*Tel: (319) 335-4900. Fax: (315) 339-4901. E-mail: [email protected].

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Present Addresses

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†

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Research Building, Iowa City, IA-52242, U.S.A.

Proteomics Facility, Carver College of Medicine, University of Iowa, 355 Eckstein Medical

ACS Paragon Plus Environment

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ABSTRACT

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Excess consumption of caffeine (>400 mg/day/adult) can lead to adverse health-effects. Recent

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introduction of caffeinated products (gums, jelly beans, energy-drinks) might lead to excessive

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consumption, especially among children and nursing mothers; hence attracted FDA attention and

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product withdrawals. An “in-home” test will aid vigilant consumers in detecting caffeine in

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beverages and milk easily and quickly, thereby restricting its consumption. Known diagnostic

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methods lack speed and sensitivity. We report a caffeine dehydrogenase (Cdh)-based test, which

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is highly sensitive (1-5 ppm) and detects caffeine in beverages and mother’s milk in one minute.

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Other components in these complex test samples do not interfere with the detection. Caffeine-

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dependent reduction of the dye INT results in shades of pink proportional to the levels in test

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samples. This test also estimates caffeine levels in pharmaceuticals, comparable to HPLC. The

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Cdh-based test is the first with the desired attributes for rapid and robust caffeine diagnostic kit.

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Keywords: caffeine, caffeine dehydrogenase, Pseudomonas sp. CBB1, diagnostic test,

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nursing mother’s milk.

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INTRODUCTION

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Caffeine (1,3,7–trimethylxanthine) is one of the most popular and commercially important plant

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derived purine alkaloid.1 It is a key component of widely consumed beverages, like coffee, tea,

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soft drinks, energy drinks, etc.2-4 In fact, it is a key additive to more than 150 food and 570 drink

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products that are currently available in the market.5 Caffeine is also an active ingredient of

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various pharmaceutical preparations and is often used as a neurological, cardiac, and respiratory

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stimulant,6 as well as an analgesic enhancer in cold, cough, headache, and asthma.7-9 In human

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diet, a low (< 200 mg/day/adult) to moderate (200-400 mg/day/adult) consumption of caffeine is

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regarded as beneficial in terms of increasing alertness and overcoming fatigue.3 According to

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International Coffee Organization (ICO), the demand for coffee (a major source of caffeine in

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food) is rapidly growing with a global consumption of 145.2 million bags (of 60 kg each) in

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calendar year 2012, which represents a 2.4% average annual growth rate over past four years.10

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Caffeine is an addictive substance.11 Recent studies suggest that long-term and excessive

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consumption of caffeine (400-700 mg/day/adult) can lead to various adverse health effects like

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changes in sleep pattern, rise in blood pressure, palpitations, anxiety, irritability, nausea,

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restlessness,12-15 to severe conditions like insomnia, auditory hallucinations, loss of appetite,

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abdominal pain and heart burn due to excess acid, vitamin deficiency, increased risk of coronary

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heart disease, and several types of cancer.16 Moreover, in a few rare instances, sudden but lethal

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doses of caffeine have also resulted in human death.17-19 Thus, safety of this widely consumed

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psychoactive drug is an issue.20

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Caffeine has received much attention recently due to two main reasons: (a) continued indiscriminate introduction into new products including instant energy/alert boosters, faster


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weight loss supplements, chewing gums and jelly beans20-22 (b) potential unregulated excessive

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consumption of this psycho-active substance among infants, children, nursing mothers and even

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adults.23,24 This has attracted FDA attention and in withdrawal of products.21,22 Unlike other

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psychoactive substances consumed worldwide, caffeine is legal and generally unregulated with a

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classification of “Multiple Purpose Generally Recognized as Safe Food Substance” by the U.S.

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Food and Drug Administration (FDA) [Under 21 Code of Federal Regulations section 182.1180].

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However, FDA does not consider it as a nutrient although it is a natural product. The FDA

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considers concentration of caffeine above 200 parts per million (ppm) equivalents to 0.02 percent

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in food and beverages as unsafe [21 Code of Federal Regulation section 182.1180(b)] and

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recommends no more than 400 milligrams of caffeine per day for adults.25 Indiscriminately

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introduced new caffeine-containing products are neither FDA approved, nor restricted to

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children, and often contain 150-250 mg of caffeine per serving. 21,22 This can be unsafe,

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particularly for infants and teenagers.23-27

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Several attempts have been made to develop a suitable assay for caffeine detection and/or

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measurement.28-36 However, none of these tests meet the attributes required for a suitable

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diagnostic test such as speed of detection, ability to detect in commercial/natural fluids, and

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sensitivity of detection. In the present work, we report a novel enzyme, caffeine dehydrogenase

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(Cdh), which is highly suitable for detection of caffeine and potential development of a

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diagnostic test. In the presence of a tetrazolium dye such as INT, Cdh is able to detect caffeine

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in less than one minute with a sensitivity of 1-5 ppm. In addition, this enzyme-based test is able

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to detect decaffeinated beverages instantly. Spiked caffeine as low as 5-20 ppm can be detected

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in mother’s milk with no interference from milk-components. Last, but not least, caffeine can be


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detected quantitatively in pharmaceuticals, nearly matching the reported values, and as detected

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by HPLC.

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MATERIALS AND METHODS

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Chemicals and Test Samples. Caffeine, DNase I, Iodonitrotetrazolium Chloride (INT),

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Tetrazolium Blue Chloride (TB), Tetrazolium Blue Tetrazolium Bromide (MTT), Tetrazolium

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Violet (TV), Nitro Blue Tetrazolium (NBT), and Tetranitro blue Tetrazolium (TNBT) were

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purchased from Sigma-Aldrich (St. Louis, MO). Yeast nitrogen base without amino acids and

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without ammonium sulfate (YNB) was purchased from ForMedium (Norfolk, United Kingdom).

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High-pressure liquid chromatography (HPLC)-grade methanol and acetonitrile were obtained

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from Sigma-Aldrich. Coffee, soft drinks and pharmaceuticals were obtained from local outlets.

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Nursing mother’s milk was obtained from Mother’s Milk Bank of Iowa (University of Iowa

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Children’s Hospital).

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Media and Growth Conditions. Strain CBB1 was grown in M9 mineral salts medium37

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with 2.5 g /L caffeine and 4 g/L YNB at 30 °C with shaking at 200 rpm. Cell-growth was

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monitored by measuring the optical density at 600 nm (OD600). Cells were harvested at late log

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phase by centrifugation (13,800g for 10 min at 4 °C) as described by Yu et al.37 Cell pellets

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were washed twice with 50 mM potassium phosphate (KPi) buffer (pH 7.5) and frozen at -80 °C

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until required.

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Purification of caffeine dehydrogenase from Pseudomonas sp. CBB1. All purification

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procedures were performed at 4 °C using an automated fast protein liquid chromatography

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system (AKTA Design, Amersham Pharmacia Biotech) as described by Yu et al.37

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Chromatography columns and column resins were from Amersham Biosciences, Piscataway, NJ.


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Amicon Ultra-15 (30K) ultrafiltration membrane (Millipore, Bedford, MA) was used for

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concentration and buffer exchange of the protein fractions containing caffeine dehydrogenase

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activity. About 8 g frozen cells were thawed and suspended in 30 mL 50mM KPi buffer (pH

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7.5) and DNase I was added to a final concentration of 10 µg/mL. The cells were lysed using a

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chilled French Press in two cycles at 138 MPa. Unbroken cells and debris were removed from

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the lysate by centrifugation (20,400g for 20 min at 4 °C) and the supernatant was designated the

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crude cell extract (CCE). A 4.0 M solution of ammonium sulfate was added drop-wise to CCE

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to obtain a final ammonium sulfate concentration of 0.8 M with constant stirring. After

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incubation with shaking on ice for 1 h, the mixture was centrifuged at 20,400g for 20 min at 4 °C

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to remove precipitated proteins. The supernatant was loaded onto a 40 mL (bed volume) phenyl

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sepharose high-performance column (Amersham) preequilibrated with 50 mM KPi buffer (pH

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7.5) containing 0.8 M ammonium sulfate. Unbound proteins were washed from the column with

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40 mL 50 mM KPi buffer containing 0.8 M ammonium sulfate. Bound proteins were eluted with

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a 200 mL reverse gradient of ammonium sulfate (0.8 to 0 M in KD buffer) at a rate of 1 mL/min.

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The column was then washed with 40 mL 50 mM KPi buffer (pH 7.5), followed by 40 mL water

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at the same flow rate to elute hydrophobic proteins tightly bound to the column. Fractions

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containing caffeine dehydrogenase were identified by the spectrophotometric enzyme activity

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assay as described below. Caffeine dehydrogenase was eluted from the column during the final

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wash step with water. Protein concentration was determined by Bradford,38 using BSA as the

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standard with a dye reagent purchased from Bio-Rad.

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Enzyme Assays: A spectrophotometric activity assay based on caffeine-specific

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reduction of NBT was developed to monitor the purification of caffeine dehydrogenase as

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described by Yu et al.37 A typical 1-mL reaction mixture contained an appropriate amount of


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enzyme (usually 1-10 µg), 0.5 mM caffeine, and 0.5 mM NBT in 50mM KPi buffer (pH 7.5).

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Caffeine dehydrogenase activity was determined by monitoring with a UV/vis spectrophotometer

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(Shimadzu UV-2450), the increase in absorbance at 566 nm (extinction coefficient of

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15,500/M/cm) due to formazan production. One unit of enzyme activity was defined as

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reduction of 1 nmol NBT/min at 25 °C and pH 7.5. All colorimetric assays in 96-well plates

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were carried out at room temperature and 400 rpm. Final reaction volume of 200 µL/well

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contained 0.5 mM tetrazolium dye in 50mM KPi buffer (pH 7.5). Reaction was started with

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addition of appropriate amount of enzyme.

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Analytical Procedures and Test Sample Preparation. Identification and quantification

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of caffeine in test samples was conducted with a Shimadzu LC-20AT HPLC system equipped

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with a SPD-M20A photodiode array detector and a hypersil BDS C18 column (4.6 by 125 mm)

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as described by Yu et al.39 20% methanol/80% water/0.5% acetic acid (20:80:0.5, v/v/v) was

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used as an isocratic mobile phase with a flow rate of 0.5 mL/min. Prior to testing of the drugs,

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one tablet from each of the listed pharmaceutical (Table 2) was powdered separately, dissolved

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in 50 mM KPi buffer (pH 7.5), and diluted appropriately to bring the caffeine concentration

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range to 5-50 ppm. Correspondingly, a reference color chart was prepared using 50 mM KPi

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buffer (pH 7.5) by spiking known concentrations of pure caffeine purchased from Sigma

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Chemicals, and developing the shades of red color using the Cdh-based detection test in the

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range 0-50 ppm. Caffeine concentration in the diluted samples was determined in triplicate,

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using HPLC as described previously.39 Coffee extract used in the validation test was obtained by

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boiling 10 g coffee powder for 10 min with 200 mL of deionized water, and filtering using a

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filter paper. HPLC method was employed to determine the caffeine content in the extract, in soft

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drinks and in pharmaceutical samples.


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RESULTS Testing Various Tetrazolium Dyes for Best Visual Suitability. Several commercially

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available tetrazolium dyes were screened as electron acceptor in the Cdh-based caffeine

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detection. Two critical attributes were (i) rapid reduction in the presence of Cdh and caffeine to

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give a bright colored product, and (ii) low or no background (minimum non-enzymatic

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reduction). Six tetrazolium dyes were tested at 0.5 mM as described in the Materials and

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Methods. As shown in Table 1, the enzyme in the presence of caffeine reduced all dyes except

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TB and TV. Of the four dyes that showed activity, MTT and TNBT showed strong background

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color (Table 1), which made it difficult to distinguish between reaction and no reaction (data not

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shown). On the other hand, INT had no background color and produced shades of light pink to

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dark red proportional to caffeine concentration. NBT upon reduction produced shades of dark

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blue to dark purple, which was readily distinguishable from the light yellow background color.

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INT was found to be most suitable for Cdh-based detection of caffeine. It also was the lowest

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priced dye (Table 1). NBT was chosen as a backup for INT. Previously, a 1:1 stoichiometry

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between caffeine and tetrazolium dye was established (Figure 1).37 Hence the dye concentration

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was fixed at 0.5 mM. This ensured that the dye-dependent color formation was not the limiting

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factor for caffeine detection.

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Cdh Required for Detection of Caffeine in One Minute. It is important that the Cdh-

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based detection of caffeine produced a visible color fast, i.e., within one minute, at the lowest

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detection limit of caffeine. In applications like detection of caffeine in breast milk, 5 ppm is

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deemed as the safe upper-limit based on the assumption that a mother consumes 500 mg of

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caffeine (100 mg higher than the safe upper-limit set by FDA for adults/day25 and about 1-1.5%

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of it partitioning into breast milk in one hour).39 Hence, 5 ppm caffeine was fixed as the lower


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limit for detection at varying enzyme load, and the corresponding time required for maximum

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color development was determined (Figure 2). As expected, increasing enzyme load decreased

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the time required for color development exponentially (Figure 2). Based on this, for maximum

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color development within a threshold of one minute, the minimum enzyme load for the present

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study was fixed at 20 U. While this enzyme load is adequate for further validation of Cdh-based

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caffeine detection in a microtiter plate, the actual enzyme load in a commercial diagnostic kit

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may have to be higher to compensate for loss of activity due to (i) immobilization of Cdh in a

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test strip (ii) formulation of the enzyme and (iii) shelf life of the enzyme and the dye. These

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parameters will have to be further optimized during the development of commercial diagnostic

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kit, like a dip stick-test. The color development in a diagnostic kit with the threshold detection

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of one minute should also be independent of caffeine concentration in the test sample. This will

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also demand higher enzyme load than 20 U.

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First Level Attributes for Cdh-Based Caffeine Detection. The first level attributes for

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further validation of the test with real world samples (caffeinated and caffeine-containing

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beverages, nursing mother’s milk and pharmaceuticals) were set as (i) minimum enzyme load of

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20 U Cdh, (ii) INT as the dye due to no background color (iii) optimal dye concentration of 0.5

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mM (equal to 97.1 ppm) (iv) visible pink color at 5 ppm caffeine to readily differentiable shades

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of red at 20, 100, and >>100 ppm), and (v) threshold time of 1 min for detection. The above

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conditions were used for caffeine detection in various samples, in 96-well microtitre plate, in a

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total reaction volume of 200 µL.

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Validation of Cdh for Caffeine Detection: Color Development at 5 ppm Caffeine

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(Lowest Limit). As discussed previously, 0-5 ppm was designated as safe range for caffeine in

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breast milk based on FDA guidelines and literature.25,40 Thus, the lowest detection limit for Cdh-


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based test should be 5 ppm. The color development for Cdh-based test in buffer, at 1 ppm

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caffeine is shown in inset of Figure 2. Light pink color is clearly distinguishable within one-

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minute reaction time, at a level lower than the safe limit of caffeine. There is no background in

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the control where caffeine is absent. These results establish that the Cdh-based test can detect

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caffeine as low as 1 ppm within the threshold time. Thus, detecting caffeine at 5 ppm should not

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be a limitation. However, this result needs to be validated with “real world samples”. These

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samples are different in that they are finished products in complex formulations. Detection of

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caffeine in buffer, under optimized conditions may not translate to the commercial samples.

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Interfering components in the formulations, pH of the test solution, background color of the test

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sample (such as cola, coffee, milk, etc.) are some of the issues. Hence, for further validation of

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Cdh for caffeine detection, three different categories of commercial samples were chosen. These

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include (i) pharmaceuticals formulated with caffeine (ii) caffeinated and caffeine-free beverages

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and (iii) milk samples, including breast milk (spiked with caffeine).

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Estimation of Caffeine in Pharmaceuticals. The first validation for Cdh-based

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detection of caffeine was done with pharmaceuticals containing caffeine. Caffeine is added in

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the final formulation of a number of drugs; although it is not an active ingredient in most of these

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drugs.41 Thus, estimating the caffeine in pharmaceuticals would be an excellent validation of the

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suitability of Cdh towards development of a diagnostic test. Caffeine concentrations in the drug

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formulations are given in the label. This was further confirmed by HPLC before estimating the

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caffeine levels in these drugs using Cdh. Six commercially available pharmaceuticals were

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chosen namely, Diurex (a diuretic), Menstrual Relief, Vanquish, Midol, Excedrin (pain

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relievers), and Vivarin (a stimulant). After appropriate dilution, caffeine concentration was

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measured in triplicate in these pharmaceutical samples using Cdh (last column in Table 2). A


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reference chart was created with standard caffeine at various concentrations, to visibly determine

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approximate caffeine levels in the pharmaceutical samples. As shown in Table 2, the caffeine

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concentration from Cdh-test matched reasonably with the HPLC estimates. The Cdh test gave

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credible estimations in the range 5-50 ppm. The components in the drug formulations did not

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interfere with Cdh for color development. Also, color development was within the threshold

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time of one minute.

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Determination of Caffeine in Commercial Beverages (Coffee and Soft-Drinks). The

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second validation of Cdh for caffeine diagnosis was performed with beverages like coffee, soft

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drinks, and their decaffeinated counter parts. For this, two sets of caffeine containing beverages

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were selected. Set-1 was regular coffee of Dunkin Donut brand, 50% decaffeinated coffee from

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Folgers, and decaffeinated coffee from Dunkin Donut, with caffeine concentration of about 600

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ppm, 330 ppm, and 20 ppm, respectively (Table 3). Set-2 was regular Coca-Cola with caffeine

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concentration of 96.4 ppm and caffeine free cola (zero caffeine); regular Pepsi with caffeine

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concentration of 107 ppm and caffeine-free Pepsi; and regular Mountain Dew with caffeine

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concentration of 152 ppm and caffeine-free diet mountain dew (Table 3). Caffeine content of

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soft drinks from the label on the cans is also shown in Table 3. The caffeine content on the label

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and that determined by HPLC were comparable (Table 3). Beverages listed in Table 3 were

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analyzed for caffeine using Cdh. The objective here was qualitative, i.e., to differentiate between

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presence, absence and low level of caffeine in decaffeinated coffee. The fact that many of these

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beverages have a background color was a concern; caffeine-dependent color formation should

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still be readily differentiable relative to background. The results of the Cdh test with the

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beverages are shown in Table 3. There was intense color production with caffeinated and 50%

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decaffeinated coffee (Table 3). In contrast, color was light with decaffeinated coffee (~20 ppm


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caffeine). The background-color of the coffee extracts did not interfere with Cdh in terms of

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clearly and instantly differentiating decaffeinated beverages from their caffeinated counterparts.

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Interestingly, the color is distinguishably lighter in 50% decaffeinated coffee (330 ppm caffeine)

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relative to regular (600 ppm caffeine), although the commercial relevance of this is not clear.

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Ingredients present in these beverages such as sugar, added chemicals, coloring agents, etc., did

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not interfere with Cdh-based test. These results further affirmed that Cdh is highly suitable and

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robust towards developing diagnostic test, and can quickly differentiate caffeinated drinks from

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decaffeinated or non-caffeinated counterparts. This test could be very useful to avoid human

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errors in restaurants and other outlets when all these beverages are served in non-labeled cups.

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Detection of Caffeine in Breast Milk. Cdh-based caffeine diagnosis would be of great

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value for rapidly detecting caffeine levels in nursing mother’s milk. Caffeine is highly restricted

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for pregnant woman and infants as documented earlier.25,26 Potential teratogenic effect of excess

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caffeine on infants was also documented.27 Hence, Cdh-based caffeine detection was tested with

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various milk samples spiked with caffeine. Milk is a complex mixture of proteins, fat, sugars,

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and other ingredients including calcium and vitamin D. In addition, nursing mother’s milk has

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immunoglobulins, metal ions, cofactors, hormones and other biochemicals. The present test

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must not have any interference from these components. The results of the Cdh-based caffeine

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detection with various milk samples (spiked with caffeine) are shown in Figure 3. For this

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specific test, both INT and NBT were used, assuming that there might be non-specific reduction

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of one or both dyes due to metal ions and other biological components in milk. The Cdh-based

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test scored very well in all the tests. Both dyes were able to differentiate no caffeine (0 ppm),

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safe level (5 ppm) and unsafe levels (>20 ppm) of caffeine (based on FDA regulations) in all

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milk samples, especially mother’s milk. The complex components in the milk did not interfere


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with caffeine detection, and the rapidity of color development. Clearly, the test is suitable to

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determine if the caffeine level is safe before nursing an infant. There is light background color in

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nursing mother’s milk with the INT dye at “0” caffeine (Figure 3). This might be due to very

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low-level reduction of the dye by milk components in this specific sample. Nevertheless, this

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does not interfere with the speed or the ability of Cdh to determine the safe and unsafe levels of

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caffeine in milk.

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DISCUSSION

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Need for a Caffeine Diagnostic Test: Caffeinated drinks and other products are

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growing in number, caffeine content, in the form of energy drinks, and even items such as

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chewing gum and jelly beans that children consume. This unregulated growth of caffeinated

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products has caught FDA attention and has also resulted in a move towards regulating

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caffeinated products.21,22 In addition, FDA has set guidelines for caffeine consumption for

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normal adults and pregnant women;25 guidelines/restriction limits for infants and teenagers is

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currently under consideration by FDA.22 Thus, there is a real need for a rapid diagnostic test at

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the consumer level. Several attempts, using different approaches, have been made to develop

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caffeine detection/diagnostic tests, which is compared in Table 4.

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Caffeine Dehydrogenase, an Ideal Candidate for Caffeine Detection. Caffeine

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dehydrogenase (Cdh, EC number 1.17.5.2; systematic name caffeine:ubiquinone oxidoreductase)

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catalyzes the first step of caffeine degradation via C-8 oxidation pathway.37,42 It is non-NAD

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dependent, but needs stoichiometric amount of an electron acceptor such as nitro blue

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tetrazolium (NBT) or quinone Q0 as electron sink.37 The enzyme is easily purified for caffeine

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detection and potential development of a diagnostic test. The optimized procedure gave a 48.6%


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yield with a specific activity of 210 units/mg of protein. Previously, Cdh was shown to be highly

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specific for caffeine with a Km of 3.7 ± 0.9 µM; 50-fold less preference for the closest analog

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theobromine and very slight or no activity with other di-, mono-methylxanthines, and xanthine.37

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Thus, none of these compounds interfered with the color development with caffeine in one

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minute (data not shown). The enzyme is robust in terms of activity at temperatures ranging from

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15 to 60 °C with linear increase in activity with temperature.37 This makes the enzyme suitable

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for measuring caffeine content in a number of cold and moderately hot beverages. The enzyme

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has activity over a broad pH range (6-8) as previously reported 37 and in the present work with

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commercial beverages in the pH range 3-7. Cdh also has other suitable attributes for

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development of a diagnostic test such as (i) In-situ (on-the-spot) detection, (ii) speed of detection

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is 1 min or less (Figure 2) (iii) easy visualization for both semi-quantitative and qualitative

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detection (Tables 2 and 3), (iv) caffeine sensitivity to the level of 5-20 ppm (FDA safe limit in

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nursing mother’s milk is 5 ppm and decaffeinated beverages have 20 ppm of caffeine).

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Comparison of the New Cdh-based Caffeine Detection with Other Reported

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Caffeine Detection Methods. Detection of caffeine in various laboratories including for quality

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control, is traditionally done by the well-established LC-MS/MS34 or HPLC method.37,39

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However, this method is tedious, instrument intensive, expensive, time consuming, and is not

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practical for a rapid diagnostic test. Various caffeine detection methods/diagnostic tests that

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have been reported are compared in Table 4 with respect to their attributes. These methods

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range from using live bacterial whole-cells,28 enzyme29,30 and antibody-based methods34-36 to

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other non-biological methods like lipid/polymer membranes,31 and fluorescent dyes.32,33 The

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immobilized bacterial cell-based assay is an electrode based amperometric test in which a

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dissolved oxygen (DO) probe measures oxygen depletion as a function of caffeine metabolism.28


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This method has major deficiencies such as bulky electronic instrumentation, low response time

306

(more than 3 min), and sensitivity (>100 ppm). Similarly, the lipid/polymer membrane based

307

method (also known as electronic tongue) is also an electrode based amperometric test.31 This

308

method is rapid (response time less than half a minute), but has other drawbacks as the bacterial

309

cell-based method. None of these tests are commercially available. Recently, two other methods

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were reported based on binding of fluorescent dyes to caffeine on a thin-layer chromatography

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(TLC) plate and visualizing using UV light.32,33 Although, these methods are simple compared

312

to bacterial-cell based method, the test failed to meet the criteria like rapid detection (more than

313

20 min for color development) and sensitivity (cannot detect 3 min

100 ppm

Method of Detection Digital

Commercial Availability NO

Sensitivity

Bacterial-cell based28

NO (DO probe)

Lipid polymer membrane based31

NO (Electrode)

Validation of Caffeine Dehydrogenase from Pseudomonas sp. Strain

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