A biosensing method for detection of caffeine incoffeeAndrea Pizzariello, Jozef Svorc, Miroslav Stred’ansky and Stanislav Miertus*POLYtech, Soc. Coop. r.l., Padriciano, 99, I-34012 Trieste, Italy

Abstract: A speci®c inhibition of 3',5'-cyclic phosphodiesterase (CPDE) from bovine heart by methyl-

xanthines was used in combination with a pH electrode to develop a new biosensing method for the

detection of caffeine in coffee. The potential response changes of the sensor were proportional to the

concentration of caffeine in the range 0±4mgmlÿ1. The response time was about 2±4min. The standard

deviation of ®ve measurements of a 0.2mgmlÿ1 caffeine solution was�7.1mgmlÿ1. The electrode gave a

detection limit of 0.6mglÿ1 caffeine. The concentration of caffeine in espresso coffee was analysed.

This model gave excellent correlation between observed and predicted caffeine values. This electrode

exhibits advantages such as fast response, short conditioning time and low cost of the instrumentation

used. We also expected to be able to perform the detection of caffeine in food and clinical analysis.

# 1999 Society of Chemical Industry

Keywords: sensor; caffeine; determination; cyclic nucleotide phosphodiesterase; coffee

INTRODUCTIONCaffeine, 1,3,7-trimethylxanthine, is a natural alkaloid

which is present, together with other trace purines, in

coffee1 and also in tea and soft drinks. Coffee is one of

the most popular drinks across the world and its

enormous commercial and social importance is ob-

vious. The total annual world production of green

coffee is about six million tons,2 with an estimated cost

of six millard US dollars. The roasting of the green

coffee beans yields the commercial coffee which is the

basis of the worldwide bitter and dark coffee bev-

erages, such as espresso. Caffeine contributes to a

particular proportion of the perceived bitterness of a

cup of coffee and also makes a small contribution to

espresso's strength and body.3 Moreover, the impor-

tance of the mild stimulating properties of caffeine

should also not be neglected: the preference between

higher and lower levels of stimulation from caffeine

may in practice dictate the blend consumed, therefore

its taste. Because of the relatively important role of

caffeine in determining the quality of coffee beverages,

the development of a sensitive, fast and cost-effective

method for monitoring caffeine is greatly needed.

Normally, high-performance liquid chromatogra-

phy separation4±8 and UV spectrophotometric detec-

tion9,10 methods are applied to both regular and

decaffeinated green and roasted coffees for caffeine

content determinations. Also, other methods such as

capillary electrophoresis,11±14 thin layer chromatogra-

phy15 and gas chromatography,16 are used for separa-

tion of caffeine in the analysis of mixtures, combined

with several other detection methods such as mass

spectroscopy16 and FTIR spectrophotometric

measurements.17±19 However, very costly instrumen-

tation, highly skilled technicians and complicated and

time-consuming procedures are required for such

methods.

Another possible technique is ¯ow injection

immunoassay using a solid phase reactor, which makes

the assay faster because no separation step is needed.20

However, the time and cost for monoclonal antibody

production and puri®cation, and the need for their

manipulation with extreme care, make this approach

not so easy to perform.

The development and application of new caffeine

detection methods remain an active area of investiga-

tion, particularly in food and clinical chemistry.

Signi®cant research and development activity has

been devoted to preparing compact analytical devices

comprising a bioactive sensing element integrated with

a suitable transducting system, also known as biosen-

sors,21 for determination of various inorganic, organic

and biological substances. The main advantages of

these devices are their speci®city, sensitivity and

simple preparation, and the fact that no other reagents

besides a buffer and a standard are usually required.

Here we report the construction and properties of a

new, simple, speci®c, rapid and low-cost caffeine

biosensing system that consists of a glass membrane

potentiometric pH-sensitive electrode with phospho-

diesterase 3',5'-cyclic nucleotide and calmodulin

(CPDE±CM) as add-on reagents. The concept of

Journal of the Science of Food and Agriculture J Sci Food Agric 79:1136±1140 (1999)

* Correspondence to: S Miertus, ICS, Unido, Padriciano, 99, I-34012 Trieste, ItalyE-mail: miertus@ics.trieste.itContract/grant sponsor: ILLYCAFFE’ SpA, Trieste(Received 24 December 1997; revised version 12 November 1998; accepted 26 January 1999)

# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50 1136

the caffeine sensor is based on the hydrolysis of

adenosine 3',5'-cyclic monophosphate (c-AMP) to

adenosine 5'-monophosphate (AMP) and H3O�

through the effect of CPDE in the presence of the

protein activator (CM) and Ca2�:

c-AMP�H2Oÿÿÿÿÿÿÿÿ!CPDE

CM;Ca2�AMP�H3O�

When this enzymatic reaction is inhibited by

caffeine,22 the H3O� production, monitored by a

potentiometric pH probe, should be reduced in an

inversely proportional way with respect to the caffeine

concentration.

MATERIALS AND METHODSApparatusA glass membrane pH electrode (Radiometer model

PHI 2401) was used as the basis of the caffeine sensor.

The electrode was connected with a pH meter

(Radiometer model PHM 85). The sensor output

was registered on a Y±t paper recorder (AMEL model

868).

A Varian Cary-3E UV-vis spectrophotometer was

employed to obtain the UV-vis spectra. All measure-

ments were carried out at room temperature in 5 or

10ml beakers with magnetic stirring.

ReagentsAdenosine 3',5'-cyclic monophosphate (cod.n.

A9501), phosphodiesterase 3',5'-cyclic nucleotide

(EC 3.1.4.17) crude complex from bovine heart

(cod.n. P 0134), calmodulin (phosphodiesterase

3',5'-cyclic nucleotide activator), phosphodiesterase

I-type VII (EC 3.1.4.1) from Crotalus atrox venom

(cod.n. P 6761), caffeine, theophylline and theo-

bromine were obtained from Sigma Chemical Co, St

Louis, MO, USA; tris(hydroxymethyl)aminomethane

hydrochloride (TRIS-HCl) was obtained from Fluka

Chemie AG, Buchs, Switzerland; other analytical

grade reagents were commercially available. Milli-Q

(18.5Mcm) water was used throughout.

Phosphodiesterase assay and protein determinationCommercial CPDE titre was assayed by measuring the

release of inorganic phosphate with use of an excess of

5'-nucleotidase.23,24 The reaction mixture contained

0.36mmoles of cyclic-3',5'-AMP, 1.8mmoles of

MgSO4 and 36mmoles of TRIS-HCl buffer, pH 7.5,

with a suitable dilution of the phosphodiesterase

sample being tested in a total volume of 0.9ml. This

mixture was incubated at 30°C for 30min. After the

®rst 20min of incubation, 0.1ml of phosphodiesterase

I-type VII from Crotalus atrox venom solution was

added, containing 0.02U of 5'-exonuclease in 10mM

TRIS-HCl, pH 7.5. The entire reaction was termi-

nated by addition of cold 550gkgÿ1 trichloroacetic

acid. The resulting precipitate was removed by

centrifugation, and aliquots of the supernatant ¯uid

were analysed for inorganic phosphate by the following

method: 0.1ml of 25gkgÿ1 ammonium molybdate in

5N H2SO4 was added to a 0.5ml aliquot of the

supernatant solution and 0.35ml of distilled water.

The colour was read at 660nm. One unit of CPDE

enzyme was de®ned as the amount of the enzyme that

hydrolyses 1.0mmole of c-AMP to AMP per minute at

pH 7.5 at 30°C. Protein content was determined by

the Bradford Bio-Rad protein assay using serum

albumin as a standard.25

Determination of caffeine in standard solutionThe determination of caffeine by the sensor was

carried out at room temperature under a nitrogen

atmosphere in a jacketed reaction vessel equipped with

magnetic stirring. The glass membrane electrode was

immersed in stirred water solution (5ml) supple-

mented with CaCl2 (0.01mM) and calmodulin

(10Umlÿ1). The substrate (c-AMP) was added to

the system (®nal concentration 0.2 or 1mM) and the

pH was adjusted to 8.00 by addition of 0.2M TRIS-

HCl buffer, pH 9. After signal stabilisation the

hydrolysis was started by addition of CPDE (®nal

concentration 0.0424Umlÿ1). After the electrode

response became constant (3±5min), the sensor

calibrations were conducted in the open cell with

intensive magnetic stirring at room temperature. The

caffeine concentration was increased stepwise by

adding de®ned volumes of a 20mgmlÿ1 stock solu-

tion. Potential difference DE vs time curves were

recorded. The decrease in the recorded line slope was

proportional to the caffeine concentration.

Determination of caffeine in espresso coffeeTo measure the caffeine content of an espresso coffee,

5ml of water supplemented with CaCl2 (0.01mM),

calmodulin (10Umlÿ1) and c-AMP (®nal concentra-

tion 0.2 or 1mM) was placed in a 10ml beaker and the

pH was adjusted to 8.00 by addition of 0.2M TRIS-

HCl buffer, pH 9, then 10ml of ¯occulated espresso

coffee was added. Typical response times ranged from

60 to 120s. Complete re-establishment of the original

electrode potential was accomplished in less than

1min by replacing the electrode in caffeine-free water

supplemented as above for a new coffee sample

analysis. Caffeine concentrations were derived from

the calibration plot.

The ¯occulation procedure was based on the

treatment of 5ml of crude espresso coffee with 500ml

of a solution of potassium hexacyanoferrate(II) tri-

hydrate (0.150gmlÿ1 K4[Fe(CN)6].3H2O) ®rst, then

500ml of a solution of zinc chloride anhydrate

(0.1422gmlÿ1 ZnCl2) was added. The suspension

was mixed and ®nally ®ltered.

RESULTS AND DISCUSSIONpH dependence and buffer capacityThe hydrolysis of c-AMP to AMP at pH values

between 7 and 8 produces a change in pH during the

J Sci Food Agric 79:1136±1140 (1999) 1137

Detection of caffeine in coffee

reaction. In this context we have evaluated the effect of

CPDE-substrate concentration (c-AMP) on the rate

of hydrolysis. We have found that with increasing

c-AMP concentration the rate of hydrolysis decreases.

Since in our system the detected species are H3O�

ions, their sensitivity depends strongly on buffer

capacity.

To adjust the pH to the optimal value for CPDE

(pH 8.0), it is necessary to add an alkaline buffer

solution (0.2M TRIS-HCl pH 9.0). If the concentra-

tion of c-AMP increases, more buffer is necessary and

the buffer capacity increases as well. Therefore a lower

sensitivity at higher substrate concentration is ob-

served.

Amount of enzyme and substrate (c-AMP)concentrationIn Fig 1 the effect of CPDE activity on the rate of

hydrolysis at constant concentration of c-AMP (1mM)

is shown. In this case the rate of hydrolysis (expressed

as the change in potential, DEdtÿ1) is a linear function

of CPDE activity.

The in¯uence of c-AMP concentration on the

sensitivity of the caffeine sensor is also due to the fact

that CPDE inhibition by methylxanthine derivatives is

apparently competitive.23 Thus a c-AMP concentra-

tion increase should cause of an additional lowering of

the sensitivity.

Effect of calmodulin (CM)The CPDE activator CM played a most important role

in the performance of the sensor. The addition of

10Umlÿ1 of CM to the assay solution resulted in a

more than threefold increase in the hydrolytic activity

of CPDE, although the enzyme used contained

`saturated activity' (declared by the supplier) of the

activator.

Analytical characteristics of the sensorThe potentiometric response of the caffeine sensor

with CPDE and CM in solution was examined using

caffeine standard solutions or directly with the solid

substance dissolved in the assay solution. The poten-

tial (E) was recorded as a function of time (t) and the

slope of the experimental curve is proportional to the

rate of c-AMP hydrolysis.

Fig 2 shows the calibration graph which was

constructed by plotting the steady state values of

DEdtÿ1 against the caffeine concentration. In addition,

Fig 3 shows another calibration plot obtained for a

higher concentration of c-AMP (1mM). While at a

c-AMP concentration of 1mM the dependence is

linear up to 4mgmlÿ1 of caffeine, at a c-AMP

concentration of 0.2mM the dependence is not linear

but the sensitivity is signi®cantly higher. Although at

the c-AMP concentration of 1mM the sensitivity is

decidedly lower, the range of linearity is doubled,

making this sensor useful for determining caffeine in

the range of 0±4mgmlÿ1.

The potentiometric response of the sensor was

reproducible. The standard deviation for ®ve measure-

Figure 1. Influence of CPDE activity on rate of c-AMP hydrolysis (DEdtÿ1)for 1mM c-AMP (points are means of five replications for eachconcentration; related SDs are in range �3.3%).

Figure 2. Influence of caffeine concentration on rate of c-AMP hydrolysis(DEdtÿ1) for 0.2mM c-AMP and 0.0424Umlÿ1 CPDE activity (points aremeans of five replications for each concentration; related SDs are in range�3.7%).

Figure 3. Influence of caffeine concentration on rate of c-AMP hydrolysis(DEdtÿ1) for 1.0mM c-AMP and 0.0424Umlÿ1 CPDE activity (points aremeans of five replications for each concentration; related SDs are in range�3.5%).

1138 J Sci Food Agric 79:1136±1140 (1999)

A Pizzariello et al

ments of a 0.2mgmlÿ1 caffeine solution (at pH 8.00)

was �7.1mgmlÿ1. The estimated detection limit was

0.6mglÿ1.

InterferencesThe inhibition of CPDE by theophylline, theobromine

and caffeine has been described in the literature.26±29

We tested the effect of theophylline and theobromine

on the rate of c-AMP hydrolysis. We have found

inhibition of c-AMP hydrolysis by both methyl-

xanthines. Fig 4 shows the relative inhibition of CPDE

hydrolytic activity as a function of theophylline,

caffeine and theobromine concentrations. At 50%

inhibition, theophylline was 16.7- and 10-fold more

potent than theobromine and caffeine respectively. In

any case, theophylline and theobromine are present

only in trace amounts, in comparison with caffeine, in

coffee products,30 so the present bioanalytical proce-

dure is applicable for caffeine content determination in

coffee beverages without signi®cant interferences.

Measurements of real samplesThe sensor was also applied for the direct determina-

tion of caffeine in real samples. Typical response

curves are shown in Fig 5, directly obtained with

espresso coffee samples.

Espresso coffee is served in a small, heavy china cup

with a capacity of 30±50ml, half-full with a dark brew

topped by a thick, clean, light reddish-brown foam of

tiny bubbles. The liquid part of the espresso is a very

complex matrix. It is in fact a concentrated solution of

salts, acids, sugars, caffeine and many other complex

substances, forming the matrix wherein three dis-

persed phases coexist (emulsion, suspension and gas

bubbles).

In our enzyme pH-based caffeine sensor, biospeci-

®city is obviously de®ned by use of CPDE, while the

electrochemical selectivity to caffeine and non-speci®c

buffering agents has to be dealt with by removal of

interfering components by gravity sedimentation,

aided by chemical ¯occulating agents. Thus, in the

sensor system presented in this paper, the major

contribution of the endogenous interferences can be

preliminarily eliminated by a simple and fast ¯occula-

tion procedure of the coffee samples. The ¯occulation

procedure leads to aggregation of large molecules or

colloidal particles, suspended in the coffee sample,

into clusters or clumps. When this occurs, by addition

of the appropriate coagulant agent, the aggregates

precipitate or separate from the dissolved state, with

complete removal of total particulate, colour and

turbidity, and thus major interfering components are

eliminated.31

The time of a single analysis in this way is as short as

6±8min, which can be considered the minimum time

attainable with a method involving also ¯occulation

pre-treatment. It is possible to perform about 10 assays

per hour. Table 1 reports a series of sample analyses

from three different kinds of coffee. The same samples

were also analysed by an HPLC method (De Palo,

unpublished). The results give a correlation plot y(caffeine sensor determination)=1.159x (HPLC

determination)ÿ0.167, with a coef®cient of correla-

tion of 0.999. This indicates good agreement between

the two methods.

CONCLUSIONSThese preliminary results demonstrate that a potentio-

Figure 4. Relative potencies of methylxanthine inhibition of c-AMPhydrolysis for 0.2mM c-AMP and 0.0424Umlÿ1 CPDE activity.

Figure 5. Profiles for potential response of caffeine electrode exposed toflocculated espresso coffee samples: (b1) reference baseline; (a) 10mlflocculated water; (b) 10ml decaffeinated coffee; (c) 10ml arabica coffee;(d) 10ml robusta coffee. Solution: 0.2mM c-AMP, 10Umlÿ1 calmodulin,0.32mM TRIS-HCl, pH 8.0.

Table 1. Analysis of espresso coffee samples by an HPLCmethod and by the caffeine sensor (mean�SD, n=3)

Caffeine (mgmlÿ1)

Coffee HPLC method a Caffeine sensor

Decaffeinated 0.35�0.05 0.27�0.04

Arabica 1.41�0.07 1.0�0.06

Robusta 2.32�0.15 2.56�0.12

a Performed at the Standard Chemical Laboratory for the

Technology of Commerce, Trieste, Italy.

J Sci Food Agric 79:1136±1140 (1999) 1139

Detection of caffeine in coffee

metric enzyme sensor can be used successfully for the

direct detection of caffeine in coffee beverage samples.

The system described here does not require any

sophisticated equipment, being based on a pH meter

with a glass membrane electrode. Up to 10 samples

can be analysed per hour. Thus the sensor system is

obtained with minimum instrumental cost and maxi-

mum speed of analysis. The manufacture of a sensor

with immobilised enzyme on the electrode surface and

the evaluation of its performance are now in progress.

ACKNOWLEDGEMENTSThis work has been carried out within a project

supported by ILLYCAFFE' SpA. (Trieste, Italy). We

also thank E Illy and M Petracco from ILLYCAFFE'

SpA for valuable discussion and scienti®c support.

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