This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 9639–9641 9639

Cite this: Chem. Commun., 2011, 47, 9639–9641

A molecular probe for the optical detection of biogenic aminesw

Boram Lee, Rosario Scopelliti and Kay Severin*

Received 17th June 2011, Accepted 1st July 2011

DOI: 10.1039/c1cc13604f

A coumarin derivative was employed for the detection of biogenic

amines in buffered aqueous solution by UV-Vis or fluorescence

spectroscopy. Incorporated in a polymeric matrix, the dye can also

be used for the optical detection of gaseous amines.

Biogenic amines (BAs) are low-molecular weight compounds

with at least one primary amine group. They can be formed

during storage and processing of food by thermal or enzymatic

decarboxylation of amino acids.1 Elevated levels of BAs

indicate spoilage of the food. BAs are therefore important

markers for food poisoning.1 BAs also display important

physiological functions. The aliphatic polyamines spermine

and spermidine, for example, are involved in cell proliferation2

whereas histamine plays an important role in gastric secretion

and as a neurotransmitter.3

Numerous methods for the qualitative and quantitative

analysis of BAs have been developed. These include chromato-

graphic techniques such as gas chromatography, capillary

electrophoresis and high-performance liquid chromatography,

as well as enzymatic and immuno-enzymatic methods.4,5 The

direct detection of BAs by optical methods (fluorescence or

UV-Vis spectroscopy) has also been explored. Sensors based

on transition metal complexes6 or supramolecular hydrogels,7 the

pattern-based analysis of amines with cross-reactive polymers8

or sensor arrays,9 and organic dyes which undergo spectral

changes upon reaction with amines in a reversible10 or

irreversible fashion have been reported.11 Below we describe

a new molecular probe which can be used for the optical

detection of BAs in buffered aqueous solution or in the gas

phase. The probe displays a very high degree of sensitivity and

selectivity for BAs in solution-based assays and its synthesis is

straightforward.

The coumarin derivative 1 was prepared by formylation of

7-(N,N-dimethylamino)-4-hydroxycoumarin (2)12 in analogy

to a published procedure (see ESIw).13 The choice of 1 as a

potential optical probe for amines was inspired by work of

Glass, who has shown that coumarin aldehydes such as 3 and

4 are able to form imines in aqueous solutions, although only

at high amine concentrations.14 4-Hydroxycoumarins, on the

other hand, are known to react with amines to give enamines.15

These nucleophilic substitution reactions typically require

forcing conditions (e.g. microwave heating).15b We anticipated

that 1 might display enhanced reactivity towards amines due

to the presence of both aldehyde and hydroxy functional

groups.

The objective of our work was the development of a

molecular probe which could detect amines in aqueous solution.

Since coumarin 1 displays low solubility in pure water, we used

small amounts of the surfactant sodium dodecyl sulfate (SDS)

as an additive. Preliminary tests showed pronounced changes

to both the UV-Vis and fluorescence spectra of a buffered

aqueous solution of coumarin 1 and SDS upon addition of

histamine. Optimization of the assay conditions resulted in the

following protocol: the amine analyte was added to a freshly

prepared solution of 1, SDS and HEPES buffer (final. conc.:

[1] = 10 mM, [amine] = 0.50 mM, [SDS]= 6.0 mM, [HEPES]=

50 mM, pH 7.4). The resulting solution was subsequently

tempered for 2 h at 50 1C and then analyzed by UV-Vis or

fluorescence spectroscopy.

The UV-Vis data for five selected amines (the amino acids

histidine and cysteine and the BAs histamine, cadaverine, and

spermine) are depicted in Fig. 1. Coumarin 1 shows an

absorption band at 451 nm. In the presence of an amine, the

absorption at 451 nm is reduced and a new peak with a

maximum between 377 and 400 nm appears. However, the

changes are different for the five analytes with the most

pronounced changes found for spermine and cadaverine.

Visually, the samples containing the three BAs (nearly colorless)

can clearly be distinguished from the samples containing the

amino acids (yellow).

We have tested several more amines and the changes in

absorption at 403 and 377 nm are depicted in Fig. 2. The

wavelength 403 nm was chosen because this wavelength

represents an apparent isosbestic point for all amines tested

with the exception of the BAs spermine, spermidine, cadaverine,

putrescine, tyramine, and tryptamine. A signal at 403 nm

therefore represents a selective indicator for the presence of

a BA. The toxicologically important BA histamine3,16 is not

detected at 403 nm. However, the low response at 403 nm is

Institut des Sciences et Ingenierie Chimiques, Ecole PolytechniqueFederale de Lausanne (EPFL), Lausanne, Switzerland.E-mail: kay.severin@epfl.ch; Fax: +41(0)21 6939305;Tel: +41(0)21 6939302w Electronic supplementary information (ESI) available. CCDC824329. For ESI and crystallographic data in CIF or other electronicformat see DOI: 10.1039/c1cc13604f

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9640 Chem. Commun., 2011, 47, 9639–9641 This journal is c The Royal Society of Chemistry 2011

misleading because histamine leads to rather pronounced

changes in the UV-Vis spectrum (Fig. 1). This fact is evident

when the absorption changes at 377 nm are compared. Here,

the response of histamine is as good as that of cadaverine and

putrescine and only slightly lower than what is found for

spermine and spermidine (Fig. 2). At 377 nm, the selectivity

for BAs (shown in red) over other amines (shown in blue) is not

as good as what was observed at 403 nm, but still remarkable.

Similar results are obtained when fluorescence spectroscopy

is used as the method of analysis. Excitation at 377 nm leads to

a fluorescence signal at around 470 nm, the precise maximum

being dependent on the nature of the analyte (see ESIw, Fig. S1).For samples containing BAs (0.5 mM) one can observe a

pronounced increase in fluorescence with IA/I0 values of up to

54 (spermine). Again, there is a very good selectivity for BAs

over other amines (see ESIw, Fig. S2). The only exception is

tryptamine, for which a weak fluorescence response was

observed.

A series of experiment was performed to obtain information

about the chemistry behind the optical response of molecular

probe 1. When the coumarin dyes 2 and 3 were used instead

of 1 for sensing experiments with histamine (0.5 mM), no

significant change in color was observed after 2 h at 50 1C.

These results provide evidence that both the hydroxy and

aldehyde functionalities in 1 are necessary for the optical

response.

Plausible products of the reaction of 1 with primary amines

include imine A and its tautomeric keto form,17 enamine B,

and the double condensation product C. When a solution of

1 (4.2 mM) in CD3OD/CDCl3 (7 : 3) was allowed to react with

i-butylamine (16.8 mM) for 10 min, two main products were

observed by 1H NMR spectroscopy. These products were

found to be the enamine B and the double-adduct C as

evidenced by comparison of the spectra of independently

synthesized samples (see ESIw, Fig. S4). In addition, the

structure of C was confirmed by a crystallographic analysis

(see ESIw). Similar results were obtained for in situ NMR

experiments with 1 and histamine. However, it should be

emphasized that the experimental conditions of the NMR

experiments are very different from the sensing condition.

For the former, we have used 4.2 mM of dye 1 in a mixture

of organic solvents, whereas the UV-Vis studies were per-

formed with 10 mM of 1 in aqueous solutions containing SDS

micelles.

The NMR experiments indicated that enamine formation by

nucleophilic substitution of the hydroxy group at the 4 posi-

tion occurred rapidly. To examine the relevance of enamine

formation under sensing conditions, we have prepared a

coumarin of type B with histamine. The compound was then

dissolved in HEPES buffer containing SDS and a UV-Vis

spectrum was recorded. The resulting spectrum was very

similar to what was obtained for reactions of 1 with histamine

(see ESIw, Fig. S6). Furthermore, the spectrum did not change

with time indicating that enamine formation is irreversible.

We therefore conclude that enamine formation is likely a

key factor for the optical response of probe 1. However, the

UV-Vis spectra also show that other products are formed, at

least in the case of some of the BAs. The additional formation

of imines such as A and C, which are stabilized by the SDS

micelles, seems plausible, but more complex reactions as

observed for other coumarins18 cannot be excluded. The

precise reason for the apparent kinetic selectivity for BAs over

amino acids and simple primary amines is presently not clear.

A plausible reaction mechanism involves the reversible forma-

tion of the condensation product A (or its tautomeric form),

which facilitate the irreversible formation of the enamine. In

the case of BAs, the nucleophilic attack of the amine could

Fig. 1 Absorption spectra of buffered aqueous solutions (50 mM

HEPES, pH 7.4) containing molecular probe 1 (10 mM), SDS

(6.0 mM) and 0.50 mM of histidine (hashed blue line), cysteine (dotted

blue line), histamine (solid red line), spermine (hashed red line),

cadaverine (dotted red line), or no amine (solid blue line). Prior to

the measurement, the solutions were tempered for 2 h at 50 1C.

Fig. 2 Changes of the absorption at 403 nm (top) and 377 nm

(bottom) of buffered aqueous solutions (50 mM HEPES, pH 7.4)

containing molecular probe 1 (10 mM) and SDS (6.0 mM) after

addition of different amines (0.5 mM). Prior to the measurement,

the solutions were tempered for 2 h at 50 1C. Important biogenic

amines are shown in red and other amines in blue.

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This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 9639–9641 9641

occur in an intramolecular fashion or could be assisted by the

basic BA side chain.

Probe 1 can also be used for quantitative measurements.

With a dye concentration of 10 mM and an equilibration time

of 2 h at 50 1C, we observed a steady increase in absorbance at

377 nm for histamine concentrations between 0.1 and 1.0 mM

(see ESIw, Fig. S7). The data could be used as a calibration

curve for quantitative analyses in this concentration range.

The advantage of a kinetically controlled sensor response is

the fact that the dynamic range can be adjusted by variation of

the equilibration conditions (time, temperature). It was thus

possible to increase the sensitivity of the sensor by extension of

the equilibration time from 2 to 6 h. Under these conditions,

quantitative histamine measurements in the low micromolar con-

centration range are possible (see ESIw, Fig. S8). Obviously,

such analyses would only work for samples containing no

other BAs. The selectivity for histamine over a simple primary

amine such as n-propylamine is reasonably good: the initial

rates for these two analytes differ by more than one order of

magnitude. It should also be noted that the reaction rates are

pH dependent: a more basic pH results in faster reactions

(see ESIw, Fig. S9). Precise pH control with a buffer is therefore

of importance for quantitative measurements.

We also examined whether coumarin 1 could be employed

for the sensing of amines in the gas phase. For this purpose,

glass sides with a poly(methyl methacrylate) layer containing

5 mol% of dye 1 were prepared by drop coating. When the

polymeric matrix was subjected to vapors of the biogenic

amine putrescine or the primary amine n-butylamine, the

yellow color of the polymer layer rapidly faded away. On

the other hand, no substantial color change was observed in

the case of the secondary amine diethylamine or ammonia (see

ESIw, Fig. S10). Again, the sensor response is cumulative and

irreversible (the system behaves as a ‘chemodosimeter’), which

could be advantageous from an application point of view.6a

In conclusion, we have shown that coumarin 1 can be used

for the selective detection of amines in buffered aqueous

solution by UV-Vis or fluorescence spectroscopy. The sensing

system displays a pronounced selectivity for important

biogenic amines. A unique feature of coumarin 1 is the fact that

a covalent connection to the amine analyte is achieved under

mild conditions in dilute aqueous solution. Consequently, it is

possible to detect biogenic amines in the micromolar concentra-

tion range. When embedded in a polymer matrix, dye 1 can also

be employed for the optical detection of amines in the gas phase.

This work was supported by the Swiss State Secretariat for

Education and Research, by the COST action CM0703, and

by the EPFL.

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