Analysis of pathogenic bacteria using exogenous volatile organic compound metabolites and optical sensor detection

Article abstract of DOI:10.1039/c4ra13914c

A novel, low-cost and simple method for the detection of pathogenic bacteria is proposed. The approach is based on the generation of an exogenous volatile organic compound (VOC) produced by the addition of an enzyme substrate to the bacterial sample. The

Full text of DOI:10.1039/c4ra13914c

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Analysis of pathogenic bacteria using exogenous volatile organic
compound metabolites and optical sensor detection
#
#
#
Emma Tait , Stephen P. Stanforth , Stephen Reed , John D. Perry* and John R.
#~
Dean
#
Department of Applied Sciences, Northumbria University, Ellison Building,
Newcastle upon Tyne, NE1 8ST, UK.
*
Department of Microbiology, Freeman Hospital, Newcastle upon Tyne, NE7 7DN,
UK.
~
corresponding author. Tel. No.: +44 191 227 3047; Fax no.: +44 191 227 3519.
Email: John.Dean@northumbria.ac.uk
Abstract
A novel, lowꢀcost and simple method for the detection of pathogenic bacteria is
proposed. The approach is based on the generation of an exogenous volatile organic
compound (VOC) produced by the addition of an enzyme substrate to the bacterial
sample. The generated VOC is then trapped in agarose gel allowing colour
development to take place; visual detection is then possible by both the naked eye
and by colorimetric analysis. Agarose gel has been evaluated as both a suitable
VOC trapping matrix and host for the colourꢀgenerating reagents. This proof of
concept method allowed for the discrimination between βꢀglucosidase and βꢀalanyl
aminopeptidase producing bacteria. Enterococcus faecium and Klebsiella
pneumoniae are both βꢀglucosidase producers and generated a yellow colour within
agarose gels upon enzymatic hydrolysis of 2ꢀnitrophenylꢀβꢀ
Dꢀglucoside.
Pseudomonas aeruginosa is a known βꢀalanyl aminopeptidase producer and was
1

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shown to hydrolyse the trifluoroacetic acid (TFA) salt of 3ꢀaminoꢀNꢀ
phenylpropanamide resulting in the development of an orange colour within agarose
gels spiked with the sodium salt of 1, 2ꢀnaphthoquinoneꢀ4ꢀsulfonic acid. 3ꢀAminoꢀNꢀ
phenylpropanamide (as its TFA salt) and 2ꢀnitrophenylꢀβꢀDꢀglucoside concentrations
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
of 20 ꢁg mL (or 72 ꢁmol L ) and 100 ꢁg mL (or 332 ꢁmol L ), respectively were
the minimum quantities required for colour production following 18 h of incubation.
The use of 3ꢀaminoꢀNꢀphenylpropanamide, TFA salt indicated that synthesised
enzyme substrates can be tailorꢀmade to liberate exogenous VOCs for colour
generation.
1. Introduction
The detection of pathogenic bacteria in clinical and food samples can often be a
timeꢀconsuming and laborious process. The rapid identification of bacteria is
essential for effective patient treatment in clinical settings and for determining the
source of contamination in food samples. Bacteria have been shown to liberate a
1,2
wide range of volatile organic compounds (VOCs). Several analytical methods
which have focussed on the detection of VOCs liberated by bacteria have been
developed. Detection of bacterial VOCs using headspace solid phase
microextraction (SPME) coupled with gas chromatography – mass spectrometry
3
(
GCꢀMS) has shown potential as a diagnostic tool for bacterial detection. Other
analytical methods used for bacterial VOC analysis include multiꢀcapillary column–
4
ion mobility spectrometry and secondary electrospray ionizationꢀmass
5
spectrometry. However, clinical and food laboratories are unlikely to possess such
instruments for VOC analysis; in addition, they are expensive to purchase, require
trained laboratory staff to operate them and regular maintenance. Alternative VOC
2

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detection methods that do not require instrumentation would be highly desirable.
Visual VOC detection methods have recently been developed. For example, a
disposable colorimetric sensing array to identify 10 strains of bacteria has been
6
developed. Dyes incorporated into the sensor changed colour upon exposure to
VOCs generated by bacteria. The use of enzyme substrates as a potential tool for
bacterial detection, via exogenous VOC evolution, has previously been described.
E. coli was detected via the generation of the exogenous 2ꢀnitrophenol using the
7
enzyme substrate 2ꢀnitrophenylꢀβꢀDꢀgalactoside; ion mobility spectrometry was used
for VOC detection. Subsequently xerogels have been used to trap and
colorimetrically detect VOCs, specifically 4ꢀnitrophenol released by enzymatic
8
hydrolysis of 4ꢀnitrophenylꢀβꢀ
D
ꢀglucuronide and pꢀdimethylaminocinnamaldehyde
9
for the detection of E. coli (a known indole producer). In the case of the latter
example, pꢀdimethylaminocinnamaldehyde reacted with indole to generate a green
colour within the xerogel.
Agarose is a polysaccharide with repeating units of
Dꢀgalactose and 3,6ꢀanhydroꢀLꢀ
galactose and upon gelation it forms a porous semiꢀsolid matrix. Agarose gels are
commonly used for separating molecules during electrophoresis; however, here it is
used as a potential matrix for VOC trapping. Agarose gel is inexpensive and its
preparation is simple. In addition, it is possible to modify the agarose gel by the
addition of specific and selective colorimetric reagents that will generate colour in the
presence of target compounds. The aim of this paper is to develop a novel, simple
optical method for bacterial identification via the detection of exogenous VOCs.
Following bacterial enzyme activity, the exogenous VOCs are trapped in modified
agarose gel and optically detected by either the naked eye or colorimetric analysis;
the latter allows quantification of the exogenous VOC. The proof of concept is
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demonstrated using two enzyme substrates: the commercially available 2ꢀ
nitrophenylꢀβꢀ
Dꢀglucoside which liberates 2ꢀnitrophenol in the presence of bacteria
with βꢀglucosidase activity and a synthesised substrate, 3ꢀaminoꢀNꢀ
phenylpropanamide, TFA salt which liberates aniline in the presence of bacteria with
βꢀalanine aminopeptidase activity. The 2ꢀnitrophenol liberated from 2ꢀnitrophenylꢀβꢀ
Dꢀglucoside is yellow coloured in alkaline conditions whereas the aniline released
from a substrate targeting βꢀalanine aminopeptidase activity can be optically
detected by its reaction with 1,2ꢀnaphthoquinoneꢀ4ꢀsulfonic acid, sodium salt (NQS)
giving a red colour. NQS has been used for the colorimetric detection of amines in
10
11
solution and in the vapour phase.
βꢀGlucosidase substrates are widely used in chromogenic media; βꢀglucosidase
activity has been demonstrated in several species, including the Gramꢀpositive
organism Enterococcus faecium and the Gramꢀnegative species Klebsiella
12
pneumoniae. Pseudomonas aeruginosa is a known βꢀalanine aminopeptidase
producer and enzyme substrates targeting βꢀalanine aminopeptidase activity have
been synthesised and successfully used in chromogenic media for the detection of
12,13
Ps. aeruginosa.
Pathogenic bacteria known to produce βꢀglucosidase and βꢀ
alanine aminopeptidase activity were selected to demonstrate this proof of concept,
illustrating the use of exogenous VOC detection, via modified agarose gels, as a
novel method of bacterial identification based on optical sensor detection. This novel
approach for identifying or detecting bacteria, via the generation of exogenous
VOCs, allows for labelꢀfree, nonꢀinvasive and continuous monitoring of a sample.
2. Experimental
2.1 Chemicals/reagents
4

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t
2
ꢀNitrophenol (98 %), aniline (≥99 %), Nꢀmethylꢀ2ꢀpyrrolidinone (99 %), bocꢀβꢀ
alanineꢀOH (≥99.0 %), Nꢀhydroxysuccinimide (98 %) and trifluoroacetic acid (99 %)
were purchased from Sigma Aldrich (Poole, UK). 2ꢀNitrophenylꢀβꢀDꢀglucoside was
obtained from Apollo Scientific (Stockport, UK). Dicyclohexylcarbodiimide (99 %)
and 1,2ꢀnaphthoquinoneꢀ4ꢀsulfonic acid sodium salt (NQS) (97 %) were purchased
from Alfa Aesar (Morecambe, UK). Sodium hydroxide (98.0 %) was obtained from
BDH Laboratory Supplies (Lutterworth, UK) and agarose (molecular biology grade)
from Melford Laboratories Ltd. (Ipswich, UK). All solvents (dichloromethane, hexane
and ethyl acetate) were of analytical reagent grade and purchased from Fisher
Scientific (Loughborough, UK). Brain heart infusion (BHI) broth and agar were
purchased from Oxoid (Basingstoke, UK).
2.2 Microbiology
Gramꢀnegative and Gramꢀpositive bacteria were obtained from the National
Collection of Type Cultures (NCTC), Colindale, UK. Gramꢀnegative bacteria:
Escherichia coli NCTC 10418, Klebsiella pneumoniae NCTC 9528, Pseudomonas
aeruginosa NCTC 10662 and Pseudomonas fluorescens NCTC 10688. Gramꢀ
positive bacteria: Enterococcus faecium NCTC 7171 and Streptococcus agalactiae
NCTC 8181. Bacteria were stored and subꢀcultured weekly on BHI agar.
2.3 Gel preparation for the detection of 2ꢀnitrophenol
Agarose gel (1 %) was prepared by dissolving 0.5 g agarose in distilled water and
microwaving until boiling (approximately 1 min). The solution was allowed to cool
ꢀ1
and then modified by addition of 0.2 mol L NaOH; the final total volume was 50 mL.
ꢀ
1
The final NaOH concentration was 2 mmol L . A subꢀsample aliquot of the NaOH
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modifiedꢀagarose gel (150 ꢁL) was added to a cuvette within a vial and allowed to
set.
2.4 Gel preparation for the detection of aniline
A 0.5 % solution of NQS was prepared by dissolving 0.025 g in 5 mL of distilled
water. This reagent was prepared fresh daily. Agarose gel (1 %) was prepared as
described in section 2.3. To 1 % agarose gel, 0.25 mL of 0.5 % NQS was added to
give a total volume of 50 mL. A subꢀsample aliquot of the NQS modified agarose gel
(150 ꢁL) was added to a cuvette within a separate vial and allowed to set.
For each gel type, a 1 mL total volume of sample in BHI media was added
aseptically to a 1.5 mL Eppendorf tube within a sterilised vial. The BHI media was
made up according to manufacturer’s guidelines. Sterilisation of media was
achieved by autoclaving at 121 °C for 11 minutes. Vials and cuvettes were sterilised
by exposure to UV radiation for 90 mins in a UV cabinet. The vial with sample was
sealed and placed overnight in a 37 °C incubator. The absorbance of the gel in the
cuvette was measured at the experimentally determined wavelengths of 415 nm for
2ꢀnitrophenol and 470 nm for the coloured complex formed by aniline / NQS,
compared against a reagent blank after 18 h incubation.
2.5 Optimisation of parameters
Aniline and 2ꢀnitrophenol stock solutions were prepared in Nꢀmethylꢀ2ꢀpyrrolidinone.
ꢀ
1
The optimum percentage agarose in gels was determined using 50 ꢁg mL of 2ꢀ
ꢀ
1
ꢀ1
ꢀ1
nitrophenol (or 359 ꢁmol L ) and 20 ꢁg mL (or 215 ꢁmol L ) of aniline in 1 mL of
BHI media. The optimum volume of gel and NaOH concentration in gel were
ꢀ
1
determined with 50 ꢁg mL 2ꢀnitrophenol in 1 mL BHI media following overnight
6

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incubation at 37 °C. [NOTE: As the yellow colour is generated by the ionic form i.e.
ꢀnitrophenolate (pKa of 2ꢀnitrophenol is 7.23) it was necessary to increase the pH
with NaOH) to generate more of the anion; this resulted in a higher molar extinction
coefficient in the presence of NaOH and a more intense colour]. Similarly, optimum
2
(
ꢀ1
NQS and NaOH concentrations in gel were determined with 20 ꢁg mL aniline in 1
mL BHI media. Absorption spectra of gels exposed to 2ꢀnitrophenol and aniline from
300 nm to 700 nm were produced and maximum absorption wavelengths for both
VOCs were determined. Calibration curves for both VOCs were produced using a
VOC concentration range in BHI media and measuring absorbance after 18 h
incubation at 37 °C. Absorbance readings below 0.03 were omitted. An absorption
spectrum of NQS measured against agarose gel with no reagents added was also
produced. A Pharmacia Biotech Ultraspec 2000 UV/visible spectrophotometer was
used for measuring the absorbance of samples and blanks.
2.6 Synthesis of 3ꢀaminoꢀNꢀphenylpropanamide, TFA salt
t
3
ꢀAminoꢀNꢀphenylpropanamide, TFA salt was synthesised using bocꢀβꢀalanineꢀOH
t
as a starting material. To a stirred solution of bocꢀβꢀalanineꢀOH (0.50 g, 2.6 mmol)
and Nꢀhydroxysuccinimide (0.30 g, 2.6 mmol) in dry dichloromethane (DCM) (10 mL)
at room temperature with a CaCl trap was added dicyclohexylcarbodiimide (DCCI)
2
(0.55 g, 2.6 mmol). The solution was stirred at room temperature for 1.5 h. To dry
DCM (10 ml) was added aniline (0.24 g, 2.6 mmol) and to this was added the
contents of the other flask slowly over 1 ꢀ 2 min. The mixture was allowed to stir
overnight at room temperature after which the precipitate was filtered and solvent
t
evaporated on a rotary evaporator. The intermediate, bocꢀ3ꢀaminoꢀNꢀ
phenylpropanamide (0.49 g, 71 % yield), was purified by column chromatography
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over silica gel using hexane : ethyl acetate (2 : 1, v/v) as the eluent. A portion (0.35
g) of the product was deprotected with trifluoroacetic acid (TFA) (used as solvent
and reagent) yielding the TFA salt of 3ꢀaminoꢀNꢀphenylpropanamide (0.31 g, 84 %
yield). For structure verification and comparison with literature, 3ꢀaminoꢀNꢀ
phenylpropanamide, TFA salt was dissolved in iceꢀcold water (2 mL), made alkaline
ꢀ
1
with 2 mmol L sodium hydroxide and extracted with chloroform (5 x 2 mL). The
combined organic phases were dried with sodium sulphate and the solvent
1
evaporated. Structure verification was achieved using H-NMR spectrum (400
13
MHz) and C-NMR spectrum (101 MHz) recorded on a Jeol ECS400 instrument.
Low resolution mass spectrum (LRMS) was recorded via direct injection of a dilute
methanolic solution into a Thermo Finnigan LCQ Advantage MS detector using
electrospray ionisation (ESI). Melting point was determined using Stuart SMP1
melting point apparatus. Analytical data for 3ꢀaminoꢀNꢀphenylpropanamide, TFA salt
and the scheme for synthesis are given in the Supplementary Information.
2.7 Preparation of bacterial samples
All bacteria were subꢀcultured overnight at 37 °C on BHI agar one day prior to the
preparation of samples. After overnight incubation, bacteria were inoculated in
sterile BHI broth and incubated at 37 °C. Samples were prepared by measuring the
absorbance of the incubated bacterial suspension at OD600nm. At an absorbance
8
ꢀ1
reading of 0.132 (equivalent to 0.5 McFarland units / 1.5 x 10 CFU mL ) an aliquot
6
of 10 ꢂL of bacterial suspension (1.5 x 10 CFU) was added to a 1.5 mL Eppendorf
containing 0.99 mL sterile BHI broth with enzyme substrate. Samples were placed in
a sterile vial alongside a cuvette containing agarose gel with added reagents and
incubated overnight (Figure 1(a)). Media with enzyme substrate was prepared by
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dissolving 10 mg of enzyme substrate in Nꢀmethylꢀ2ꢀpyrrolidinone then transferring
aliquots aseptically to BHI broth.
2.8 Application of optimised methods to bacterial samples
E. faecium, S. agalactiae, E. coli and K. pneumoniae were tested with 2ꢀnitrophenylꢀ
βꢀ ꢀglucoside. Ps. aeruginosa, Ps. fluorescens and E. coli were evaluated with 3ꢀ
aminoꢀNꢀphenylpropanamide, TFA salt. Parameters investigated with E. faecium
and 2ꢀnitrophenylꢀβꢀ ꢀglucoside and Ps. aeruginosa with 3ꢀaminoꢀNꢀ
D
D
phenylpropanamide, TFA salt were substrate concentration and sensitivity in terms
of initial inoculum used. All samples were tested after overnight incubation at 37 °C,
after which the absorbance of the gel in the cuvette was measured at 415 nm with 2ꢀ
nitrophenylꢀβꢀDꢀglucoside and at 470 nm with 3ꢀaminoꢀNꢀphenylpropanamide, TFA
salt, both against reagent blanks. Uninoculated broth with added substrate and
bacteria inoculated in broth without substrate were tested to ensure no colour
developed due to spontaneous breakdown of the substrate or interfering VOCs. All
bacterial samples, as well as uninoculated BHI samples with substrate and
uninoculated BHI samples without substrate, were tested in triplicate.
3. Results and Discussion
Modified agarose gel has been identified as a suitable matrix for the trapping of
exogenous VOCs. It was found that colour development was homogenous
throughout the gel, allowing accurate absorbance measurements to be recorded.
The reagents required to generate colour were easily added to agarose gels and
their optimisation for increased colour generation was straightforward (see
Supplementary Information).
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3.1 Application of optimised methods to bacterial samples
The structures of both enzyme substrates are shown in Figure 2. 3ꢀAminoꢀNꢀ
14,15
phenylpropanamide has previously been synthesised.
A comparison of the NMR
data is given in the Supplementary Information. Bacterial samples were prepared
ꢀ
1
ꢀ1
with a final 2ꢀnitrophenylꢀβꢀ
There was an increase in absorbance and the development of a yellow colour after
8 h incubation with both E. faecium and K. pneumoniae (known producers of ßꢀ
Dꢀglucoside concentration of 200 ꢁg mL (664 ꢁmol L ).
1
glucosidase). The generation of a yellow colour indicated that 2ꢀnitrophenol had
been trapped in the modified agarose gel. Both S. agalactiae and E. coli showed no
activity with 2ꢀnitrophenylꢀβꢀDꢀglucoside and no yellow colour was generated (Table
1). It was noted that all species were able to grow in the presence of enzyme
substrate. 2ꢀNitrophenol generating bacteria could be visually differentiated from
those species negative for 2ꢀnitrophenol production (Figure 1(b)).
Bacterial samples were tested with a final 3ꢀaminoꢀNꢀphenylpropanamide, TFA salt
ꢀ
1
ꢀ1
concentration of 100 ꢁg mL (or 359 ꢁmol L ). Ps. aeruginosa hydrolysed 3ꢀaminoꢀ
Nꢀphenylpropanamide, TFA salt and this was detected by an increase in absorbance
of the gel and the generation of an orange colour within the gel. No hydrolysis
occurred with Ps. fluorescens and E. coli and no orange colour developed with these
species (Table 2). All species grew in the presence of 3ꢀaminoꢀNꢀ
phenylpropanamide, TFA salt. Aniline generating bacteria, and therefore βꢀalanine
aminopeptidase producing species, could be differentiated visually from species that
did not liberate aniline (Figure 1(b)). No substrate hydrolysis occurred in
uninoculated BHI controls with either enzyme substrate. In addition, there was no
colour development in bacterial samples in BHI without the addition of enzyme
substrates.
10

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ꢀ
1
A 2ꢀnitrophenylꢀβꢀ
Dꢀglucoside concentration range of 50 ꢀ 200 ꢁg mL was tested
with E. faecium (Table 3); the optimum substrate concentration was determined to
ꢀ
1
ꢀ1
be 200 ꢁg mL (or 664 ꢁmol L ). An increase in absorbance readings and a yellow
colour were detected from a 2ꢀnitrophenylꢀβꢀ ꢀglucoside concentration of 100 ꢁg
mL (or 332 ꢁmol L ); 2ꢀnitrophenol generation continued to increase as the
D
ꢀ1
ꢀ1
ꢀ
1
substrate concentration increased up to 200 ꢁg mL . A 3ꢀaminoꢀNꢀ
ꢀ
1
phenylpropanamide, TFA salt concentration range of 10 ꢀ 200 ꢁg mL was tested
with Ps. aeruginosa (Table 3). Aniline was detected from a 3ꢀaminoꢀNꢀ
ꢀ1
ꢀ1
phenylpropanamide, TFA salt concentration of 20 ꢁg mL (or 72 ꢁmol L ); the
ꢀ1
optimum 3ꢀaminoꢀNꢀphenylpropanamide, TFA salt concentration was 100 ꢁg mL (or
ꢀ
1
3
59 ꢁmol L ). At a 3ꢀaminoꢀNꢀphenylpropanamide, TFA salt concentration of 200 ꢁg
ꢀ1
ꢀ1
mL (or 719 ꢁmol L ), aniline generation decreased from that detected at a
ꢀ
1
4
ꢀ1
substrate concentration of 100 ꢁg mL . An initial inoculum of 1ꢀ1.5 x 10 CFU mL
was required for detectable levels of colour to develop with both enzyme substrates
after 18 h incubation (Table 4).
The developed method allowed for the discrimination of βꢀglucosidase producing
bacteria, as well as βꢀalanine aminopeptidase producing species. Bacterial VOC
analysis has previously suffered from a lack of specificity; in addition, experimental
variables such as choice of growth medium have been shown to alter the VOCs
3
generated. Use of enzyme substrates increases the specificity of bacterial VOC
profiles as VOCs released from substrates would act as markers for a particular
species.
3ꢀAminoꢀNꢀphenylpropanamide, TFA salt demonstrated potential as a suitable
substrate for the detection of Ps. aeruginosa. The development of an orange colour
indicated aniline liberation and its subsequent coupling with NQS, and thereby
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signifying βꢀalanine aminopeptidase activity. A 3ꢀaminoꢀNꢀphenylpropanamide, TFA
ꢀ
1
salt concentration of between 20 ꢀ 100 ꢁg mL was sufficient for colour generation;
this was comparable to βꢀalanine aminopeptidase enzyme substrate concentration
13
used in chromogenic media. However, the absorbance of gels decreased at 3ꢀ
ꢀ
1
aminoꢀNꢀphenylpropanamide, TFA salt concentrations above 100 ꢁg ml , possibly
due to the increased amount of solvent at higher substrate concentrations as 3ꢀ
aminoꢀNꢀphenylpropanamide, TFA salt was less soluble in Nꢀmethylꢀ2ꢀpyrrolidinone
than 2ꢀnitrophenylꢀβꢀDꢀglucoside.
Limitations of the current method include use of a small sample size, testing with a
limited range of species and only one strain of each species used. Applying the
method to more species is required. As a result of the size of vials used (30 mL), the
headspace volume in the vial was large. Use of a smaller vial would decrease the
headspace volume, could lead to an improvement in the linear range for
quantification purposes, as well as improving method sensitivity and hence
4
increasing the amount of colour developed. An initial inoculum of 1ꢀ1.5 x 10 CFU
ꢀ1
mL was required for colour generation after 18 h incubation with both enzyme
substrates. However, using a xerogel, pꢀnitrophenol was detected after 16 h
5
ꢀ1 8
incubation with an initial E. coli inoculum of 10 CFU mL . The initial inoculum
required to produce a detectable amount of colour could be reduced by decreasing
the headspace volume. Following the implementation of a new experimental setꢀup
with a smaller sample headspace volume, a study investigating the minimum time
required for sufficient exogenous VOC liberation and detection would be useful.
Pure bacterial cultures inoculated in a liquid culture medium were used to
demonstrate the proof of concept; however, this method could potentially be applied
to the identification of pathogenic bacteria in “real” clinical and food samples.
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Producers of ßꢀglucosidase and/or ßꢀalanyl aminopeptidase can be readily detected
by inclusion of chromogenic or fluorogenic enzyme substrates in the medium that
contains the bacterial cells without the need to detect released VOCs in the
headspace, and such methods are well established. However, our approach has
several possible advantages. For example, inoculation of foodstuffs or other test
samples into enrichment broths may lead to opacity or discolouration that could
confound the interpretation of whether a chromogenic substrate has been hydrolysed
(or lead to the quenching of released fluorogens, if fluorogenic substrates are used).
Furthermore, development of this technology could lead to the design of a sensor
that is divided into compartments, each of which contains specific reagents for
trapping and visualizing distinct VOCs. This could potentially allow generation of a
biochemical profile showing the presence (or absence) of multiple bacterial enzymes
within an enrichment broth allowing the detection of bacterial pathogens with high
specificity.
In addition, it is also possible to compare the theoretical trapping efficiency of 2ꢀ
nitrophenol in agarose. This would allow this approach to be directly compared with
8
other trapping media (e.g. xerogels). Unfortunately some uncertainty exists in the
scientific literature on the values for the molar extinction coefficient and Henry’s
constant for 2ꢀnitrophenol. For example, the molar extinction coefficient (molar
absorptivity) for 2ꢀnitrophenol has been reported to have values that range from
ꢀ
1
ꢀ1 16
2
,150 – 21,300 mol L cm . In addition, the Henry’s constant for 2ꢀnitrophenol also
ꢀ
5
ꢀ5
have some variability in their numerical values (e.g. 4.13 x 10 and 1.28 x 10 atmꢀ
3
ꢀ1 8,17
m mol ).
Similar variability is also evident for aniline. On that basis it is not
possible to determine an accurate trapping efficiency for either VOC.
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4. Conclusions
This work highlights the potential of designing enzyme substrates to liberate
exogenous VOCs for bacterial identification, as well as the suitability of agarose gel
as a matrix for VOC trapping. In addition, the ability to tailor reagents present in the
gel for the colorimetric detection of specific exogenous VOCs is demonstrated and
reagents added to gel can be optimised for maximum colour generation. This
innovative method displays potential for the development as a novel, simple and lowꢀ
cost optical VOC sensor for the detection of bacteria in clinical and food samples.
Supplementary Information
The Supplementary Information contains details of the optimisation processes for
determination of percentage agarose gel, 2ꢀnitrophenol detection, aniline detection,
analytical calibration data for 2ꢀnitrophenol and aniline. In addition, experimental
details for the synthesis of 3ꢀaminoꢀNꢀphenylpropanamide, TFA and its analytical
data are included as well as analytical data for 3ꢀaminoꢀNꢀphenylpropanamide. In
total eight Figures and three Tables are provided.
Acknowledgements
The financial support of Northumbria University is gratefully acknowledged.
References
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Chemosensors, 2014, 2, 171ꢀ181.
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15

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Wu, T.Y. Tsai, W.C. Chen, H.P. Hseih, Y.S. Chao and W.T. Jiaang, J. Med. Chem.,
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6. M. Soundararajan, C.P. Bailey, and J. Markwell, Biochem. Mol. Biol. Educ.,
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2
17. ChemIDplus. A Toxnet Database. US National Library of Medicine, Bethesda,
MD, USA (http://chem.sis.nlm.nih.gov/chemidpus/name/2ꢀnitrophenol). (last
accessed on 15/01/2015).
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Table 1. 2ꢀNitrophenylꢀβꢀDꢀglucoside with bacterial samples.
Mean 2ꢀnitrophenol
Species
Growth*
†
ꢀ1 §
concentration ± 1SD (ꢁg mL )
¶
E. faecium
+
+
+
+
33.50 ± 2.32
¶
‡
S. agalactiae
ND
K. pneumoniae**
E. coli**
17.31 ± 1.95
ND
†
‡
§
*
+, growth observed; SD, 1 standard deviation (n = 3); ND, not detected; absorbance
¶
measured at 415 nm; Gramꢀpositive organism; **Gramꢀnegative organism.
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Table 2. 3ꢀAminoꢀNꢀphenylpropanamide, TFA salt with bacterial samples.
Mean aniline
†
Species
Growth*
concentration ± 1SD
ꢀ
1 §
(
ꢁg mL )
Ps. aeruginosa
Ps. fluorescens
E. coli
+
+
+
6.12 ± 0.75
‡
ND
ND
†
‡
§
*
+, growth observed; SD, 1 standard deviation (n = 3); ND, not detected; absorbance
measured at 470 nm.
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Table 3. Exogenous VOC detection over an enzyme substrate concentration
range.
Substrate concentration
2ꢀNitrophenol concentration
Aniline concentration
ꢀ1
ꢀ1 †
ꢀ1 ‡
(
ꢁg mL )
(ꢁg mL )
(ꢁg mL )
2
1
1
00
50
00
33.48
23.92
10.46
ND*
ND
5.32
§
ꢀ
9.63
ꢀ
7
5
0
0
0
5
8.37
5.84
ND
2
1
ꢀ
ꢀ
†
*
4
ND, not detected; E. faecium with 2ꢀnitrophenylꢀβꢀDꢀglucoside, absorbance measured at
‡
15 nm; Ps. aeruginosa with 3ꢀaminoꢀNꢀphenylpropanamide, TFA salt, absorbance
measured at 470 nm; not tested at this concentration.
§
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Table 4. Sensitivity of modified agarose gels method in terms of initial
inoculum used.
ꢀ1
Initial inoculum (CFU mL
2ꢀNitrophenol
Aniline concentration
ꢀ1 †
ꢀ1 ‡
BHI)
concentration (ꢁg mL )
(ꢁg mL )
6
1
1
1
1
1
1
ꢀ1.5 x 10
ꢀ1.5 x 10
ꢀ1.5 x 10
ꢀ1.5 x 10
ꢀ1.5 x 10
ꢀ1.5 x 10
31.5
27.8
16.4
ND*
ND
8.25
6.48
5.03
ND
5
4
3
2
1
ND
ND
ND
†
ꢀ1
*
ND, not detected; E. faecium with 200 ꢁg mL 2ꢀnitrophenylꢀβꢀDꢀglucoside, absorbance
‡
measured at 415 nm; Ps. aeruginosa with 3ꢀaminoꢀNꢀphenylpropanamide, TFA salt,
absorbance measured at 470 nm.
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Fig. 1. Exogenous volatile organic compound detection (a) schematic diagram
of experimental setꢀup and (b) application to bacterial samples.
(a)
(b)
Uninoculated
BHI*
Uninoculated
†
†
E. faecium*
S. agalactiae*
Ps. fluorescens Ps. aeruginosa
†
BHI
ꢀ
1
ꢀ1
†
*
Enzyme substrate: 200 ꢁg mL (or 664 ꢁmol L ) 2ꢀnitrophenylꢀβꢀ
D
ꢀglucoside; Enzyme
ꢀ1
ꢀ1
substrate: 100 ꢁg mL (or 359 ꢁmol L ) 3ꢀaminoꢀNꢀphenylpropanamide, TFA salt
.
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Fig. 2. Structures of 3ꢀaminoꢀNꢀphenylpropanamide, TFA salt and 2ꢀ
nitrophenylꢀβꢀ ꢀglucoside.
D
NOTE:
3ꢀaminoꢀNꢀphenylpropanamide, TFA salt: empirical formula C11
13 3 2 3
H F N O ; molecular
weight 278.23
8
2ꢀnitrophenylꢀβꢀDꢀglucoside: empirical formula C12H15NO ; molecular weight 301.25
22