Evaluation of Various Factors Affecting Fluorescence Emission Behavior of Ochratoxin A: Effect of pH, Solvent and Salt Composition
Abstract
In the present work, a design of customized portable fluorescence
imaging system was developed (in-house) to quantify the change in
fluorescence emission of fungal toxin ochratoxin A (OTA). OTA is
naturally fluorescent, and the fluorescence properties of OTA solutions
depend on the pH, solvent polarity and the presence of ligand molecule.
In this work, the influence of solvent, pH and salt concentration on OTA
fluorescence emission was investigated. The fluorescence properties of
OTA in aqueous solutions have been investigated by means of steady-state
fluorescence at different pH (range 6.8-8.4), Na+ salt ranges from 10
120mM, buffer solutions and in organic solvents. All the fluorescence
measurements were performed through the fluorescence imaging system
consists of an ultraviolet light at 365nm and a CMOS sensor controlled
by an executable interface designed in MATLAB. The obtained image is
decomposed into its red, green and blue component (RGB components) and
analyzed.
For each solution, spiked at a concentration of 20 ^g L-1
OTA with different conditions (pH, solvent and salt composition), the
red, green and blue (RGB) coordinates were obtained and plotted to
quantify the effect of the fluorescence emitted by the OTA. The higher
fluorescence yielding conditions were identified and results were
compared with the commonly used fluoroskan microplate reader. The
developed design of fluorescence instrument was successfully employed to
study of fluorescence behavior of OTA in different environments. As a
potential, the proposed design instrument could be employed to quantify
the fluorescence behavior of fluorescence exhibiting target molecules.
Abbreviations: IARC: International Agency
for Research on Cancer; MRL: Maximum Residue Limit; PBS: Phosphate
Buffered Saline; UV: Ultraviolet; PMMA: Polymethylmethacrylate; RGB:
Red, Green and Blue; OTA: ochratoxin A; MgCl2: Magnesium Chloride; NaCl: Sodium Chloride; Na2HPO4: Di Sodium Hydrogen Phosphate, KH2PO4: Potassium Di Hydrogen Phosphate
Introduction
Figure 1: S)-2-((R)-5-chloro-8-hydroxy-3-methyl-1-oxoi-sochroman-7-carboxamido)-3-phenylpropanoic acid.
Mycotoxins are the secondary metabolites, which are produced by the several fungal species belonging to the family of Aspergillus, Fusarium, Penicillum [1,2]. Among them, the Aspergillus ochraceus and Penicillium citrinum
are the main producers of ochratoxin A (OTA), which is the most widely
occurring fungal toxin. Biosynthetically, OTA is the pentaketide derived
from the dihydrocoumarins coupled to β-phenylalanine i.e. (S)-2-((R)-5-
chloro-8-hydroxy-3-methyl-1-oxoisochroman-7-carboxamido)-3
phenylpropanoic acid (Figure 1) and as contaminant, present in the
varieties of foodstuffs and beverages such as cereals, spices, wine
grapes [3,4]. The presence of OTA in animal tissue, human blood and milk
increases the risk of nephrotoxicity, immunosuppressant, teratogenicity
[5,6]. Recently, the International Agency for Research on Cancer (IARC)
has been classified the OTA in group 2B (possible carcinogenic agent in
human). The maximum residue limit (MRL) for OTA has been set by the
European Community in several foodstuffs such as the MRL of OTA in wine
is 2μg kg -1 or 5μg kg'1 in unprocessed cereal (European Commission 2006 [7]).
Moreover, due to the chronic cases and occurrence of OTA incidences
and exposure from contaminated food, there is a need to develop a
faster, sensitive, robust and portable method for quantification of OTA.
Chromatographic methods, such as high-performance liquid chromatography
[8] and thin-layer chromatography [9], are the mainly used for OTA
determination. Recently, the advancement in OTA detection methods is
based on electropolymerization [10], surface plasmon resonance [11]
aptasensors [12,13] utilizing electrochemical and fluorescence- based
signal generating principle. Among all, the fluorescence based detection
methods gained significant attention due to the ease of reaction, label
free detection, diverse measurement methods [14]. The intrinsic
fluorescence emission behavior, intensity and sensitivity of the
analytical method strongly depends on solution composition, such as
concentration and buffer ions, pH of solvent, aqueous-organic phase
ratio, temperature etc [14]. Based on the above assumption, in the
present work the focus of study was to quantify the effect of pH,
solvent composition and temperature variables on the fluorescence
emission behavior of OTA by exploration of designing a field portable
fluorescence measuring platform. The present results strongly suggested
the potential of designed instrument as a portable and affordable cost-
effective system for rapid screening and quantification of OTA in real
samples. Based on our results, as a generalized fluorescence measuring
platform, the proposed platform can be further employed for other target
analyte poses fluorescence behavior or in fluorescence-based sensing
platforms.
Material and Methods
Chemicals and Reagents
Spectrophotometric cuvettes for fluorescence measurement were
procured from Ratiolab (Germany). HEPES (4-(2-hydroxyeth-
yl)-1-piperazineethanesulfonic acid) buffer was purchased from Fisher
Scientific (USA). All other chemicals and reagents di-sodium hydrogen
phosphate (Na2HPO4), potassium di-hydrogen phosphate (KH2PO4), magnesium chloride (MgCl2),
potassium chloride (KCl) and sodium chloride (NaCl), acetic acid,
ethanol, methanol and acetonitrile of analytical grade were procured
from Sigma-Aldrich (France). Ochratoxin A (OTA) derived from Aspergillus
ochraceus was purchased from Sigma Aldrich (France).
Solutions Preparation
Phosphate buffered saline (PBS) was prepared be dissolving appropriate amount of 1.44g Na2HPO4, 0.24g KH2PO4
containing 8g NaCl, 0.2g KCl in deionized Milli-Q water. The pH of the
buffer was adjusted to 7.4. Similarly, the HEPES binding buffer (HBB,
50mM) was prepared by dissolving appropriate amount of HEPES salt
containing 5mM MgCl2, 120mM NaCl and 5mM KCl. The pH of the
buffer was adjusted from 6.8 to 8.2. For HPLC, a mixture of
acetonitrile/water/acetic acid (48/51/1) sonicated for 20 minutes was
used. A stock standard stock solution of OTA was prepared by dissolving 1
mg of pure crystalline OTA in methanol at a concentration of 1 mg L-1. For working solution, the OTA stock was further diluted in different solvent before fluorescence measurements.
Instrumentation
A lab based customized (in-house designed) fluorescence image
capturing device consist of an Ultraviolet emitter to 360370nm (NICHIA
Corporation) was supplied by Power light systems (Germany). Electronic
components were procured from Farnell Element14 and Mouser Electronics
(France). Arduino UNO board and a serial port color camera module with
CMOS sensor with TTL interface was purchased from Sparkfun (Spain). For
image capturing and measurement, a graphical user interface was created
in MATLAB R2011a. A fluorescence instrument, Fluoroskan Ascent FL 2.6
(Thermo Scientific, Finland) equipped with Ascent software version 2.6
was used for fluorescence measurements.
Design of Developed System
The developed device based on a bracket designed to allocate the
sensing module, the serial port camera module and the cuvette into the
chamber was used in the proposed work. Polymethylmethacrylate (PMMA)
cuvette holder was used for holding cuvette for photometric measurements
in the ultraviolet (UV) range. The cuvette outer dimensions are
12.5*12.5*45mm; one hole for the light emitter was drilled into black
poly methyl methacrylate cuvette holder at 900 from the serial port camera module as illustrated in the (Figure 2) The developed system comprises:
Figure 2: Sensing module connected to the final device and its control with the computer.
Sensing Module
The serial port camera module captures high resolution pictures and
transmits over a serial TTL interface, which captures JPEG (Joint
Photographic Experts Group) images with a CMOS sensor from a serial port
with communication via UART. The sensing camera required, 5VDC power
supply with a low current consumption of 80 to 100mA. The ultraviolet
emitter produces high power light with an emission peak at 365nm with
2.7 mW of radiant power. The concentration of the ochratoxin A was
determined by exciting UV light. LED voltage regulator (LM317) that is
powered by a constant current through the USB port of Arduino UNO was
used as power source. The circuit was designed and placed in an
electrolytic plate with dimensions of 30x20 mm.
Software
A graphical interface was created in MATLAB R2011a. The developed
platform facilitates the acquisition of the data generated by the
emission of light and allows a first analysis of data with a choice for
automatic file saving for further processing and comparison studies.
Processing
The user does not need to install MATLAB since the interface is an
executable file, hence the user need to install the executable file
generate in its computer. The developer interface is easy to use,
automation of file processing (text file, images, figures, names of
files) allowing us to use a portable system.
Recording
The fluorescence emission from the excited sample passes through a
lens, to a serial port camera module controlled by the computer.
Fluorescence image data from the serial port camera module are analyzed
by application software. Images with two dimensions such as photograph
captured by optical devices like cameras are considered digital images,
which are a numeric representation of a two-dimensional image. The
digital image contains a fixed number of rows and columns of pixels.
Each pixel is specified by three values for the red, blue and green
components of the pixel array. The position of the pixel is described by
a pair of coordinates (xi,xj), which indicate the intensity of the
pixel (x,y) in the red, green and blue (RGB) component.
Fluorescence Measurements
Effect of Porogen (Solvent) on Fluorescence Intensity: To study the effect of solvent on the fluorescence behavior of OTA, a 20 ng L-1
OTA concentration was prepared in different solvent such as methanol,
ethanol, PBS buffer, acetonitrile, distilled water (Millipore filtered),
mobile phase (for HPLC), tap water and HEPES buffer. Similarly, blank
was prepared without spiking OTA in the different solvents. Then, 1 mL
of volume from each composition was transferred into cuvette and
inserted in the chamber for fluorescence measurement. The ultraviolet
led excited the sample and the serial port camera module captures the
image when the interface design was executed. With the option multiples
images, its RGB values, the image to JPEG format and the mean of the RGB
components in a text file were obtained automatically and the results
were saved.
Effect of pH on Fluorescence Intensity: Under optimized condition, a 20 μg L-1
OTA concentration was prepared in different buffer ranging pH from
6.8-8.4. All fluorescence measurements were performed in triplicate.
Similarly, the blank was prepared without spiking OTA and considered for
fluorescence measurements.All fluorescence measurements were performed
by designing a fluorescence measuring instrument and compare with the
fluorescence intensity measured through Fluoroskan Multiplate reader.
Effect of Salt Concentration Intensity: To study the salt
effect, different concentrations of saline salt (sodium chloride) range
from 0-120mM NaCl was studied. The effect of HEPES buffer containing a
different salt concentration was studied against concentration of 20 μg L-1
OTA. The fluorescence measurements were performed using the developer
portable system and compare against the result obtained from Fluoroskan
measurements. Then, measurements were repeated after 1h. Control
measurements were performed without the addition of OTA in buffer under
same optimized conditions.
Results and Discussion
Principle Design: In the proposed work, a design of proposed
fluorescence imaging platform was used for the fluorescence measurements
as shown in Figure 2. Based on the concept of fluorescence imaging and
the RGB color model, the developer interface built a RBG diagram using
the recorded fluorescence image. Further, the recorded fluorescence
image is decomposed into its red, green and blue component (RGB
components) and analyzed by computer. In the present platform, the blue
component of RGB diagram was considered because OTA exhibit strong
fluorescence in the ultraviolet light Bueno [15]. When the solution
containing OTA is excited using UV light, the solution illuminates a
blue fluorescence due to the presence of OTA and blank (no fluorescence)
respectively as depicted in the Figure 3. For qualitative analysis, the
blue component of RGB diagram was taken into consideration. The reason
could be attributed to the presence of the high blue component,
establishing a direct relation between the blue component and OTA
concentration. Control measurements showed the lower value of blue
component comparing to sample.
Figure 3: Fluorescence imaging using developed platform.
a. Blank
b. With 20 μg L-1 OTA
a. Blank
b. With 20 μg L-1 OTA
Effect of Solvent on Fluorescence Intensity of OTA: It has been
already reported that the fluorescence behavior (such as the
fluorescence maximum) of OTA, strongly dependent on the porogen used. It
was observed that the phenolic group of OTA undergoes an excited state
proton transfer reaction depending upon the solvent used [16,17].
Fluorescence intensity of OTA obtained in different solvent system at
20μg L-1 OTA using the developed platform as shown in the
Figure 4. The buffer solutions (PBS, HEPES) and the alcohols (Methanol,
Ethanol) show the best results with the greatest fluorescence intensity
for the OTA. As depicted in the Figure 4a, the OTA showed less
fluorescence in distilled water which is due to the opening of the
lactone ring of OTA molecule causing slow decomposition of OTA in water
Brow [18]. The significantly higher fluorescence was observed in the
buffer and methanol in comparison to the acetonitrile Turner [19]. The
obtained results further compared with the fluorescence intensity
response recorded using the fluoroskan microplate reader at 360nm. It
was observed that the design fluorescence imaging platform has similar
type of results as depicted in the Figure 4a & 4b.
No comments:
Post a Comment
Note: Only a member of this blog may post a comment.