The screening of complex mixtures such as foodstuffs, beverages, dietary supplements, and wastewater for
toxins and adulterants or potential biological activity
is often expensive and time consuming. To identify
the active or toxic constituents in such mixtures
requires the tedious isolation of single components
followed by assays of their biological effects.
Alternatively, standard rapid screening tests only
establish the overall toxicity of a mixture disregarding
the identification of the active culprit. Additionally,
when these materials are tested in their mixture condition
there is a high risk of artifacts or false data due
to interferences and interactions with other components
in the mixture. Optimally, a rapid screening
assay that analyzes the activity of the individual components
of a mixture in an economical and efficient
fashion would be of great value. The Bioluminex™
assay (ChromaDex, Santa Ana, CA), which is based
on direct bioautography, is one such technique.
The Bioluminex rapid screening assay employs direct
bioautography detection by coupling the separation
power of thin layer chromatography (TLC) with the
biosensor properties of bioluminescent microorganisms.1–5 This technology provides a characteristic
chemical and biological toxicity profile or “fingerprint”
for each mixture analyzed and can be used to
identify potentially bioactive compounds or adulterants.
In addition, the profile can be used to help support
material identity. The TLC–bioluminescence
assay offers several advantages over standard toxicity
screening assays. First, activity is assigned to single
components of a mixture, thereby identifying the
active constituent and eliminating interferences
from other analytes. Second, the sample medium is
evaporated from the TLC plate prior to introducing
the biosensor organism, allowing for the analysis of
nonaqueous samples. Third, the need for a secondary
visualization agent is eliminated by employing a variety
of bioluminescent organisms as the primary
detection reagent. Fourth, the assay can simultaneously
analyze up to 20 samples, with the organism
toxicity results produced within 2 min. Last, the
TLC–bioluminescence assay has been designed to be
kit compatible, providing a rapid and inexpensive
analysis for many complex samples.
Bioluminescent organisms of interest range from
wild-type naturally bioluminescent bacteria to genetically
modified (GM) bacteria or yeast, including
dark variants that express the lux or GFP (green fluorescent
protein) reporters under the control of a variety
of promoters. Perhaps the most applicable organism
for general, broad-range toxicity testing and
which is used in the Bioluminex assay is the bacterium
Vibrio fischeri. The bioluminescent marine
bacterium V. fischeri (Beijerinck, 1889; Lehmann and
Neumann, 1896) is a nonpathogenic, Gram-negative
species of bacterium that thrives in the marine environment
and is an ideal biosensor organism for
TLC–bioluminescence applications. This bacterium
has been well characterized and used in standardized
and validated ecotoxicity assays for over 25 years.6 It
is robust and easily cultured, and provides a consistent
and reproducible response with an apparent and
quantifiable endpoint. The V. fischeri bacteria are also
very suitable for use in a rapid screening kit assay. The
bacteria can be freeze-dried and subsequently stored
at 4 °C for more than 1.5 years. Additionally, the
freeze-dried bacteria can be directly inoculated and
liquid cultured without an intermediate culture plate.
They can be liquid cultured at room temperature
using a flask, stir bar, and stir plate, eliminating the
need for expensive laboratory equipment.
As V. fischeri cells reach a crucial cellular density, their
lux operon expresses the reaction catalyst luciferase. In
the presence of O2 and luciferase, a reduced nicotinamide
adenine dinucleotide (NADH) riboflavin
phosphate (FMNH2) and a long-chain fatty aldehyde
are oxidized. The resulting interaction forms an
excited yet highly stable intermediate, which decays
slowly, resulting in the release of excess free energy in
the form of a blue-green light (490 nm).7–10 The
observed bioluminescence reflects the metabolic status
of the cell and will decrease for cells exposed to
toxic substances. Thus, a reduction in light emission is
a measure of toxicity toward V. fischeri and can be
selectively viewed and quantitated directly on the
TLC or high-performance thin layer chromatography
(HPTLC) plate. Typical limits of detection for toxic
substances are in the picomole range.
In the TLC–bioluminescence assay, complex mixtures
are first separated by TLC or HPTLC. These
effective, inexpensive, and rapid techniques use
adsorption and capillary action to separate up to 20
(20 × 10 cm plate) complex mixtures simultaneously.
The matrices provide a characteristic
distribution pattern of compounds that is
dependent on sample composition and are routinely
used to support the identity of a compound
in a mixture when the retention factor
(Rf) or migration distance of a compound is
compared with the Rf of a known compound.
After compound separation, the mobile phase is
evaporated and the matrix plate is coated with a
broth of bioluminescent bacteria employing a
simple dipping procedure. The broth can be
used to dip multiple plates and can be buffered
to use with mobile phases containing acids or
bases that do not fully evaporate during the drying
phase. Results occur within seconds and last
until the plate dries, approximately 30 min or
more with the use of the BioLuminizer
(CAMAG, Muttenz, Switzerland), which optimizes
the plate compartment for prolonged bacterial
activity. Results identify single compounds,
which inhibit luminescence, resulting
in dark zones (quenched bioluminescence) on a
luminescent background where the bacteria
remain viable. Data can be documented by
direct contact of photographic film such as X-ray
and Polaroid film (Waltham, MA) or indirectly,
such as with a cooled charge-coupled device (CCD) camera, video imaging, Polaroid
documentation system, or 35-mm camera.
Experimental
Methanol (HPLC grade), toluene (OmniSolv), and
isopropyl ether (HPLC grade) were purchased from
VWR (West Chester, PA). Ethyl acetate (biotech
grade) and formic acid (purum 98%) were purchased
from Sigma Aldrich (St. Louis, MO). The standards
ochratoxin A, Capsicum annuum, capsaicin, and
Bioluminex positive and negative controls were
obtained from ChromaDex. 4-Androstene-3,17-dione (≥98% purity) (Sigma Aldrich), 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(-2-methoxy-1-methylethyl)acetamide (Metolachlor–Pestanal®,
Riedel-de-Haën, Seelze, Germany), and arsenic(III)
oxide (99.995%) (Aldrich Chemical Co., Milwaukee,
WI) were obtained as indicated.
Lyophilized V. fischeri cultures (ChromaDex
Analytics, Inc., Boulder, CO) stored at 4 °C were
inoculated and grown overnight (30 hr) in a 200-mL
batch culture of complex medium (Bioluminex
medium), and 6 mL/L 50% aqueous solution of glycerol
(Fisher Scientific, Pittsburgh, PA), and H2O
(Millipore, Billerica, MA) adjusted to a pH of 7.2 ±
0.2 at 120 rpm and 28 °C under atmospheric conditions. Directly before assay, TLC–bioluminescence
buffer (Bioluminex buffer) was added to fully luminescent
bacteria and dissolved at 120 rpm and 28 °C
under atmospheric conditions.
Samples and standards were applied in water or
methanol to prewashed (methanol elution) 10 × 10
cm HPTLC silica gel 60 F254 plates (ChromaDex) via an ATS-4 automatic TLC sampler 4 (CAMAG) at y
= 8 mm using band spray application set to a
methanol or water application mode as appropriate.
After sample application, the plates were air dried for
15 min. Plates were then developed to 70 mm using
appropriate mobile phases in a preequilibrated (30
min) 10 × 10 cm ridged-bottom TLC chamber.
Postdevelopment mobile phases were evaporated from
the plates in a mechanical oven at 40 °C for 2 hr.
Dried plates were coated with buffered luminescent
V. fischeri (200 mL) using an automatic immersion
device (CAMAG). Excess bacteria were removed
from the plate using a squeegee device, and images
were immediately recorded over a 10-min period
using an exposure time of 120 sec with a cooled (–30
°C absolute) CCD camera and dark box (Fluorchem®
8900, Alpha Innotech, San Leandro, CA).
Discussion
Figure 1 - TLC–bioluminescence image of increasing concentrations
(0.6, 1.1, and 1.6 μg of each analyte per lane) of 4-androstene-3,17-dione, arsenic oxide, and Metolachlor spiked tap water in tracks 1, 2, and
3, respectively. Corresponding application volume (8, 14, and 20 μL) of
unadulterated water in tracks 4, 5, and 6, and 2 μg of each analyte, 4-androstene-3,17-dione, arsenic oxide, and Metolachlor in tracks 8, 9, and
10, respectively. Chromatogram was developed with toluene:ethyl
acetate:formic acid:water (4:8:1.1:0.2, v/v/v/v) and analyzed using the
Bioluminex assay.
Figure
2 - TLC–bioluminescence image of increasing concentrations (0.9, 1.8, 2.6, 3.5, 8.8,
and 17.5 μg) of C. annuum (Cayenne pepper) extract (10 mL biomass, 10 mL CHCl3) in
tracks 2–7, respectively, and 2 μg of capsaicin in track 1 (track 8 is a blank). Chromatogram was
developed with isopropyl ether and analyzed using the Bioluminex assay.
Figure 3 - TLC–bioluminescence image of increasing concentrations (1 μL = 0.5 μg analyte,
3 μL = 1.5 μg analyte, and 3 μL = 3.0 μg analyte) of ochratoxin A spiked corn extract (1 g
freeze-dried corn, 10 mL CH3OH) in tracks 1–3, respectively; equivalent amounts of unspiked
corn extract in tracks 4–6; 2 μg of ochratoxin A standard in track 8; 4 μg and 8 μg of Bioluminex
negative and positive controls in tracks 9 and 10, respectively (track 7 is a blank). Chromatogram
was developed with ethyl acetate:methanol:formic acid:water (50:2:5:3, v/v/v) and analyzed
using the Bioluminex assay.
The TLC–bioluminescence assay has been shown to be
effective in the analysis of pesticides (fungicides, insecticides,
herbicides), heavy metals, organic pollutants,
pharmaceuticals, and mycotoxins in a variety of complex
matrices such as dietary supplements, natural products,
foodstuffs, beverages, and wastewater. For example,
in Figure 1, tap water was spiked with the anabolic
steroid 4-androstene-3,17-dione, the heavy metal
arsenic(III) oxide, and the chloroacetanilide herbicide
Metolachlor. Various concentrations of these samples
in conjunction with unspiked tap water and the individual
analytes were examined via TLC–bioluminescence.
Each separated analyte showed a concentration-dependent
decrease in bioluminescence (tracks 1–3)
and can be identified by comparing the Rf (compound
distance relative to baseline/solvent front distance relative
to baseline) of the standard analytes in tracks 8, 9,
and 10 to the Rf of the compound in the mixtures in
lanes 1, 2, and 3 (i.e., 4-androstene-3,17-dione Rf ≅
0.71, arsenic oxide Rf ≅ 0 , and Metolachlor Rf ≅ 0.80).
The assay is also effective for obtaining a fingerprint
profile that can be applied to the support of material
identity. For example, in Figure 2 increasing concentrations
of C. annuum (Cayenne pepper) extract and
2 μg of capsaicin were analyzed
using TLC–bioluminescence.
At lower extract
concentrations, compounds
in the C. annuum extract
that readily inhibit V. fischeri bioluminescence can be
easily identified, such as the unidentified compound
detected at Rf ≅ 0.87 in track 2. At higher extract
concentrations, a fingerprint identity emerges. This
fingerprint is a characteristic profile that can be used
to simultaneously support material identity and
detect sample adulteration.
Detecting mycotoxins in complex mixtures such as
agricultural commodities can also be accomplished
with the method presented. The third application
example shown in Figure 3 displays the TLC–bioluminescence image of increasing concentrations of
corn extracts (tracks 4–6), corn extracts spiked with
the mycotoxin ochratoxin A (tracks 1–3), and 2 μg
of the ochratoxin A standard (track 8). Most
markedly, in lane 6, a fingerprint of the analyzed
corn extract is detected. In lane 3, the characteristic
corn extract fingerprint is also observed, but with an
extra band at Rf ≅ 0.76. This band correlates well to
the Rf of the ochratoxin A standard in track 8.
Conclusion
The TLC–bioluminescence assay is an effective,
rapid, and versatile tool that has the potential to
become an industry standard. It can be used to support
material identity, detect toxins and chemical adulterants,
and control manufacturing procedures. It may
also prove to be a potent research tool used to identify
biologically active constituents of complex mixtures
such as natural products. Furthermore, the rugged
technology is kit compatible to provide a quick and
inexpensive analysis of many complex samples.
References
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FASEB1993, 7, 1016–22.
Dr. Verbitski is Bioluminex Manager, Mr. Gourdin is Bioluminex
Project Investigator, Ms. Ikenouye is Bioluminex Research
Associate, and Dr. McChesney is Consulting Chief Scientific
Officer, ChromaDex Analytics, Inc., 2830 Wilderness Pl.,
Boulder, CO 80301, U.S.A.; tel.: 303-442-4281, ext. 226;
fax: 303-442-4237; e-mail: [email protected].