Food Control 21 (2010) 155–161
Contents lists available at ScienceDirect
Food Control
journal homepage: www.elsevier.com/locate/foodcont
Self-indicating nanobiosensor for detection of 2,4-dinitrophenol
Sanghoon Ko a, Sundaram Gunasekaran b,*, Jaehyuck Yu c
a
b
c
Department of Food Science and Technology, Sejong University, 98 Gunja-Dong, Gwangjin-Gu, Seoul 143-747, Republic of Korea
Department of Biological Systems Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
Department of Food Microbiology and Toxicology, University of Wisconsin-Madison, Madison, WI 53706, USA
a r t i c l e
i n f o
Article history:
Received 28 April 2008
Received in revised form 24 April 2009
Accepted 1 May 2009
Keywords:
Biosensor
2,4-Dinitrophenol (DNP)
Gold nanoparticle
Toxin detection
a b s t r a c t
A colorimetric nanobiosensor was developed based on the chromogenic effect of latex microspheres
hybridization with gold nanoparticles. A toxin analog, 2,4-dinitrophenol–bovine serum albumin (DNP–
BSA) was attached to gold nanoparticles and was allowed to hybridize via antigen–antibody association
with an anti-DNP antibody on latex microspheres, changing the color of latex microspheres pinkish-red.
A model toxin, DNP–glycine (GLY), was detected and quantified via a competition that occurs between
the analog-conjugated-gold nanoparticles and the toxin molecules for the binding pocket in the antiDNP antibody. When the gold nanoparticles were displaced from host latex microspheres in the presence
of toxin molecules, a visible color change occurred from pinkish-red to white. This proof-of-concept selfindicating nanobiosensor detected the model toxin rapidly, and the results were quantifiable. When further developed, this nanobiosensor system will be useful at the consumer end to help improve food safety
and counter possible bioterrorism attacks.
Ó 2009 Elsevier Ltd. All rights reserved.
0. Introduction
Food safety is a significant global issue with public health and
international trade implications (Satcher, 2000). Foodborne illness
associated with toxins, pathogens or other food contaminants is a
serious health threat in the US and elsewhere. According to the
Centers for Disease Control and Prevention (CDC), in the US alone
foodborne toxins, pathogens or contaminants account for about
76 million cases of illness, 325,000 hospitalizations, and 5000
deaths annually (Mead et al., 1999). This concern for food safety
has recently been further exacerbated by the growing threat of
bioterrorism.
A major part of our efforts to improve food safety is to enable
detecting foodborne toxins rapidly, on site, and in situ. Biosensors
are devices that use biological components to react or bind with
a target molecule and transduce this event into a detectable signal
(Nath & Chilkoti, 2004a, 2004b). They can play a vital role in assuring food safety since they provide accurate results rapidly for preventive immediate actions by users who are close to the site of
contamination. Thus, simple visual tests are highly desirable as color changes are easily detected by even unskilled users.
Nanotechnology is playing an increasingly important role in the
development of biosensors. Various approaches to exploit the advances in nanoscience and nanotechnology for bioanalyses have
been recently reported (Chen, Miao, He, Wu, & Li, 2004; Haruyama,
* Corresponding author. Tel.: +1 608 262 1019; fax: +1 608 262 1228.
E-mail address: guna@wisc.edu (S. Gunasekaran).
0956-7135/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2009.05.006
2003; Jain, 2003; Vo-Dinh, Cullum, & Stokes, 2001). Such bioanalytical nanosensors can be used for detection of pathogens, toxins,
nutrients, environmental characteristics, heavy metals, particulates, allergens, etc. Sensitivity, specificity, rapidity of testing and
other necessary attributes of biosensors can be improved by using
nanomaterials in their construction (Jin, Wu, Li, Mirkin, & Schatz,
2003).
Gold nanoparticles have been particularly effective labels for
sensors because a variety of analytical techniques can be used to
detect them; they have various functional ligands, and form their
assemblies and complexes with various conjugates. Therefore, gold
nanoparticles have versatile uses as biosensors, catalysts, and other
nanoscale devices. Extensive studies have reported the use of gold
nanoparticles as sensing platforms including colorimetric sensors
for biospecific interaction analyses (Lee & Perez-Luna, 2005; Mulvaney, 1996). Colorimetric systems are especially attractive option
for biosensing since they are simple to monitor, relatively inexpensive, and can be designed to be self-indicating.
Chromogenic effect of gold nanoparticles facilitates many options to detect bioanalytes. For example, gold nanoparticles change
color depending on the solution conditions such as aggregation
(Daniel & Astruc, 2004; Liu & Lu, 2004) or complex formation with
other materials such as latex (Reynolds, Mirkin, & Letsinger, 2000).
Accordingly, gold nanoparticles have been used as host metal to
develop an optical biosensor for recognizing and detecting specific
structure such as DNA sequences (Maxwell, Taylor, & Nie, 2002).
The target DNA sequences were detected by the gold nanoparticles
aggregation process, which occurs with a concomitant color
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S. Ko et al. / Food Control 21 (2010) 155–161
change from red1 (the color of dispersed gold nanoparticles) to
blue (the color of aggregated networks), which can be monitored
spectrophotometrically or with the naked eye. In another study,
a colorimetric detection method for Hg2+ using DNP-gold nanoparticles yielded purple-to-red color change when two complementary DNA-gold nanoparticles were combined to form DNA-linked
aggregates (Lee, Han, & Mirkin, 2007).
2,4-Dinitrophenol (DNP) was formerly used in body weight control since it increases fat metabolism. In living cells, DNP loses the
energy of the proton gradient as heat instead of producing ATP.
DNP was used extensively in the 1930s in diet pills. Concerns about
dangerous side-effects such as a fatal fever forced DNP being discontinued in the United States by the end of 1938. However some bodybuilders and athletes who wish to rapidly lose body fat still use DNP.
With global bioterrorism is being perceived as real threat to our
safety and security, the traditional methods for detecting toxins are
inadequate. We need a measurement method that is easy to perform and rapid so that the consumers or first responders can identify the presence of toxic substances quickly and take necessary
corrective actions. Such rapid detection of toxins is a significant
step toward securing safety of our food supply from both inadvertent and intentional contamination compared to time-consuming
detection methods such as chromatography and ELISA that are
used today. Therefore, we focused on developing a low-cost, selfindicating nanobiosensor to continually monitor the presence of
toxins in food systems. Since our study is a proof-of-concept investigation of the colorimetric biosensing based on competitive displacement of gold nanoparticles by toxin, it is important that we
use materials that are guaranteed to produce verifiable results.
We selected DNP–GLY as model toxin since all critical materials
including anti-DNP antibody, DNP–BSA (toxin analog), and DNP–
GLY (toxin) are commercially available.
The specific aims of this study were to fabricate a colorimetric
nanobiosensor taking advantage of the chromogenic effect of latex
surface when hybridized with gold nanoparticles and to investigate the effectiveness and robustness of the biosensor using a
model toxin system.
1. Proof-of-concept
The colorimetric, self-indicating nanobiosensor we developed
comprises indicator gold nanoparticles and host latex microspheres. Latex microspheres are optically white; when gold nanoparticles bind into receptors attached to latex microspheres, the
color of latex microspheres change from white to red, the color
of gold nanoparticles. We selected gold nanoparticles as signal
indicators because they can attach to various functional ligands,
and form their assemblies and complexes with various conjugates.
Nanotechnologies using gold nanoparticles can easily accommodate such demands as real-time detection, high sensitivity, high
throughput, and low sample volume. Gold nanoparticles at nanomolar concentration can be clearly observed allowing sensitive
detection with minimal consumption of materials (Jin et al., 2003).
Gold nanoparticles functionalized with antibody attachable
molecules (antibody analogs) have the potential for use in indicator-analyte displacement reaction. Therefore, we pursued the indicator-analyte displacement reaction for the detection of toxins.
Since the affinity of toxins to the binding site is much higher than
that of analog-conjugated gold nanoparticles, the analog segments
associated with the segments in the binding sites are dissociated in
the presence of toxin, and subsequently the toxin molecules displace the analog-conjugated gold nanoparticles and then attach
1
For interpretation of color in Fig. 1, the reader is referred to the web version of
this article.
to the binding sites. Fig. 1 is an illustration of the indicator-analyte
reaction, where in analog-conjugated gold nanoparticles are displaced by toxin molecules. The color change is schematically illustrated from bright red (when there is no contamination) to white
(when the contamination threshold has exceeded). The intermediate pink color indicates the presence of contamination at a level
below a preset threshold. In our nanobiosensor system, toxin analog DNP–BSA was attached to gold nanoparticles and was allowed
to hybridize via antigen–antibody association with anti-DNP antibody on the latex microspheres. Thus, the gold nanoparticles attached non-covalently with the binding site of the latex
microspheres appeared red. When toxin molecules are present in
the system a competition occurs between the analog-conjugatedgold nanoparticles and the toxin molecules for the binding pocket.
When toxins present in food system displace all gold nanoparticles
the latex regains its white color.
2. Materials and methods
2.1. Fabrication of citrate-stabilized gold nanoparticles
We followed published methods to stabilize gold nanoparticles
by preventing them from aggregating (Brown, Walter, & Natan,
2000; Grabar, Freeman, Hommer, & Natan, 1995). Hydrogen tetrachloroaurate (III) (HAuCl4, Acros Organics, Morris Plains, NJ) was
dissolved in the Milli-Q water with 1% (w/v) concentration. Under
stirring, 1 mL of the hydrogen tetrachloroaurate solution was
added to 90 mL of Milli-Q water and stirred at 25 °C for 1 min. Further, 2.0 mL of 38.8 mM sodium citrate (Trisodium salt, Dihydrate,
Sigma-Aldrich, Inc., St. Louis, MO) solution was added, and 1 min
later 1 mL of 0.075% sodium borohydride (NaBH4, Fisher Scientific,
Inc., Pittsburgh, PA) in 38.8 mM sodium citrate solution was added.
This seed solution was stirred for an additional 5 min and stored in
a dark bottle at 4 °C.
Three milliliters of the seed solution was added, along with
71 mL of 38.8 mM sodium citrate solution, to 100 mL of 0.01%
(w/v) HAuCl4 solution. Under stirring, this mixture was boiled for
15 min and then cooled down to 25 °C. This produced purple-color
citrate-stabilized gold nanoparticles.
2.2. Particle size and zeta-potential measurement
Size and zeta-potential of the gold nanoparticles were measured
using a particle analyzer (90Plus + ZetaPlus, Brookhaven Instruments Corp., Holtsville, NY) at 25 °C with a scattering angle of
90°. The dispersion of the gold nanoparticles was sonicated in an
ultrasound bath (Bransonic 1510, Branson Ultrasonic Corp., Danbury, CT) for 5 min and stirred continuously at 25 °C for 30 min.
The measurements were repeated three times for each sample.
2.3. Optical measurement
A UV–visible spectrophotometer (Multispec-1510 Spectrophotometer, Shimadzu, Japan) was used to measure the spectra of
the nanoparticles from 200 to 800 nm. The sample preparation
methods for optical measurement were the same as that for particle size measurement. To eliminate the effect of the cuvette, same
cuvette was used after washing with Milli-Q water after every
measurement.
2.4. Confocal laser scanning microscopy
A confocal laser scanning microscope (MRC-1024, Bio-Rad Inc.,
Hercules, CA) attached to an inverted camera (Eclipse TE300, Nikon
Inc., Japan) was used to verify the binding among latex micro-
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S. Ko et al. / Food Control 21 (2010) 155–161
Toxin
Red
Toxin
White
Pink
Fig. 1. Indicator-analyte displacement between analog-conjugated gold nanoparticles and toxin molecules (
nanoparticles;
, analog;
, toxin;
, latex microsphere;
, toxin antibody;
, gold
, latex microspheres).
sphere, functionalized antibody, and non-covalently bound analog.
A krypton/argon laser with excitation wavelengths of 488, 568, and
647 nm and emission collected above 522 ± 17, 605 ± 16 and
680 ± 16 nm, respectively, with oil-coupled differential-interference contrast objective lens (60 magnification) was used for
imaging.
Latex microspheres or their conjugates with antibodies and
analogs were spread onto a microscope slide (Fisher Scientific,
Inc., Pittsburgh, PA). A drop of Rhodamine B solution (0.05%, laser
grade 99+%, excitation 540 nm; emission 625 nm, ACROS Organics,
New Jersey) was added to facilitate easy visualization during imaging. After placing a cover slip on the slide glass, the specimen was
observed under the microscope.
2.5. Functionalization of gold nanoparticles with toxin analog
Gold nanoparticles were functionalized with a DNP analog (Park
et al., 2003; Park, Kurosawa, Aizawa et al., 2003). A 2,4-dinitrophenol–bovine serum albumin (DNP–BSA, Molecular Probes, Eugene,
OR) was selected as the DNP analog due to its minor affinity to
the antibody. Two milliliters of 5% (v/v) glutaraldehyde/Milli-Q
water was added dropwise to 4 mL of the citrate-stabilized gold
nanoparticles solution. The mixture was stirred at 25 °C for 2 h to
pre-activate the gold nanoparticles for crosslinking. Four milliliters
of 200 lg/mL DNP–BSA solution in a borate buffer (pH 9.5) was
added to pre-activated gold solution, and stirred continuously at
25 °C for 2 h. This yielded DNP–BSA conjugated gold nanoparticles.
the redispersed antibody conjugated latex microsphere dispersion.
After 30 min, the tube was centrifuged at 2650 g for 10 min to remove unbound DNP–BSA conjugated gold nanoparticles and to
precipitate bound DNP–BSA conjugated gold nanoparticles on
anti-DNP antibody/latex microsphere complexes. After centrifugation, the pellet was redispersed in the 0.7 mL of PBS.
2.8. Determining sensor effectiveness in detecting toxin
Model toxin, DNP–GLYcin (DNP–GLY, MP Biomedicals, Inc.,
Solon, OH) was used to study the effectiveness of the nanobiosensor fabricated. The DNP–GLY solution of 0.5 mL at various concentrations was mixed in 0.7 mL of the solution with DNP–BSA
conjugated gold nanoparticles on the anti-DNP antibody conjugated latex microsphere. After 1 min, the mixture containing the
model toxin was centrifuged at 420 g for 10 min to detect the displacement of DNP–BSA conjugated gold nanoparticles by DNP–GLY
and at the binding pockets of the anti-DNP antibody. Absorbance of
the supernatant after the centrifugation was measured using UV
spectrophotometer to quantify the extent of displacement of
DNP–BSA conjugated gold nanoparticles by DNP–GLY molecules.
The absorbance of 0.8 mL supernatant was measured at wavelengths from 200 to 800 nm.
3. Results and discussion
3.1. Concept of self-indicating nanobiosensor for toxin detection
2.6. Immobilization of toxin–antibody on latex microsphere
In Eppendorf tube, 0.05 mL of amino-derivated latex microsphere dispersion (Polybead Amino Microspheres, 3.00 lm, Polysciences, Inc., Warrington, PA) was pipetted and subsequently
0.2 mL of 5% (v/v) glutaraldehyde/Milli-Q water was added. The
tubes were shaken at 60 shakes/min for 2 h to pre-activate the primary amino groups on the latex microspheres. Anti-DNP antibody
(goat, 1 mg/mL, Biomeda Corp., Foster City, CA) was diluted into
0.1 mg/mL stock solution, which consisted of 0.025 M borate buffer
containing 0.15 M NaCl and 0.002% NaN3. The pH of the stock solution was adjusted to 8.0 using 1 M HCl. In the Eppendorf tube containing the pre-activated latex microspheres, 0.5 mL of 0.1 mg/mL
anti-DNP antibody solution was added, and then the tube was shaken at 60 shakes/min for 2 h. This yielded anti-DNP antibody conjugated latex microspheres.
The conjugates so produced were harvested after centrifugation
at 20,800g for 10 min. The pellet was redispersed in 1 mL of phosphate buffered saline (PBS, pH 7.4, Fisher Scientific, Inc., Pittsburgh,
PA), and sonicated in the ultrasound bath for 10 min to remove
loosely bound materials. The particles were recentrifuged at
20,800g for 10 min, and the pellet was redispersed in 0.1 mL of PBS.
2.7. Non-covalent attachment of analog-gold nanoparticles conjugate
on antibody–latex microspheres
To attach via antigen–antibody interaction, 0.05 mL of the DNP–
BSA conjugated gold nanoparticles solution was added to 0.1 mL of
In our self-indicating nanobiosensor the intensity of the color
change corresponded to the extent of contamination present. Thus,
model toxin DNP–GLY was detected and quantified using this selfindicating colorimetric gold nanoparticle/latex microsphere-based
system. Fig. 2 is a flowchart of our self-indicating nanobiosensor
for toxin detection.
3.2. Properties of citrate-stabilized gold nanoparticles
Gold nanoparticles were prepared via particle growth from gold
seeds. First, seeded gold nanoparticles were made by the reduction
of HAuCl4 using citrate. Then, existing gold seeds were increased by
mixing with additional citrate and gold ions. This approach to use
the seeded particle growth is attractive. Since gold ions can be made
limiting, it should be possible to grow particles to a predefined size.
The particle size can be controlled by changing the amount of gold
seeds and citrate used in the mixture (Brown et al., 2000).
The physical properties of the gold nanoparticles prepared are
listed in Table 1. The wavelength of maximum absorbance of the
optical spectra measured by the UV–visible spectrophotometer
was 530 nm. This result was in agreement with that previously reported on gold nanoparticles (Brown et al., 2000; Nath & Chilkoti,
2002, 2004b). Mean particle size was 49 ± 11 nm. We made an effort to prepare about 50 nm gold nanoparticles since sufficient surface area was needed to functionalize enough toxin analogs. The
size of gold nanoparticles is controllable by changing the amount
of gold seeds and citrate as mentioned above. Mean zeta-potential
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S. Ko et al. / Food Control 21 (2010) 155–161
Fabrication of citrated-gold
nanoparticles
Functionalization of gold
nanoparticles with toxin analog
Immobilization of toxin antibody on
latex microspheres
binding to citrate-coated gold nanoparticles occurs by an electrostatic mechanism (Brewer et al., 2005). As verified in our experiments, negatively charged BSA bound to negatively charged gold
nanoparticles. It is somewhat bewildering since overall zeta-potential values of both BSA and gold nanoparticles are negative.
However, it is known that positively charged amino acids such as
lysine in the negatively charged BSA can have electrostatic interactions with negatively charged moieties of the gold nanoparticles
(Brewer et al., 2005). We added glutaraldehyde to secure the
attachment between DNP–BSA and gold nanoparticles and prevent
the detachment between them due to the environmental changes
such as electrostatic condition.
3.4. Immobilization of toxin–antibody on latex microsphere
Non-covalent attachment of analoggold nanoparticle on antibody-latex
microsphere
Introduction of model toxin to
observe color change
Detection and quantification of
model toxin
Fig. 2. Self-indicating nanobiosensor fabrication schemes.
Table 1
Physical properties of citrate-stabilized gold nanoparticles and their functionalized
product.
Product
Mean particle
size (nm)
Gold nanoparticle
Anti-DNP conjugated
gold nanoparticle
49 ± 11
88 ± 1
Zetapotential
(mV)
26 ± 7
26 ± 6
Wavelength at maximum
absorbance (nm)
530
–
of the gold nanoparticles was 26 ± 7 mV. The BSA is known to
bind well on the surface of the gold nanoparticles in the negative
zeta-potential (Brewer, Glomm, Johnson, Knag, & Franzen, 2005).
3.3. Functionalization of gold nanoparticles with toxin-analog
The DNP–BSA has less affinity to the anti-DNP antibody than
DNP–GLY, which is a model toxin (Park, Kurosawa, Aizawa et al.,
2003; Park et al., 2003). Thus, it can occupy the binding site of
the antibody with less strongly and hence it is readily displaced
by DNP–GLY, which has stronger affinity to the binding site of
the anti-DNP antibody.
We studied the effect of the functionalization protocols on the
attachment of DNP–BSA on the surface of the gold nanoparticles.
Mean particle diameter and zeta-potential were used to prove
the functionalization. The physical properties of the citrate-stabilized gold nanoparticles and their functionalized derivatives are
listed in Table 1. Gold nanoparticle + DNP–BSA binding was confirmed by an increase in particle diameter to 88 ± 1 nm and change
in zeta-potential value to 26 ± 6 mV. While the particle size changed significantly, zeta-potential value did not change much before
and after attaching the toxin analog.
The mechanism of BSA and citrate-stabilized gold nanoparticles
binding has not yet been elucidated, but so far it is known that BSA
Anti-DNP antibody was immobilized on the latex microspheres.
Latex microspheres contained surface primary amine groups at the
outer surface. Pre-activation of the latex-bound amino groups with
glutaraldehyde as a coupling agent, and consecutive protein coupling resulted in protein binding from the surface of the microspheres. Aqueous solution of glutaraldehyde has been used for
the stabilization of proteins reacting with their lysinyl residues
(Korn, Feairhel, & Filachio, 1972; Monsan, Puzo, & Mazarguil,
1975). It is well known that reaction of glutaraldehyde with primary amines gives rise to so-called Schiff’s bases. The anti-DNP
antibodies were immobilized onto the latex microspheres by passive adsorption but also by covalent coupling.
We studied how well the antibody immobilization protocols
succeeded in attaching anti-DNP antibody on the surface of the latex microspheres. Attachment of anti-DNP antibody on the latex
microspheres was verified by zeta-potential measurement and by
confocal laser scanning microscopy. Zeta-potential of amino-derivated latex microspheres at pH 7.4 PBS buffer was 24 ± 4 mV
while that of anti-DNP antibody conjugated latex microspheres
was 7 ± 3 mV (Fig. 3). This result indicates that negative charge
on the surface decreases after functionalizing the antibody onto latex microspheres. The binding of anti-DNP antibodies on latex
microspheres was observed using the confocal laser scanning
micrographs shown in Fig. 4. An optical tag, Rhodamine B solution,
was added to the dispersion of latex/antibody conjugates to facilitate easy visualization. The edge of the latex microspheres before
attaching the antibodies appeared blurry and dull as shown in
Fig. 4a. However, the edge of the latex microspheres functionalized
with the antibodies was sharper and brighter since Rhodamine B
molecules were coupled with the antibodies (Fig. 4b). Thus, these
micrographs are evidence that anti-DNP antibodies were attached
successfully on the surface of the latex microspheres using our
immobilization protocols.
3.5. Sensor effectiveness in detecting toxin
The DNP–BSA conjugated gold nanoparticles and the anti-DNP
antibody conjugated latex microsphere formed a complex via antigen–antibody interaction. The DNP–BSA conjugated gold nanoparticles were attached non-covalently with anti-DNP antibody bound
latex microspheres. The observation of their binding by confocal laser scanning microscope is shown in Fig. 4c. The DNP–BSA conjugated gold nanoparticles are displaced by model toxin DNP–GLY
due to the difference of affinity in the binding pocket of the antiDNP antibody. Under the microscope, the edge of the latex microspheres functionalized with the antibodies was brighter while the
background was cloudy due to the scattering of light by gold
nanoparticles.
A series of experiments were performed to observe color change
of the self-indicating nanobiosensor from red to white due to selec-
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S. Ko et al. / Food Control 21 (2010) 155–161
0
-10
-10
Indicator fabrication steps
Zeta-potential (mV)
Zeta-potential (mV)
0
-20
-30
-20
-30
Host fabrication steps
-40
Gold
Gold+Analogue Sensor unit Latex+Antibody
Latex
Fig. 3. Zeta-potential at each fabrication step: (left) indicator fabrication steps of DNP–BSA conjugated gold nanoparticles, (right) host fabrication steps of anti-DNP antibody
conjugated latex microspheres, (center) indicator and host complexes.
Fig. 4. Confocal laser scanning micrographs of (a) latex microspheres, (b) anti-DNP antibody conjugated latex microspheres, and (c) complexes of anti-DNP antibody
conjugated latex microspheres and DNP–BSA conjugated gold nanoparticles.
tive displacement of DNP–GLY. The DNP–GLY displaced the DNP–
BSA conjugated gold nanoparticle from the anti-DNP antibody conjugated latex microsphere complex as shown in Fig. 5. Fig. 6 shows
the absorbance of supernatant in the visible range after centrifugation at 2650 g. The stronger absorbance means more displacement
has occurred. The lowest absorbance was observed in the absence
of DNP–GLY since the displacement did not occur at all. Absorbance was the largest at 5 lg/mL of DNP–GLY. As concentration
of DNP–GLY increased, absorbance increased in proportion to the
DNP–GLY content. Absorbance at 633 nm of supernatant is a function of DNP–GLY concentration shows non-linearity of the absorbance and the DNP–GLY content as shown in Fig. 7. However, the
plot was linearized using a semi-log transformation. Based on a
model ABS = a + b log C, where, C, the independent variable is
DNP–GLY concentration and ABS, the dependent variable, is absorbance, and a and b are constants, resulted in the following model:
ABS = 0.31 + 0.026 log C (R2 = 0.85). This predictive equation helped
to quantify DNP–GLY content between 0.0005 and 5 lg/mL.
A successful nanobiosensor should be highly sensitive and specific and suitable for rapid and accurate detection of food toxins
and other contamination of interest. Some of the potential pitfalls
include false negatives and false positives. Under unfavorable conditions, the analog-conjugated gold nanoparticles might not dissociate completely from the antibody which may decrease sensor
Fig. 5. Stages of analyte-indicator displacement reaction. (a) solution of latex microspheres + DNP antibody, (b) addition of gold nanoparticles (conjugated with DNP–BSA)
turns latex microsphere solution red, (c) solution in b after mild centrifugation (420g) shows good binding of latex microspheres and gold nanoparticles (observe the dark red
precipitate at the bottom), (d) solution b after addition of model toxin (DNP–GLY) and light centrifugation shows that some of the gold nanoparticles have been displaced by
DNP–GLY as observed by the very light color precipitate. (The solution color is still yellow due to the strong yellow color of the DNP–GLY.) (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
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S. Ko et al. / Food Control 21 (2010) 155–161
0.50
The self-indicating feature enables development of this novel
biosensor for use at the consumer-end (e.g., at grocery stores) both
to improve food safety and to counter possible bioterrorism attacks. Although our sensor can provide quantitative information
on the extent of contamination, the resolution and the detection
limit are not as good as the conventional chromatography and
other classical methods. Thus, after the initial screening with our
nanobiosensor, the contaminated samples may be analyzed by an
accredited laboratory for acquiring more precise quantitative data.
Additionally, opportunities exist for our nanobiosensor to be fabricated for simultaneously detecting multiple analytes.
20
0.45
0.40
Absorbance
0.35
0.30
0 µg/mL
0.0005 µg/mL
0.001 µg/mL
0.005 µg/mL
0.01 µg/mL
0.05 µg/mL
0.1 µg/mL
0.5 µg/mL
1 µg/mL
5 µg/mL
0.25
0.20
0.15
0.10
0.05
0.00
625
650
675
700
725
750
775
4. Conclusions
800
Wavelength (nm)
Fig. 6. Absorbance sweep of supernatants including gold nanoparticles at 420g
centrifugation after displacement of DNP–BSA conjugated gold nanoparticles with
different concentrations of DNP–GLY (more displacement is, stronger absorbance
is).
0.40
0.35
Absorbance
0.30
0.25
0.20
We have developed a colorimetric nanobiosensor based on the
chromogenic effect of latex microspheres hybridized with gold
nanoparticles. This proof-of-concept DNP–GLY detection method
was rapid (1 min), straightforward, and inexpensive (no special
equipment is required). The properties of the attachment between
gold nanoparticles and toxin antibodies determine the response
time, sensitivity, and dynamic range since they affect the displacement interactions between gold nanoparticles and toxin molecules.
Type of antibody, solution concentration of analog-conjugated gold
nanoparticles, time of incubation, and temperature are critical variables, which control the attachment of the analog-conjugated gold
nanoparticles in the binding sites of the antibodies. In addition, this
system establishes a general detection methodology that can be applied to a variety of material compositions and particle sizes. Future
experiments to improve sensor specificity (non-specific interactions), sensitivity, and accuracy (reduction of false positive and false
negative) would enable extending this novel technique to detect
various toxins, allergens, pathogens, and other contaminants.
0.15
Acknowledgements
0.10
0.05
0.0001
Supernatant after displacement by DNP-GLY
0.001
0.01
0.1
1
10
DNP-GLY Concentration (mg/mL)
Fig. 7. Absorbance profiles of supernatants including gold nanoparticles at 2650g
centrifugation after displacement of DNP–BSA conjugated gold nanoparticles with
different contents of DNP–GLY.
sensitivity (false negative) and in some instances the attached analog-conjugated gold nanoparticles may dissociate even when toxin
molecules were not present (false positive). These factors need to
be fully investigated under extreme conditions to ensure highly
reliable results.
3.6. Future applications
Due to its simplicity, ease of use, and self-indicating uniqueness
our nanobiosensor design has tremendous potential as simple,
low-cost, and highly-sensitive biosensors for rapid detection of a
wide variety of biological and biochemical entities: various toxins,
allergens, pathogens, and other contaminants. Furthermore, these
sensors are likely to be useful in diverse operating environments
ranging from at-home to on-field conditions. They can also be used
in industries in conjunction with process machinery in-line. Thus,
we envision our system being used in a variety of food processing,
packaging, and handling lines so contaminated foods can be detected and disposed off even before they leave the manufacturing
facility.
This work was supported in part by Hatch grant in the College
of Agricultural and Life Sciences at the University of WisconsinMadison and by Korea Research Foundation Grant by the Korean
Government (MOEHRD, Basic Research Promotion Fund) (KRF2007-331-F00051).
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