polymer-coated fluorescent cdse-based quantum dots for application in immunoassay
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Author's Accepted Manuscript
Polymer-coated fluorescent CdSe-based quan-tum dots for application in immunoassay
Elena S. Speranskaya, Natalia V. Beloglazova,Pieterjan Lenain, Sarah De Saeger, ZhanhuiWang, Suxia Zhang, Zeger Hens, DietmarKnopp, Reinhard Niessner, Dmitry V. Potapkin,Irina Yu. Goryacheva
PII: S0956-5663(13)00663-5DOI: http://dx.doi.org/10.1016/j.bios.2013.09.045Reference: BIOS6229
To appear in: Biosensors and Bioelectronics
Received date: 6 August 2013Revised date: 10 September 2013Accepted date: 20 September 2013
Cite this article as: Elena S. Speranskaya, Natalia V. Beloglazova, PieterjanLenain, Sarah De Saeger, Zhanhui Wang, Suxia Zhang, Zeger Hens, DietmarKnopp, Reinhard Niessner, Dmitry V. Potapkin, Irina Yu. Goryacheva, Polymer-coated fluorescent CdSe-based quantum dots for application in immunoassay,Biosensors and Bioelectronics, http://dx.doi.org/10.1016/j.bios.2013.09.045
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Polymer-coated fluorescent CdSe-based quantum dots for application in immunoassay
Elena S. Speranskayaa,b, Natalia V. Beloglazovab, Pieterjan Lenainb, Sarah De Saegerb,
Zhanhui Wangc, Suxia Zhangc, Zeger Hensd, Dietmar Knoppe, Reinhard Niessnere, Dmitry V.
Potapkina, Irina Yu. Goryachevaa
a Department of General and Inorganic Chemistry, Chemistry Institute, Saratov State
University, Astrakhanskaya 83, 410012 Saratov, Russia b Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University,
Harelbekestraat 72, 9000 Ghent, Belgium c College of Veterinary Medicine, National Center for Veterinary Drug Safety Evaluation,
China Agricultural University (CAU), Yuanmingyuan Western Road 2, 100094, Beijing,
China d Department of Inorganic and Physical Chemistry, Ghent University, Krijgslaan 281 S3,
9000 Ghent, Belgium e Technical University of Munich, Institute of Hydrochemistry and Chemical Balneology,
Chair of Analytical Chemistry, D-81377 Munich, Germany
Corresponding author : Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium. Tel: 09/2648137; 09/2648134. E-mail address: [email protected], [email protected] (Elena S. Speranskaya).
Abstract
The paper describes all stages of synthesis and characterization of biocompatible CdSe-based
core/shell quantum dots (QDs) and their application as fluorescent label for immunoassay. A
special attention was focused on development of maleic anhydride-based amphiphilic
polymers for QDs solubilization in aqueous media. In this work two PEG-amines were tried
for polymer modification: monoamine Jeffamine M1000 used previously in some researches
and diamine Jeffamine ED-2003 applied for the first time for QDs solubilization. The use of
different Jeffamines allows to obtain QDs with carboxyl or amine functional groups available
for conjugation. The influence of polymer composition on optical properties of the
nanocrystals and their stability in aqueous solutions as well as on their conjugation with
biomolecules was studied. QDs with different coatings were used as biolabels in quantitative
fluorescence microtiter plate immunoassay and qualitative on-site column test. It was found
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that quantum dots covered with amphiphilic polymer prepared from poly(maleic anhydride-
alt-1-octadecene) and Jeffamine ED-2003 retained up to 90% of their initial brightness, easily
conjugated with protein and showed low non-specific adsorption. In optimized conditions the
obtained QDs were successfully used for determination of mycotoxin deoxynivalenol in
wheat and maize samples by fluorescence microtiter plate immunoassay with an IC50 of 220
�g�kg�1 and by on-site column test with cut-off of 500 �g�kg�1.
Keywords: Quantum dots. Amphiphilic polymers. Fluorescent nanoparticles. Immunoassay.
Deoxynivalenol.
1. Introduction
Semiconductor fluorescent nanocrystals (e.g. quantum dots – QDs) are well-known materials
for use in different fields such as biolabelling, electroluminescent and photovoltaic devices,
and light-emitting diodes due to their unique optical properties (Algar et al., 2011; Frasco and
Chaniotakis, 2009; Petryayeva et al., 2013; Sapsford et al., 2013). QDs have size-tunable
emission with narrow band-width and broad excitation spectra and are characterized by high
photostability compared to conventional organic dyes (Resch-Genger et al., 2008; Rogach,
2008). Due to QDs’ high stability and easily detectable analytical signal they were recently
applied as sensitive fluorescent label in immunoassay (Pinwattana et al., 2010; Trapiella-
Alfonso et al., 2011). Some recently published articles describe development of the QD-label
immunochemical rapid tests for analysis of mycotoxins (Beloglazova et al., 2012; Wang et al.,
2011).
Quantum dots used as biolabels have to meet the following requirements: (i) stability in
aqueous solutions at wide pH range; (ii) high quantum yield (QY) and photostability; (iii) low
non-specific interaction and (iv) presence of functional groups available for conjugation
(Algar et al., 2011; Bruchez and Hotz, 2007; Rogach, 2008). As a rule QDs with high QY and
narrow size distribution are prepared at high temperature in organic solvents (Blackman et al.,
2008; Chen et al., 2010; Medintz et al., 2005). To transfer such hydrophobic QDs to aqueous
solution, modification of the hydrophobic ligand layer is necessary. The two main approaches
for QDs hydrophilization are ligand exchange and encapsulation of QDs with amphiphilic
molecules (Medintz et al., 2005; Yu et al., 2006a). Most of the used hydrophilic ligands for
ligand exchange contain mercapto groups due to high binding affinity of mercapto groups to
the QDs’ surface (Lees et al., 2009; Willard et al., 2001). The problem is the thiol groups
quench fluorescence when directly bound to bare fluorescent cores. So, in this case the
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brightness of QDs after ligand exchange depends directly on the uniformity of the isolating
shell of wider band gap semiconductor (for CdSe cores, ZnS is typically used as an external
shell) and often decreases dramatically (Blackman et al., 2008; Gao et al., 2002). The
encapsulation of QDs with amphiphilic molecules is preferred for QDs hydrophilization
because the original hydrophobic ligands are not removed from the QDs surface in this
process, thus better maintaining the initial QD brightness (Lees et al., 2009; Lin et al., 2008;
Yu et al., 2007). The use of amphiphilic polymers, not molecules or di- or triblock systems, is
preferable because single polymer chains contain multiple hydrophobic units which lead to
strong interaction with the initial organic coating (Sperling et al., 2006; Yu et al., 2006a). It
was proven that introduction of polyethylene glycol (PEG) chains into amphiphilic molecules
leads to improved colloidal stability of QDs over a wide pH range and reduces non-specific
binding to biomolecules and carriers (Bentzen et al., 2005; Susumu et al., 2007). The first
amphiphilic polymers used for QDs encapsulation were not commercially available and their
modification with PEG chains was expensive and laborious. But then it was demonstrated that
application of polymers with reactive maleic anhydride groups allowed to obtain high-quality
polymer coatings (Lees et al., 2009; Yu et al., 2007). The easiest way to modify such
polymers with PEG is a reaction with PEG-NH2: primary amines easily react with maleic
anhydride groups at room temperature. The disadvantage of this approach is the relatively
high price of PEG-derivatives, especially bi-functional PEG-derivatives, and the labor-
intensive modification of PEG under laboratory conditions (Gao et al., 2004; Lin et al., 2008).
Some researchers (Yu et al., 2006b, 2007) proposed to use inexpensive poly(ethylene glycol)
methyl ether (CH3O-(PEG)-OH) to modify polymers. Hydroxyl group can react with maleic
anhydride group with the formation of ester bonding but the reaction only occurs in the
presence of a catalyst (concentrated H2SO4) and after prolonged refluxing. Recently it was
proposed to use the low-cost polyether amine Jeffamine M1000 for modification of maleic
anhydride-based polymers (Lees et al., 2009; Muir et al., 2009; Pichaandi et al., 2013).
Jeffamines are a group of polyethers based on mixed propylene oxide and ethylene oxide,
containing amine groups. Until now, to the best of our knowledge, there exist no data about
conjugation and real application of QDs covered with Jeffamine-based polymers.
In this work CdSe-based core-shell QDs were prepared in an organic solvent. The initially
hydrophobic quantum dots were transferred to aqueous solution by polymer encapsulation.
Amphiphilic polymers were prepared from maleic anhydride-based polymers and Jeffamines
and the optimal conditions for their preparation were established. In this investigation, we
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used two PEG-amines: monoamine Jeffamine M1000 used previously in some researches and
diamine Jeffamine ED-2003 was applied for the first time for QDs solubilization. The use of
different Jeffamines allows to obtain QDs with carboxyl or amine functional groups available
for conjugation.The obtained hydrophilic QDs covered with the polymers retain up to 90% of
their fluorescence after the phase transfer and show stability over a wide pH range. The
fluorescent QDs with different polymer coatings were compared as biolabeles in two assays: a
fluorescence-labeled immunosorbent assay (FLISA) and a rapid column test. Both types of
immunoassays were developed for determination of the mycotoxin deoxynivalenol (DON),
known as “vomitoxin”, a trichothecene mycotoxin and a common cereal contaminant. Various
methods for DON determination were described earlier, such as chromatographic (Monbaliu
et al., 2010; Simsek et al., 2012) and immunochemical techniques (Kolosova et al., 2008;
Schneider et al., 2004). To the best of our knowledge, there exist no QDs-based
immunochemical techniques for DON determination.
2. Experimental
The list of the used reagents can be found in the supporting information (SI1).
2.1. Synthesis of hydrophilic core-shell quantum dots
All syntheses of hydrophobic CdSe cores and CdSe/CdS/CdZnS/ZnS core/multishell QDs as
well as the methods of QDs characterization are described in the supporting information
(SI2).
Water-solubilization of QDs
Amphiphilic polymer synthesis. For amphiphilic polymer preparation, poly(maleic
anhydride-alt-1-octadecene) (PMAO) or poly(styrene-co-maleic anhydride) (PSMA)
polymers containing maleic anhydride groups were conjugated with polyoxyethylene
/polyoxypropylene block-copolymers, contained one primary amine group (Jeffamine M1000)
or two primary amine groups (Jeffamine ED-2003)
Amphiphilic polymers with carboxyl functional groups were synthesized by dissolving
Jeffamine M1000 (2 g) in chloroform (~15 mL) and then added drop by drop to a flask
containing 1 g of PMAO (or 0.425 g of PSMA) powder. PMAO (or PSMA) was readily
dissolved in the solution and the mixture was left to stir overnight. The synthesis of
amphiphilic polymer with amine functional groups was done similarly by dissolving PMAO
powder (0.25 g) in chloroform (10 ml) and then added in drops to Jeffamine ED-2003 powder
(2.5 g). Again, the mixture was left to stir overnight. A large excess of amine was used
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(n(PMAO):n(Jeffamine ED-2003)~1:200 molar ratio) to avoid cross-linking of polymer
chains.
Encapsulation of QDs. For encapsulation, QDs and the amphiphilic polymer were mixed in
chloroform and stirred overnight at room temperature (molar ratio of QD:polymer was ~1:40).
Two different techniques were used to transfer QDs to aqueous solution: thin-film hydration
and reverse-phase evaporation. The first one consists of slow chloroform evaporation under
vacuum resulting in a fluorescent film. Then it was dissolved in an aqueous KOH solution
(pH 12) followed by sonication (20 min). To transfer QDs to aqueous solution by the second
approach the same volume of KOH solution was added to the QD-polymer chloroform
solution. Afterwards, the chloroform was slowly evaporated by a Bunsen’s water-air-jet pump
and a clear fluorescent solution was obtained.
To remove the polymer, an excess of chloroform was added drop by drop to an aqueous QDs
solution (V(CHCl3):V(QDs solution)=1:1). As a result, a white precipitate of amphiphilic
polymer excess at the chloroform-water interface was formed. The sample was centrifuged at
2700 g, and aqueous phase was taken away. The procedure was repeated 3-4 times until the
next chloroform addition did not provoke the formation of white precipitate. After this, the
solution was placed under reduced pressure (with water-jet pump) to remove chloroform
residues. To completely remove any polymer residues, a method developed by Bronstein et al.
(2010) and Yu et al. (2006b) was used: an aqueous QDs solution was ultracentrifuged at
200 000 g, and the QDs precipitate was suspended in water followed by sonication.
2.2. Conjugation of QDs with DON
First, deoxynivalenol-ovalbumin (DON-OVA) was obtained via synthesis of a DON-
carboxymethyloxime derivative according to the modified technique described by Burkin et
al. (2000). For coupling of DON-OVA with NH2-containing QDs, thiolation of DON-OVA
with 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) was done. In
parallel, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-
hydroxysuccinimide ester sodium salt (sulfo-SMCC) was used to modify NH2-containing
QDs. The conditions used for conjugation are described in the supporting information (SI3).
Conjugation of COOH-contained QD was done according to Beloglazova et al. (2012). The
prepared conjugates DON-NH2-QDs and DON-COOH-QDs were kept at 4 °C.
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The description of FLISA, gel-based immunoassay procedure, sample preparation, analytical
performance of the column test and LC-MC/MC procedure can be found in the supporting
information (SI4 – SI8).
3. Results and discussion
3.1. Synthesis of fluorescent QDs
CdSe quantum dots were prepared via a rapid hot-injection method in octadecene. To improve
fluorescence intensity and stability, CdSe nanocrystals were covered with an inorganic shell
of the wider band-gap semiconductor. The most suitable shell for CdSe cores is zinc sulfide
due to its largest band gap between Cd-Zn chalcogenides (Palmer, 2008; Xie et al., 2005). An
additional layer of CdS semiconductor with intermediate lattice parameters is grown between
the CdSe core and the ZnS outer shell to reduce the strain inside nanocrystals (Li et al., 2003;
Reiss et al., 2009). CdSe cores with initial d~2.4 nm (�fl = 524 nm) were used to obtain core-
shell CdSe/CdS/CdZnS/ZnS QDs (�fl = 594). The formation of a CdS shell around the CdSe
cores resulted in a sufficient red shift of both the absorption and fluorescence maxima (Fig.
S1) due to the band gap of CdS which is not large enough to provide the potential barrier
necessary to block both electrons and holes inside the CdSe core. The fluorescence QY of
CdSe cores did not exceed 1.5%, but QY was sufficiently improved up to 40% after coating
with wider band-gap semiconductor (Fig. 1). Transmission electron microscopy (TEM)
images of initial CdSe cores and core-shell QDs are presented in Fig. 2. The nanocrystals
have a spherical shape, the average diameter of core-shell QDs (CdSe/CdS/CdZnS/ZnS)
calculated in ImajeJ program was 7.6 nm.
3.2. Preparation of amphiphilic polymers
Two different commercially available polymers were used: PMAO and PSMA. They differ in
hydrophobic chain composition and molar mass. The polymers contain reactive maleic
anhydride groups which are ready for modification (Fig. S2). PMAO and PSMA polymers
without any derivatization could transfer QDs from chloroform to water solution: maleic
anhydride rings open in alkaline solution with the formation of hydrophilic carboxyl ions
which can stabilize an aqueous QD solution by electrostatic repulsion (Fig S2, A).
However it was shown that it is preferable to introduce PEG chains into the QD ligand layer
to increase the QD’s stability at different pH and ionic strength and to sufficiently decrease
QD non-specific interaction with biomolecules (Lees et al., 2009; Yu et al., 2007). In this
work we used low-cost polyether amine Jeffamines as PEGylated agents: the primary amine
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groups react with maleic anhydride groups easily at room temperature according to scheme
(Fig. S2). In the Jeffamines M1000 and ED-2003 which were used in the present work,
ethylene oxide fragments are dominant and the polyethers easily dissolve in both water and
chloroform. We used Jeffamine M1000 to produce amphiphilic polymers with carboxyl
functional groups and Jeffamine ED-2003 to produce polymers with primary amine groups
for further conjugation (Fig. S2, B).
The formation of polymers was monitored by FTIR characterization (Fig. 3). Peaks at 1780
cm-1, 1850 cm-1 correspond to C=O vibrations; the 1710 cm-1 peak corresponds to the C=O
vibration of carboxylic acid. During the reaction, 1780 cm-1 and 1850 cm-1 peaks intensities
should decrease, thus indicating the disappearance of the anhydride ring. As shown in Fig. 3,
the PMAO polymer initially has carboxylic groups that could have been formed through
partial hydrolysis of maleic anhydride groups during polymer preparation or storage while the
initial PSMA polymer doesn’t contain carboxyl groups (Fig. 3B).
In addition, the water solubility of the modified polymers was checked by the following
method: chloroform was completely removed by rotor evaporation from the polymer solution
and the KOH aqueous solution was added, followed by sonication. To determine the optimal
composition of amphiphilic polymer, different Jeffamine M1000:PMAO molar ratios (30:1,
40:1, 80:1, 90:1) were tested (suggesting 40000 g/mol as the average molecular weight of
PMAO). The polymers with n(Jeffamine M1000):n(PMAO) molar ratio less than ~80 were
not water-soluble. As is apparent from the FTIR-spectra (Fig. 3A), the obtained water-soluble
polymer (n(Jeffamine M1000):n(PMAO) ~�80) does not contain unreacted maleic anhydride
groups. The PMAO-Jeffamine M1000 amphiphilic polymer was used for QDs encapsulation.
In the case of PSMA-Jeffamine M1000 polymer, the same approach gave
Jeffamine/PSMA=1:8 molar ratio as optimal (Fig. 3B). PMAO-Jeffamine ED-2003 polymer
was prepared with a high excess of Jeffamine ED-2003 (Jeffamine:PMAO=200:1 molar
ratio). PMAO solution was slowly added to Jeffamine powder in order to avoid cross-linking
of PMAO polymer chains by diamine. The obtained polymer easily dissolved in an aqueous
KOH solution and its FTIR-spectrum doesn’t contain peaks corresponding to maleic
anhydride group.
3.3. Quantum dots water solubilization
The hydrophobic ligands on QDs surface intercalate with hydrocarbon chains of amphiphilic
polymer by hydrophobic interactions, and the hydrophilic parts of the amphiphilic polymer
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are exposed to solution and provide colloidal stability in aqueous solutions (Lin et al., 2008;
Yu et al., 2007; Zhao et al., 2010). The main advantage of this approach is that the initial
hydrophobic ligands (octadecylamine molecules in our case) are not removed from the QD
surface and the surface is not being disturbed during this process (Fig. 4). So, QDs do not
suffer a dramatic loss in brightness after solubilizing in water.
QDs covered only with PMAO polymer were obtained by the reverse-phase evaporation
technique. QDs covered with PMAO showed reduced fluorescence brightness (Fig. 1) and
formed stable solution only at basic pH because nanocrystals are stabilized by electrostatic
repulsion of deprotonated carboxylic groups (Fig. S2).
In the case of Jeffamine-modified polymers (PMAO-Jeffamine M1000 and PMAO-Jeffamine
ED-2003) both approaches to transfer QDs to water solution were used. But the reverse-phase
evaporation technique showed a higher reproducibility. QDs covered with PSMA-Jeffamine
M1000 were not stable during storage and upon dilution – therefore it was impossible to
measure their QY. We suppose this is due to weak interaction between hydrophobic ligands
on QDs surface and hydrocarbon chains of polymer originating from the big difference in
chain lengths (Fig. 4).
After water solubilization there was no shift in fluorescence and absorbance spectra (Fig. S1);
the fluorescence brightness of QDs covered with PMAO-Jeffamine decreased only slightly in
optimized conditions after solubilization in water (Fig. 1). It was established that QDs covered
with PMAO-Jeffamine M1000 did not precipitate in the pH range 4-12. Carboxyl groups
formed during the reaction were then used for QDs conjugation with biomolecules (Fig. 4).
Similar results for fluorescence and stability were received for QDs covered with PMAO-
Jeffamine ED-2003. The primary amine groups of the polymer were used for conjugation of
such QDs with biomolecules. TEM images of the polymer-coated QDs did not show the
presence of aggregates (Fig. 2, C-D). The high-resolution TEM (HRTEM) image of QDs
covered with PMAO-Jeffamine ED-2003 (�fl = 594 nm) is presented in Fig. 2, E: a well-
developed lattice structure in the QDs was observed.
3.4. Preparation of the QD-labeled conjugates
The obtained QDs were applied as label in two variants of solid-phase immunochemical assay
(FLISA and on-site column test). A main challenge in the synthesis of QD-labeled conjugates
is the determination of the optimal ratio of QDs and analyte-protein. A high ratio (i.e. high
QDs amount) might result in an elevated luminescence of the test-zone which could lead to a
false interpretation of the obtained results. Whereas too low QDs concentration leads to a
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weak analytical signal, which impedes a decrease in antibody concentration and an increase in
sensitivity of the developed method.
Two different kinds of water-soluble QDs covered with PMAO-Jeffamine M1000 (carboxyl
groups present on their surface, COOH-QDs) and PMAO-Jeffamine ED-2003 (NH2 groups
are available for coupling, NH2-QDs) were compared in terms of conjugation with the
analyte-protein complex. First, the optimal conjugation parameters were selected by coupling
QDs just with OVA, and afterwards they were applied for the labeling of DON-OVA. After
synthesis, the presence of QD-OVA conjugates was confirmed by gel electrophoresis: the
obtained QD-OVA conjugates and free QD moved with different rates through the gel.
Two binding techniques for the COOH-QD were evaluated: application of just 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) as linker and combining of N-
hydroxysulfosuccinimide sodium salt (sulfo-NHS) and EDC. The addition of sulfo-NHS is
essential to stabilize the amino-reactive intermediate by converting it to an ester, thus
increasing the efficiency of the EDC-mediated coupling reaction. According to Fig. S3 the
reaction product made by use of EDC (OVA-COOH-QDs 1) migrated through the agarose gel
with the same speed as free COOH-QD. On the other hand, the conjugate synthesized using
the mixture of linkers (OVA-COOH-QDs- 2) was successfully obtained and further syntheses
were performed with this two-steps protocol. For coupling of NH2-QD with OVA, the
heterobifunctional cross-linking reagents sulfo-SMCC and SPDP were used. In this case
conjugating NH2-QD with OVA comprised several steps. QD and OVA were activated with
sulfo-SMCC and SPDP, respectively, afterwards QDs were coupled with OVA. This
conjugation technique is originally based on the modification of proteins. NH2-QD contains
carboxyl groups as well, but they are enclosed by the PEG chains, impeding their contribution
to the conjugation with OVA. Anyway, use of sulfo-SMCC prevents this possible weak
binding of amino and carboxyl groups of the QD. Different ratios of OVA and QD were
verified and it was found that a 25-fold molar excess of OVA compared to QD was suitable
for application in immunoassay.
Finding the optimal conjugate allowed to identify an important feature. In our previous
publication, the water-soluble QDs covered with denaturated bovine serum albumin (dBSA)
(carboxyl groups present on the surface of QD) was used (Beloglazova et al., 2012), and the
conjugation of the QDs covered with dBSA to zearalenone-OVA was obtained via EDC.
Comparison of QDs covered with dBSA with COOH-QD and NH2-QD showed higher
adsorption of dBSA-coated QDs during assay onto the polyethylene frits used for fixation of
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the gel in an on-site column test (Fig. S4, a). Replacing dBSA by PMAO-Jeffamine M1000
resulted in the decreasing of this undesirable adsorption compared with dBSA-coated QDs,
however, non-specific interaction with the polyethylene frits was still observed (Fig. S4, b),
whereas NH2-labeled conjugates did not show it (Fig. S4, c). This can be related to the
conjugation technique, since SPDP is less likely to polymerize the protein. For all further
experiments DON-OVA labeled with NH2-QD (DON-NH2-QD) was used.
3.5. Development of immunochemical techniques based on QD labels
The anti-DON antibody pairing with DON-horseradish peroxidase showed an IC50 of 540 μg
kg-1 with direct competition enzyme-linked immunoassay (data not published). Application of
DON-NH2-QD as a label resulted in a 2.5-times decrease in IC50 value with FLISA (220 μg
kg-1) with a detection limit of 1,2 μg kg-1. Validation of the developed FLISA was done using
naturally contaminated cereal samples (7 maize and 2 wheat samples for checking of
applicability for two different matrices) and results were compared with liquid
chromatography with tandem mass spectrometry detection (LC-MS/MS). For the wheat
samples with 436 and 217 μg kg-1 of DON (according to LC-MS/MS data), 553 and 261 μg
kg-1, respectively, were found by FLISA. Results obtained after screening of maize samples
by FLISA are presented in Fig 5. These data show the good correlation between the two
techniques.
DON is one of the most frequently occurring mycotoxins in cereals and contamination of
cereals products with DON has increased (De Boevre et al., 2012). The maximum level for
DON in unprocessed cereals (other than durum wheat, oats and maize) set by Commission
Regulation (1126/2007) is 1250 �g kg-1. So, an on-site test with cut-off levels of 500 �g kg-1
(bearing in mind the sample pretreatment procedure, the sensitivity of test should be around
25 ng mL-1) was developed. As previously mentioned, application of DON-NH2-QD did not
show any kind of strong non-specific interaction with carrier materials, no special tricks are
essential to avoid it and no special blocking reagents should be applied. All possible
undesirable effects were quite easily avoided by the dilution of the labeled conjugate in
phosphate-buffered saline containing 0.05 % (v/v) Tween 20 (PBST) (1/25) and using PBST
as washing solution (3 mL). By observation with the naked eye, in the absence of DON or its
presence in concentrations less than 25 ng mL-1, a luminescence of the test layer was seen.
DON concentrations equal or higher than 25 ng mL-1 resulted in a reproducible absence of
luminescence (Fig. 6). The detection time was set at 6 min.
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An intra-laboratory validation was performed with blank maize extracts (absence of DON was
confirmed by LC-MS/MS) spiked with DON at concentrations below, equal and above the
cut-off level. Analytical parameters were determined for the cut-off level of 25 ng mL–1 based
on the summarized data after a fivefold repetition of the experiments. Obtained false-positive
and false negative rates were less than 2% each, the specificity rate was 97% and the
sensitivity rate 98% thus complying to the requirements set by the Commission Decision
(2002/657/EC) for a screening method.
The developed on-site test was applied for DON determination in seven maize samples (DON
concentration in the range of 76 – 436 �g kg-1, Fig. 5). To prevent possible false results due to
matrix interferences, non-spiked and spiked (DON 500 �g kg-1) portions were analyzed for
each sample. No false results were observed for spiked samples. Comparison of the
immunoassay and chromatography results showed good agreement for both non-spiked and
spiked samples.
Conclusions
In summary, the initially hydrophobic CdSe-based QDs were transferred to aqueous solutions
by coating with amphiphilic polymers. The amphiphilic polymers were synthesized from
cheap and stable reagents; optimal ratios of initial components were found. It was shown that
QDs covered with the synthesized polymers retained up to 90% of their initial brightness.
QDs covered with the polymers containing different groups for conjugation were compared as
biolabels in two variants of solid-phase immunochemical assay: FLISA and on-site column
test. It was shown that QDs covered with the polymer prepared from poly(maleic anhydride-
alt-1-octadecene) and Jeffamine ED-2003 almost did not show any non-specific adsorption on
the microtiter plates or polyethylene frits. Such QDs were successfully used in column on-site
test for fast detection of DON with a cut-off of 500 �g�kg�1. Application of these QDs as a
label in FLISA resulted in a 2.5-times decrease in IC50 value (220 �g�kg�1) as compared to the
traditional enzyme-labeled immunosorbent assay.
Acknowledgment
This research was financially supported by the Belgian Federal Science Policy Office
(BELSPO) in order to promote the S&T cooperation with China (contract No BL/02/C58), the
Special Research Fund (BOF), Ghent University (01SB2510) and the Russian Foundation of
Basic Research (RFBR, project 12-03-91167). E.S. Speranskaya also benefits from a
fellowship granted by the Belgian Federal Science Policy Office (BELSPO). We thank our
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collaborator Prof. Soumyo Mukherji of IIT Bombay for the TEM images which were acquired
in the FEG-TEM central facility at CRNTS, IIT Bombay
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Figure captions
Fig. 1. The relative fluorescence quantum yields: A: initial CdSe cores covered with OA (toluene); B: core-shell QDs covered with ODA/OLA (toluene); C: core-shell QDs covered with PMAO (aqua); D: core-shell QDs covered with PMAO-Jeffamine M1000 (aqua); E: core-shell QDs covered with PMAO-Jeffamine ED 2003 (aqua) Fig. 2. TEM images of hydrophobic (A,B) and water-soluble (C-E) QDs: CdSe cores (A), CdSe/CdS/CdZnS/ZnS (B), CdSe/CdS/CdZnS/ZnS covered with PMAO-Jeffamie M1000 polymer (C) and PMAO-Jeffamine ED2003 polymer (D) and HRTEM of the sample shown in (D)
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Fig. 3. FTIR spectra of initial reagents and the amphiphilic PMAO-Jeffamine M1000 (A) and PSMA-Jeffamine M1000 (B) polymers Fig. 4. Schemes of encapsulation of hydrophobic QDs (I) with different amphiphilic polymers: II: PSMA (R = O-) or PSMA-Jeffamine M1000 (R = NH(C2H4O)19(C3H6O)3CH3); III: PMAO-Jeffamine M1000; IV: PMAO-Jeffamine ED-2003 Fig. 5. Linear regression equation derived using FLISA and LC–MS/MS data for deoxynivalenol screening in naturally contaminated maize samples (n = 5)
Fig. 6. DON detection in maize samples by on-site column test. DON concentration in spiked sample is presented below the tests. Cut-off level (= no fluorescence) was at DON concentration 500 �g�kg-1
Highlights
� The amphiphilic polymers from stable and inexpensive reagents were produced � The influence of polymer composition on QDs’ properties was studied � Polymer-coated quantum dots retained up to 90% of the initial brightness � QDs-based immunoassay for determination of deoxynivalenol was developed � The detailed description of all steps of QDs synthesis and application is presented
0
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