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: Elena.Speranskaya@UGent.be, speranskaya_es@yahoo.com (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

9

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

12

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

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