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Silver Nanolabels-Assisted Ion-Exchange Reaction with CdTe Quantum Dots Mediated Exciton Trapping for SignalOn Photoelectrochemical Immunoassay of Mycotoxins Youxiu Lin, Qian Zhou, Dianping Tang, Reinhard Niessner, Huang-Hao Yang, and Dietmar Knopp Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02124 • Publication Date (Web): 27 Jun 2016 Downloaded from http://pubs.acs.org on June 30, 2016

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Analytical Chemistry

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Silver Nanolabels-Assisted Ion-Exchange Reaction with CdTe

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Quantum Dots Mediated Exciton Trapping for Signal-On

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Photoelectrochemical Immunoassay of Mycotoxins

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Youxiu Lin,† Qian Zhou,† Dianping Tang,*,† Reinhard Niessner,‡ Huanghao Yang,† and Dietmar

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Knopp*,‡

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†

Key Laboratory of Analysis and Detection for Food Safety (MOE & Fujian Province), Department of Chemistry,

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Fuzhou University, Fuzhou 350108, People's Republic of China ‡

Chair for Analytical Chemistry, Institute of Hydrochemistry, Technische Universität München, Marchioninistrasse 17, D-81377 München, Germany

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CORRESPONDING AUTHOR INFORMATION

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Phone: +86-591-2286 6125; fax: +86-591-2286 6135; e-mail: [email protected] (D. Tang)

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Phone: +49-89-2180 78252; fax: +49-89-2180 78255; e-mail: [email protected] (D. Knopp)

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ACS Paragon Plus Environment


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ABSTRACT: Mycotoxins, highly toxic secondary metabolites produced by many invading species of filamentous fungi, contaminate different agricultural commodities under favorable temperature and humidity conditions. Herein we successfully devised a novel signal-on photoelectrochemical immunosensing platform for the quantitative monitoring of mycotoxins (aflatoxin B1, AFB1, used as a model) in foodstuffs on the basis of silver nanolabels-assisted ion-exchange reaction with CdTe quantum dots (QDs) mediated the hole-trapping. Initially, a competitive-type immunoreaction was carried out on a high-binding microplate by using silver nanoparticle (AgNP)-labeled AFB1-bovine serum albumin (AFB1-BSA) conjugates as the tags. Then, the carried AgNPs with AFB1-BSA were dissolved by acid to release numerous silver ions, which could induce ion-exchange reaction with the CdTe QDs immobilized on the electrode, thus resulting in formation of surface exciton trapping. Relative to pure CdTe QDs, the formed exciton trapping decreased the photocurrent of the modified electrode. By contrast, the detectable photocurrent increased with the increasing of target AFB1 in a dynamic working ranging from 10 pg mL-1 to 15 ng mL-1 at a low detection limit (LOD) of 3.0 pg mL-1 under optimal conditions. In addition, the as-prepared photoelectrochemical immunosensing platform also displayed high specificity, good reproducibility, and acceptable method accuracy for detecting naturally contaminated/spiked blank peanut samples with consistent results obtained from the referenced enzyme-linked immunosorbent assay (ELISA) method.

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Ion-exchange technologies are non-hazardous in nature between two electrolytes/between an

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electrolyte solution and a complex, and being widely used to denote processes of purification

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and separation.1,2 Generally speaking, this technique is employed to improve the sensitivity

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and the selectivity of the detection method by using the pre-concentration process of target

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analyte.3,4 Typically, ion-exchange reactions with inorganic/organic exchangers are applied to

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chemical analysis, recovery of useful ions, and water purification including the preparation of

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'ultrapure' water.5 Apart from these applications, ion-exchange technologies have been also

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utilized to design sensors, e.g., humidity sensor,6 drug sensor,7 carbon monoxide sensor,8 solid

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polyelectrolytes,9 and generation of photovoltage and photocurrent.10,11 To the best of our

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knowledge, however, there are no reports focusing on design of the immunosensors coupling

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with ion-exchange reaction until now.

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Inspiringly, the rapidly emerging research field of ion-exchange-based nanotechnology, and

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the processes used to generate, manipulate and deploy nanomaterials, provides excitingly new

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possibilities for the advanced development of new analytical tools and instrumentation.12-14

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Exchange on the nanocrystals exhibits some unique properties, e.g., rapid kinetics at room

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temperature (orders of magnitude faster than in the bulk), and the tuning of reactivity via

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control of nanocrystal size, shape and surface faceting.15,16 These advantages make it a useful

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method for synthesis of new ionic nanocrystals which may not have conventional synthesis

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approaches.17-19 Manzi et al. innovatively developed a common synthesis route for Cu2S via a

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novel cation exchange reaction from CdS nanocrystals, taking advantage of the reducing

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potential of photoexcited electrons in the conduction band of CdS.20 The Lee's group

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synthesized PbSe nanorods through direct Cd-to-Pb cation exchange in CdSe nanorods.21

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Smith et al. found that carrier locations could be quantitatively mapped and visualized during

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shell growth or cation exchange simply using absorption transition strengths.22 Significantly,

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the Wang's group found evolution of hollow TiO2 nanostructures via the Kirkendall effect

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driven through cation exchange reaction between TiCl4 and ZnO with enhanced photocurrent

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performance.23 To this end, our motivation is exploiting a novel photoelectrochemical

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immunosensing platform via the ion-exchange reaction with the nanocrystals in this work.

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To develop a high-efficient photoelectrochemical (PEC) sensing platform, ongoing progress

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on semiconductor nanocrystals has brought the extensive application in PEC fields,24 especial 3



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quantum dots (QDs),25 which have been confirmed as an attractive photoelectrochemical

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active materials due to their remarkable properties of high photocurrent conversion efficiency,

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generation of multiple charge carriers with a single photon, attractive opto-electronic property,

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large optical cross sections and tunable band gap.26-29 CdTe, a widely used semiconductor in

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thin film solar cell modules with exceptional photostability, has a bulk bandgap of 1.54 eV

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with the conduction and valence band energies at -1.0 and 0.54 V (vs. NHE), respectively,30

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which rendered CdTe as an ideal material for harvesting near-infrared and visible photons.

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Huang et al. used CdTe QDs for sensitive visual detection of Selenium though ion-exchange

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reaction between Cd and Se elements.31 Meanwhile, the same group also utilized CdTe QDs

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for speciation analysis of silver ion based on room temperature cation exchange reaction in

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nanocrystals.32 Experimental results revealed that about 40-fold of Cd2+ ions in the CdTe ionic

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nanocrystals could be exchanged in less than 1.0 min by mixing the nanocrystals with Ag+

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solution at room temperature. This observation provides a novel ideal for the immunoassay

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development using silver nanoparticles as the labels and CdTe QDs as photoactive receptors.

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Scheme 1 Schematic illustration of signal-on photoelectrochemical immunoassay toward target AFB1 on

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the basis of silver nanolabels (AgNPs)-induced ion-exchange reaction with the immobilized CdTe QDs on

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PDDA-modified glassy carbon electrode (GCE): (A) Silver nanoparticle (AgNP)-labeled competitive-type

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immunoassay protocol and (B) silver ion (Ag+)-induced ion-exchange reaction with CdTe quantum dot

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(QD) for photoelectrochemical measurement.

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Aflatoxins (AF) are fungal secondary toxic metabolites including 12 structures, and the

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most common four forms with a toxicity order of AFB1 > AFG1 > AFB2 > AFG2.33 They are

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produced by some certain fungi, especially Aspergillus parasiticus, Aspergillus nomius and

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Aspergillus flavus.34 Aflatoxin B1 is regarded as one of the most harmful contaminants in feed,

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because of their carcinogenic, hepatotoxic, teratogenic and mutagenic adverse effects toward

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animals.35 As a proof-of-concept, herein we design a novel PEC immunosensing protocol for

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sensitive detection of AFB1 based on silver nanolabels-assisted ion-exchange reaction with

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CdTe QDs, mediated the exciton trapping (Scheme 1). The assay is executed with a split-type

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detection mode. Silver nanoparticles (AgNPs) are used for the labeling of AFB1-bovine serum

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albumin (AFB1-BSA) conjugate. Target AFB1 is detected in a competitive-type assay format

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on anti-AFB1 antibody-coated microplates. Introduction of AgNPs is expected to produce

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numerous silver ions with the aid of HNO3. In this case, the released silver ions can exchange

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with CdTe QDs to form the weak photoactive Ag2Te nanocrystals (Ag+ + CdTe → Ag2Te +

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Cd2+). Moreover, the conjugated AgNPs accompanying the specific antigen-antibody reaction

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decrease with the increasing of target AFB1, which can cause the decrease of the exchanged

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CdTe QDs, thereby resulting in the enhancement of photocurrent. In this way, this system

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exhibits a signal-on photocurrent response with the increment of AFB1 concentration in the

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sample. Our aim of this study is looking to develop innovative and powerful signal-on PEC

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immunoassay toward small-molecular mycotoxins to change the signal-off situation of the

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conventional competitive-type immunoassays.

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■ EXPERIMENTAL SECTION

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Materials and Chemicals. Monoclonal anti-AFB1 antibody (mAb 62, clone no. 2B7) was

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synthesized and characterized in our laboratory.36 AFB1-BSA conjugate was a gift from Oil

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Crops Research Institute of the Chinese Academy of Agricultural Sciences (Wuhan, China).

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AFB1 (0.5 µg mL-1 in acetonitrile, analytical standard), AFB2, AFG1, AFG2 and AFB1 ELISA

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kit (cat. no. SE120002; inter-assay CV < 15%, intra-assay CV < 10%, sensitivity: 0 – 4.0 ng

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mL-1, standard curve range: 0.02 – 4.0 ng mL-1) were purchased from Sigma-Aldrich (St.

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Louis, MO 63103 USA). Sodium borohydride (NaBH4), cadmium nitrate [Cd(NO3)2·4H2O],

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bovine serum albumin (BSA) and silver nitrate (AgNO3) were acquired from Sinopharm

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Chem. Re. Inc. (Shanghai, China). 3-Mercaptopropionic acid (MPA) was obtained from Alfa

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Aesar. Sodium tellurite (Na2TeO3) was purchased from Aladdin (Shanghai, China). All other

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reagents were of analytical grade and were used without further purification. Ultrapure water

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obtained from a Millipore water purification system (18.2 MΩ cm-1, Milli-Q) was used in all

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runs. Phosphate-buffered saline (PBS) solutions with various pH values were prepared by

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mixing different volumes of NaH2PO4 and Na2HPO4, and 0.1 M KNO3 was added as the

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supporting electrolyte.

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Preparation and Bioconjugates of Silver Nanoparticles. Silver nanoparticles (AgNPs)

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were synthesized referring to the literature with minor modification.37 All glassware used in

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the work was cleaned in a bath of freshly prepared solution (3:1 K2Cr2O7-H2SO4), thoroughly

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rinsed with ultrapure water and dried prior to use. Following that, silver nanoparticles were

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synthesized by adding 2.0 mL of 1.0 wt % trisodium citrate to 50 mL of boiling ultrapure

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water containing 1.0 mM AgNO3 under vigorous stirring. Afterwards, the resulting mixture

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maintained for about 30 min under the same conditions. During this process, the color of the

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solution became from bright yellow to brown-yellow. Finally, the obtained silver colloids

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were cooled to room temperature, and stored at 4 °C when not in use.

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Next, the as-prepared AgNPs were utilized for the labeling of AFB1-BSA conjugate similar

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to our previous reports.38,39 Before labeling, silver colloids were initially adjusted to pH 9.0 –

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9.5 by using 0.1 M Na2CO3 aqueous solution to avoid the precipitation after addition of

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proteins. Thereafter, 300 µL of 0.5 mg mL-1 AFB1-BSA conjugate was injected into the

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colloidal silver nanoparticles (4.5 mL). The resultant suspension was gently shaken for 12 h at

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room temperature on a shaker (MS, IKA GmbH, Staufen, Germany). During this process,

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partial AFB1-BSA ligands were covalently conjugated to AgNPs through the dative binding

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between silver nanoparticles and free –SH on the BSA, whilst another partial conjugates were

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bound onto the AgNPs via the electrostatic and hydrophobic interaction between proteins and

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AgNPs. Following that, 200 µL of polyethylene glycol (1.0 wt %) was added into the

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suspension to enhance the dispersivity. Finally, the mixture was centrifuged for 10 min at

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14,000 g, and the obtained precipitate, AFB1-BSA-AgNP, was dispersed in 2.0 mL of pH 7.4

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PBS containing 1.0 wt % BSA and 0.1% sodium azide for further use. 6


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Preparation of CdTe QDs-Modified Photosensitive Electrode. Prior to modification, the

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water-soluble MPA-capped CdTe QDs were prepared, based on a typical one-pot synthesis

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method.40,41 Initially, 118 mg of Cd(NO3)2 (≈0.5 mmol) and 200 mg of trisodium citrate were

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dissolved into 50-mL ultrapure water, followed by instant addition of 55 µL MPA. Then the

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resulting mixture was adjusted to pH 10.5 by using 1.0 M NaOH aqueous solution. Following

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that, 22.2 mg of Na2TeO3 (≈0.1 mmol) and 50 mg of KBH4 were added into the mixture in

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sequence, which was refluxed for 60 min at room temperature. The obtained suspension was

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precipitated with n-propanol and centrifuged for 10 min at 8,000g in order to remove the

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impurities. Finally, the carboxylated CdTe QDs were re-dispersed into 1.0 mL of ultrapure

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water.

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CdTe QDs-modified photosensitive electrode was prepared by using poly(diallydimethyl-

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ammonium chloride) (PDDA) as the matrix. A cleaned glassy carbon electrode (GCE, 3.0 mm

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in diameter) was first electrochemically activated by holding it at +2.0 V for 30 s and -1.0 V

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for 10 s in 0.1 M H2SO4 and then pretreated by potential cycling in the same solution within

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the potential range from 0.0 to +1.0 V at 100 mV s-1 until a stable cyclic voltammogram was

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obtained. Following that, 5 µL of 1.0 wt % PDDA aqueous solution was dropped onto the

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treated GCE and dried at room temperature. Finally, CdTe QDs-modified GCE was prepared

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by throwing 5 µL of the above-prepared CdTe QDs on the electrode (QD/PDDA/GCE). In

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this case, the negatively charged CdTe QDs were adsorbed to the positively charged PDDA.

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Immunoreaction Protocol and Photocurrent Measurement. Scheme 1 gives schematic

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illustration of silver nanolabels-induced ion-exchange reaction with CdTe QDs for signal-on

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photoelectrochemical immunoassay of target AFB1. The immunoreaction was carried out in a

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high-binding polystyrene 96-well microtiter plate by coating monoclonal anti-AFB1 antibody

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(50 µL per well, 10 µg mL-1 mAb in 50 mM sodium carbonate buffer, pH 9.6) in the wells (12

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h, 4 °C). To avoid evaporation, the microplate was covered with an adhesive plate sealing

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film during the incubation process. Following that, the microplate was washed three times

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with the washing buffer (10 mM PBS containing 0.05% Tween 20, pH 7.4) and then blocked

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with the blocking buffer (300 µL per well, 10 mM PBS containing 1.0 wt % BSA, pH 7.4) for

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1 h at 37 °C with gentle shaking. The microplate was washed as before. Following that, AFB1

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standard/sample and the above-prepared AFB1-BSA-AgNP at the same volume (25 µL) were 7



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simultaneously injected to the well, and reacted for 60 min at 37 °C under gentle shaking.

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After the microplate was washed again, a 20-µL aliquot of 0.1 mM HNO3 was added to each

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well to release silver ions from the captured AgNP labels (≈10 min). Afterwards, the acidic

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solution containing the dissolved silver ions was transferred onto the CdTe QD-modified

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GCE (reaction for ~15 min). The photocurrent of the resulting electrode was determined in 10

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mL PBS (pH 7.5) at an applied potential of -0.15 V with a conventional three-electrode

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system by using QD/PDDA/GCE as the working electrode, a platinum wire electrode as the

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auxiliary and a saturated calomel electrode (SCE) as the reference electrode. The detection

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cell was equipped with a 500 W Xe lamp (as the excitation light source) and a 420 nm cutoff

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filter (NBET, Beijing, China). All photocurrent measurements were carried out on an

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AutoLab electrochemical workstation (µAUTIII.FRA2.v, Eco Chemie, Netherlands). All

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determinations were made at least in duplicate. A baseline correction of the resulting

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photocurrent was performed with the self-software. The calibration curves were calculated by

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mathematically fitting experimental points using the Rodbard's four-parameter function with

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Origin 6.0 software. Graphs were plotted in the form of photocurrent against the logarithm of

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AFB1 concentration.

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■ RESULTS AND DISCUSSION

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Design of Ion-Exchange Reaction-Induced PEC Immunosensing Platform. Usually,

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most competitive-type immunoassays exhibit a signal-off sigmoidal 'S' relationship with the

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increasing target analytes. In this case, establishment of a relatively strong initial (background)

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signal would be preferable in order to achieve a high sensitivity and a wide dynamic working

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range. Unfavorably, such a high-background signal is unsuitable for the detection of low-level

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target molecules because the signal change caused by a low-concentration analyte is smaller

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relative to the strong initial signal. Hence, the first starting point of this work is to replace the

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conventional signal-off competitive-type immunoassay with a signal-on detection system by

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coupling with two competitive/ion-exchange reactions based on the dialectic point of view

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(i.e., double negation equals affirmation). Another important issue for obtaining low limits of

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detection and quantification lies in the signal-transduction labels (tags). Compared with

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commercial enzyme labels, one major merit of using nanolabels is that one can control and

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tailor their properties in a predictable manner to meet the requirements of specific applications.

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Typically, natural enzymes are relatively expensive, and the signal-generation stage of active

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enzymes is susceptible to interference and the assay conditions. To this end, our secondary

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innovative point relies on silver nanolables-induced ion-exchange reaction with CdTe QDs

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mediated exciton trapping without the participation of bioactive enzymes. Under the visible

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light irradiation, the formed exciton on the surface of CdTe QDs releases electrons into the

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vacant conduction band to promote the dissolved oxygen for the generation of O2- ion. The

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hole in the valence band of CdTe QDs acquires an electron from the electrode, thus producing

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the sensitive photocurrent.

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To interpret and efficiently operate the ion-exchange processes, it is necessary to theorize

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about or model ion-exchange reactions to understand and predict the extent of reactions under

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given conditions. Density Functional Theory (DFT) calculation was employed to demonstrate

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the strong binding affinity between CdTe QDs and Ag+ ions in this reaction by using Cd33Te33

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cluster as a model referring to the literature.42 The adsorption energies were calculated

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according to the equation: ∆Eads = Etotal – EQD – EAg+ (where Etotal, EQD, EAg+ are the electronic

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energies of the Ag+ on the Cd33Te33 QDs, Cd33Te33 QDs and free Ag+ ions, respectively). The

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more negative ∆Eads represents the high adsorption energy. As shown in Figure 1 and Figure

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S1 in the Supporting Information, the largest adsorption energy for Ag+ on the Cd33Te33 was

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-4.91 eV calculated from DFT, indicating that Ag+ ion could chemically adsorb on the

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Cd33Te33 to implement the ion-exchange reaction and form Ag2Te, thereby increasing the

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surface defects of quantum dots. The formed exciton trapping on the surface could decrease

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the photocurrent of CdTe QDs. Meanwhile, these results further revealed that the binding

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force of Ag-Te bond was stronger than that of Cd-Te, while the Ksp of Ag2Te was lower than

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that of CdTe. As a result, Cd2+ ion on the surface of CdTe QD could be chemically replaced

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by Ag+ ion as follows: CdmTen + 2xAg+ → Cdm-xAg2xTen + xCd2+. The formed Ag2Te on the

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surface created new energy level in the band gap. The band gap of Ag2Te was reported to be

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around 0.67 eV, which was lower than the band energy of the CdTe QD, and created a new

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central of electron-hole recombination, thus resulting in formation of the trapping sites.43,44

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The trapping sites on the surface of CdTe QDs inhibited the formation of the exciton to block 9



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the escape of photoelectron, thereby leading to the decrease of cathode photocurrent.

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Figure 1. Structures of Cd33Te33 cluster and its reaction with Ag+ ion (Note: Cd, Te and Ag atoms are

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colored in milk-white, orange and blue, respectively).

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Logically, one important question arises to whether the as-synthesized CdTe QDs could be

6

readily exchanged by silver ions in practical application. To verify this issue, the synthesized

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CdTe nanocrystals were characterized by using different techniques before and after reaction

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with excess Ag+ ions. Figure 2A represents high-solution transmission electron microscope

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(HRTEM, H-7650, Hitachi, Japan) image of the as-synthesized CdTe QDs, and the mean size

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was 5 nm (Note: The size of the as-prepared AgNPs was ~7 nm as shown from the inset in

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Figure 2A). When CdTe QDs reacted with excess Ag+, we vaguely seemed to observe that the

12

lattice and shape of the resulting crystals were different from CdTe QDs alone (Figure 2B).

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Also, the microscopic structures could be further firmed by using X-ray diffraction pattern

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(XRD, PANalytical X'Pet spectrometer) and X-ray photoelectron spectroscopy (XPS, Thermo

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Fisher Scientific, Model Escalab 250 spectrometer). The given XRD pattern (Figure 2C, curve

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'a') was typical of CdTe QDs with high crystallinity, which displayed three characteristic

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peaks occurred at 2θ of 24.9°, 41.3° and 47.8° for 111, 220 and 311, confirming a cubic

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structure of the zino blend type.45,46 However, the XRD's results could not verify the existence

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of silver ions in the nanocrystals because the characteristic peaks were gently changed before

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and after reaction with Ag+ ions (curve 'b' vs. curve 'a'). Significantly, the XPS signature of the

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Ag3d doublet for the resulting silver ion could be clearly appeared (Figure 2D, curve 'b' vs.

8

curve 'a'), suggesting the existence of silver element in the nanocrystals. Moreover, the

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characteristic absorption peak of CdTe QDs was disappeared after reaction with excess Ag+

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ions (Figure 2E, curve 'b' vs. curve 'a'), and the fluorescence was also completely quenched

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(Figure 2F, curve 'b' vs. curve 'a'). The reason was attributed to the formation of Ag2Te after

12

reaction with CdTe QDs and Ag+ ions. These results also revealed that (i) CdTe QDs could be

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successfully synthesized via the one-pot synthesis method, and (ii) the ion-exchange reaction

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could be occurred between CdTe QDs and Ag+ ions.

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Figure 2. HRTEM images of (A) CdTe QDs [insets: (top) magnification image and (bottom) silver

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nanoparticles] and (B) CdTe QDs + excess Ag+ (inset: magnification image); (C) XRD patterns, (D) XPS

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analyses (inset: Ag3d core level XPS spectra), (E) UV-vis absorption spectra (insets: the corresponding

19

photographs) and (D) fluorescence spectra of CdTe QDs before (a) and after (b) reaction with excess Ag+.

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Characterization of Photosensitive Electrode. As an effective tool for characterizing the 11



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interface properties of the modified electrodes, electrochemical impedance spectroscopy (EIS)

2

was used to monitor the fabrication process of the photosensitive electrode. In the Nyquist

3

diagram, the diameter of the semicircle equals the resistance of the electron transfer. As seen

4

from diagram 'a' in Figure 3A, a relatively small resistance was observed at the cleaned GCE.

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With the assembly of PDDA and CdTe QD in sequence, the resistance gradually increased

6

(Figure 3A, diagrams 'b-c'), indicating that PDDA and QDs inhibited the electron transfer

7

between the solution and the base electrode. Further we also monitored the change in the

8

photocurrent after each step (Figure 3B). Almost no photocurrents were acquired at bare GCE

9

(curve 'a') and PDDA/GCE (curve 'b'). When CdTe QDs were modified onto the PDDA/GCE,

10

significantly, a strong photocurrent was appeared (curve 'c'), indicating that the photosensitive

11

electrode was successfully fabricated by using our developed strategy.

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Figure 3. (A) Nyquist diagrams and (B) photocurrent responses for (a) bare GCE, (b) PDDA/GCE and (c)

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QD/PDDA/GCE (note: Nyqusit diagrams obtained in 5 mM Fe(CN)64-/3- + 0.1 KCl with the range from 10-2

15

Hz to 105 Hz at an alternate voltage of 5 mV, photocurrents obtained in 0.1 M pH 7.5 PBS), and (C)

16

photocurrent responses of CdTe QD-modified electrode (a) before and (b) after ion-exchange reaction with

17

silver ions.

18

To realize our design, one precondition for the development of the signal-on immunoassay

19

was whether silver ions could cause the change in the photocurrent of CdTe QDs. Curve 'a' in

20

Figure 3C gives the photocurrent response of CdTe QD-modified GCE. Significantly, the

21

photocurrent decreased after the modified electrode reacted with Ag+ ions (curve 'b'). For

22

comparison, photocurrent responses of PDDA-modified GCE were monitored in the presence

23

of Ag+. Experimental results indicated that almost no photocurrent was acquired in this case,

24

suggesting that the photocurrent derived from the immobilized CdTe QDs. As control test, 12


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1

AgNPs were directly used for the incubation with CdTe QD-modified GCE. Inspiringly, the

2

obtained photocurrents did not change before and after reaction with AgNPs. The results

3

revealed that the decrease in the photocurrent originated from the interaction of CdTe QDs

4

with Ag+, which was in accordance with the previous report.32

5

Optimization of Experimental Conditions. As is well-known, the surface-to-volume ratio

6

and quantum effect of nanocrystals increase with their decreasing size. Doron et al. confirmed

7

that the bigger-size nanocrystals could discontinuously assemble, while the smaller-sized

8

nanostructures might generate continuous particle arrays on the monolayer.47 The packing of

9

small-sized nanocrystals was denser than that of big size due to high surface-to-volume ratio.

10

Unfortunately, the Coulomb repulsion became stronger with the decreasing size. To this end,

11

we prepared four kinds of CdTe QDs with different sizes including 2, 4, 5 and 6 nm referred

12

to previous report.31 Figure 4A shows the effect of the sizes on the photocurrent of CdTe

13

QDs-modified GCE, and the modified electrode with 5-nm CdTe QDs displayed a stronger

14

photocurrent response than those of other sizes. Meanwhile, we also noticed that 5-nm CdTe

15

QDs could exhibit a red fluorescence (Figure 4A, inset). So, 5-nm CdTe QDs were used for

16

preparation of the photosensitive electrode.

17

To enhance the stability of CdTe QDs on the GCE, the nanocrystals were immobilized by

18

combination of two or more phases of different natures, which acted not only as a support for

19

the nanostructures but also a transducer. Owing to introduction of carboxylated CdTe QDs,

20

several positively charged species including polyetherimide (PEI), PAMAM dendrimer and

21

PDDA were used as the immobilized matrices of quantum dots. During the preparation, all the

22

matrices and CdTe QDs used were at the same concentration. As indicated in Figure 4B, the

23

stable and strong photocurrent was obtained by using PDDA as the matrix. The reason might

24

be attributed the fact that PDDA had thermal and photochemical stability under illumination,

25

which could couple with CdTe QDs as photoactive matrix to make the PEC signal more stable

26

and stronger.48 Hence, PDDA was utilized as the support for the immobilization of CdTe QDs

27

on the GCE in this work.

28

As described above, the change in the photocurrent derived from the ion-exchange reaction

29

between CdTe QDs and the released Ag+ from the labeled AgNP. Although a low-pH acidic

30

solution was favorable for the release of silver ions during the PEC immunoassay, it might 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

inhibit the generation of photocurrent of CdTe QDs. Meanwhile, a too low-pH solution might

2

dissolve the quantum dots. More unfavorably, the exposed Cd2+ at the surface defect sites of

3

the CdTe QDs would adsorb the OH- and hindered the electron transfer from QDs to O2, thus

4

lowering the photocurrent. Considering this concern, we investigated the effect of the used pH

5

solution on the photocurrent of PEC immunoassay by using 0.1 ng mL-1 AFB1 as an example.

6

As shown in Figure 4C, the maximum photocurrent was achieved at pH 7.5 PBS, which was

7

used as the supporting electrolyte for PEC measurement. Further, we also investigated the

8

effect of the ion-exchange reaction time between Ag+ ions and CdTe QDs on the photocurrent

9

(Figure 4D). An optimal signal was obtained after 15 min. To save the assay time, 15 min was

10

selected as the ion-exchange reaction time.

11 12

Figure 4. Effects of (A) different-size CdTe QDs (a: 2 nm, b: 4 nm, c: 5 nm and d: 6 nm; Inset: the

13

corresponding fluorescence photographs) and (B) the immobilized matrices with the same concentration (a:

14

PEI, b: PAMAM, c: PDDA) on the photocurrent of the photosensitive electrode in the absence of silver ion;

15

Effects of (C) pH of PBS detection solution and (D) ion-exchange reaction time on the photocurrents of the

16

PEC immunoassay by using 0.1 ng mL-1 AFB1 as an example. 14


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1

Calibration Plots toward Target AFB1. Under optimal conditions, ion-exchange reaction-

2

based immunosensing system was utilized for photoelectrochemical detection of target AFB1

3

standards with different concentrations with a split-type detection mode. The competitive-type

4

immunoassay was carried out in anti-AFB1 (mAb)-coated microplates by using AgNP-labeled

5

AFB1-BSA conjugate as the competitors, and the photocurrent was determined on CdTe QDs

6

-modified GCE through the released Ag+ ions to induce ion-exchange reaction with QDs. As

7

shown in Figure 5A, the corresponding photocurrent increased with the increasing of target

8

AFB1 concentration. A good linear correlation between the photocurrents and the logarithm of

9

AFB1 levels was obtained within the dynamic range from 10 pg mL-1 to 15 ng mL-1 with a

10

correlation coefficient (r) of 0.996 (n = 8) (Figure 5B). Each data represents the average value

11

obtained from three different measurements. The maximum relative standard deviation (RSD)

12

was 9.32%, indicating a good reproducibility. The equation of linear regression could be fitted

13

to y (nA) = 115.92 × logC[AFB1] + 42.34 (ng mL-1). The limit of detection (LOD) was

14

calculated to 3.0 pg mL-1 (S/N = 3), which was obviously lower than those of commercialized

15

available AFB1 ELISA kits for different companies, e.g., 100 ppt from Quicking Biotech, 50

16

pg mL-1 from MaxSignal, 250 pg mL-1 from MyBioSource and 5 pg mL-1 from Diagnostic

17

Automation Inc. Since the threshold value for AFB1 in food is ~2.0 ng mL-1, our strategy

18

could meet the requirement of AFB1 detection.

19

Stability and Selectivity. As mentioned above, the major purpose of using PDDA was to

20

enhance the stability of the photosensitive electrode. To verify this issue, the photocurrents of

21

the as-prepared sensor were detected by controlling the light irradiation, on or off, during a

22

period of 250 seconds. As seen from Figure 5C, the photocurrents were reproducible and

23

stable, and the RSD was ~9.16% (n = 17), thus suggesting an acceptable stability.

24

In addition to reproducibility and stability, specificity is also a key criterion for new assay

25

development because non-specific identification can cause the error of detection results. To

26

clarify this point, some possible simultaneous co-existence mycotoxins in foodstuff, e.g.,

27

AFB2, AFG1, AFG2, ochratoxin A (OTA), brevetoxin B (BTB) and okadaic acid (OA), were

28

evaluated in the absence and presence of target AFB1. As shown in Figure 5D, AFG1, AFG2,

29

OTA, BTB and OA alone did not nearly cause the increase of the photocurrent in comparison

30

with the background signal. Moreover, their co-existence for each other did not result in the 15



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1

significant change of the photocurrent yet. Nevertheless, AFB2 has a high cross-reactivity with

2

anti-AFB1 antibody due to the similar structures between AFB1 and AFB2. Considering this

3

issue, the selectivity of our system was satisfactory.

4 5

Figure 5. (A) Photocurrent responses of ion-exchange reaction-based immunosensor toward target AFB1

6

with different concentrations, (B) the corresponding calibration curve, (C) the stability of the as-prepared

7

photosensitive electrode, and (D) the specificity of PEC immunoassay (AFB1: 0.1 ng mL-1; AFB2: 100 ng

8

mL-1; AFG1: 100 ng mL-1; AFG2: 100 ng mL-1, BTB: 100 ng mL-1, OTA: 100 ng mL-1; OA: 100 ng mL-1).

9

Analysis of Real Samples and Evaluation of Method Accuracy. Actually, the feasibility

10

of a newly building immunoassay is very important for analysis of real samples, especially in

11

the complex matrix. To evaluate them, the naturally contaminated and spiked peanut samples

12

were determined by using our developed ion-exchange reaction-based immunoassay. Note:

13

Naturally contaminated and spiked peanut samples were prepared and described in detail in

14

our previous reports.39 Meanwhile, these samples were measured by using commercialized

15

available AFB1 ELISA kit purchased from Sigma-Aldrich. All data are summarized in Table 1.

16


Page 16 of 22

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1

Method accuracy between PEC immunoassay and AFB1 ELISA kit was evaluated on the basis

2

of a t test and a regression equation. As shown in Table 1, all texptl values in these 12 samples

3

were lower than tcrit (tcrit[4,0.05] = 2.77), and the slope of the regression equation between the

4

average values of two methods was close to the ideal unit '1'. The reproducibility of the PEC

5

immunoassay was further studied by using RSD (< 10.9%, n = 3). Therefore, ion-exchange

6

reaction-based PEC immunoassay could be preliminarily applied for quantitative monitoring

7

of target AFB1 with a good accuracy relative to the referenced enzyme-linked immunosorbent

8

assay method.

9

■ CONCLUSIONS

10

In summary, this work reports on a novel photoelectrochemical immunoassay method for the

11

sensitive detection of mycotoxins (AFB1 used in this case) with a split-type reaction mode.

12

Experimental results indicated that ion-exchange reaction-based immunoassay coupling with

13

silver nanolables could exhibit better detectable sensitivity in comparison with commercial

14

ELISA kits. Compared with our previous reports,39,49,50 highlights of the present system lie in

15

the following issues: (i) Fabrication of the split-type detection platform for immunoreaction

16

and PEC measurement could efficiently avoid the damage of the biomolecules (e.g., antibody

17

or antigen) by the light radiation; (ii) The signal-generation tags through the silver nanolabels

18

were easily operated due to the absence of natural enzymes (i.e., enzyme-free immunoassay),

19

and (iii) the exciton trapping was readily mediated by the ion-exchange reaction between the

20

dissolved silver ions and CdTe QDs. These fundamental advantages make the new technique

21

attractive for further development of photoelectrochemical immunoassays on the basis of the

22

nanolabels and ion-exchange reaction without the needs of natural enzymes. Nevertheless,

23

one disadvantage of ion-exchange reaction-based PEC immunoassay relied on use of 0.1 mM

24

HNO3 for the releasing of silver ions, and the acidic solution affected the photocurrent of the

25

sensing platform. So, future work should focus on improvement of the releasing protocol.

26

■ ACKNOWLEDGEMENT

27

This work was financially supported by the National Natural Science Foundation of China (Grant nos.:

28

41176079 and 21475025), the National Science Foundation of Fujian Province (Grant no.: 2014J07001),

17



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1

the Program for Changjiang Scholars and Innovative Research Team in University (Grant no.: IRT15R11),

2

and the Alexander von Humboldt-Foundation of Germany.

3

■ ASSOCIATED CONTENT

4

Supporting Information

5

Additional information as noted in the text. The Supporting Information contains the computational details

6

on the DFT and Figure S1, which is available free of charge on the ACS Publications website at

7

DOI:10.1021/acs.analchem.0000.

8

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Table 1. Comparison of the Results Obtained by Ion-Exchange Reaction-Based PEC Immunoassay and AFB1 ELISA Kit for Spiked and Naturally Contaminated Peanut Samples method; concentration [mean ± SD (RSD), ng mL-1, n = 3]a type

spiked

no.

PEC immunoassay

AFB1 ELISA kit

texptl

1

43.22 ± 3.67 (8.49%)

46.56 ± 2.43 (5.22%)

1.32

2

1.31 ± 0.12 (9.16%)

1.17 ± 0.08 (6.84%)

1.68

3

12.3 ± 1.21 (9.84%)

14.76 ± 1.21 (8.19%)

2.49

4

19.54 ± 2.01 (10.29%)

22.31 ± 2.12 (9.50%)

1.64

5

7.89 ± 0.69 (8.75%)

7.31 ± 0.53 (7.25%)

1.15

6

0.23 ± 0.02 (8.69%)

0.27 ± 0.02 (7.41%)

2.45

7

14.53 ± 1.53 (10.53%)

16.35 ± 1.57 (9.60%)

1.44

8

8.89 ± 0.72 (8.09%)

9.01 ± 0.91 (10.09%)

0.18

9

43.56 ± 4.54 (10.42%)

46.72 ± 4.32 (9.25%)

0.87

10

23.52 ± 1.76 (7.48%)

20.32 ± 1.92 (9.45%)

2.13

11

19.89 ± 1.42 (7.14%)

21.37 ± 1.87 (8.75%)

1.09

12

6.54 ± 0.71 (10.86%)

6.98 ± 0.53 (7.59%)

0.86

peanut

naturally contaminated peanut

a

Each sample was determined in triplicate, and the high-concentration AFB1 sample was assayed with an appropriate dilution.

The regression equation for the average values obtained by the two methods was as follows: y = 1.0661x – 0.1339 (r = 0.994, n = 12, x axis: the photoelectrochemical immunoassay; y axis: AFB1 ELISA kit).

21



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■ FOR ONLY TOC

22


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