Rapid and sensitive label-free determination of aflatoxin M1 levels in milk through a White Light Reflectance Spectroscopy immunosensor

Accepted Manuscript Title: Rapid and sensitive label-free determination of aflatoxin M1 levels in milk through a White Light Reflectance Spectroscopy immunosensor Authors: Dimitra Tsounidi, Georgios Koukouvinos, Panagiota Petrou, Konstantinos Misiakos, Grigorios Zisis, Dimitris Goustouridis, Ioannis Raptis, Sotirios E. Kakabakos PII: DOI: Reference:

S0925-4005(18)31980-4 https://doi.org/10.1016/j.snb.2018.11.026 SNB 25624

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

28 June 2018 17 October 2018 6 November 2018

Please cite this article as: Tsounidi D, Koukouvinos G, Petrou P, Misiakos K, Zisis G, Goustouridis D, Raptis I, Kakabakos SE, Rapid and sensitive label-free determination of aflatoxin M1 levels in milk through a White Light Reflectance Spectroscopy immunosensor, Sensors and amp; Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.11.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Rapid and sensitive label-free determination of aflatoxin M1 levels in milk through a White Light Reflectance Spectroscopy immunosensor

Dimitra Tsounidia, Georgios Koukouvinosa, Panagiota Petroua*, Konstantinos

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Misiakosb, Grigorios Zisisc, Dimitris Goustouridisc,d, Ioannis Raptisc, Sotirios E.

a

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Kakabakosa*

Immunoassays/Immunosensors Lab, Institute of Nuclear & Radiological Sciences &

Technology, Energy & Safety, NCSR “Demokritos”, 15341 Aghia Paraskevi, Greece Optical Sensors Lab, Institute of Nanoscience and Nanotechnology, NCSR

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ThetaMetrisis S.A., 12132 Athens, Greece

Department of Electronics Engineering TEI of Piraeus, 12244 Egaleo, Greece

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d

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c

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“Demokritos”, 15341Aghia Paraskevi, Greece

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Panagiota Petrou

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Corresponding author:

Immunoassay/Immunosensors Lab

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INRASTES

NCSR “Demokritos” Neapoleos 27 & Patriarchou Gregoriou

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15341 Aghia Paraskevi Greece E-mail: [email protected] Tel: ++30 210 6503819, Fax: ++30 210 6543526

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GRAPHICAL ABSTRACT

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Abstract

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Α miniaturized optical immunosensor based on White Light Reflectance Spectroscopy (WLRS) for the rapid and label-free detection of aflatoxin M1 (AFM1) in milk samples

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has been developed. WLRS sensing system consists of the measurement set-up and the

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biochip. The first encompasses the reflection probe, the light source and the spectrometer, while the latter is a Si chip with a SiO2 layer on top where an AFM1bovine serum albumin conjugate has been immobilized. The assay was performed by

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running mixtures of rabbit polyclonal anti-AFM1 antibody with the calibrators or the samples, followed by reaction with biotinylated anti-rabbit IgG antibody and streptavidin. The assay cycle was completed in 25 min, the limit of detection was 6 pg/mL, and the linear working range extended from 0.012 to 2.0 ng/mL. The assay was repeatable (intra-and inter-assay coefficients of variation ranged from 2.1 to 6.3% and 2

3.5 to 8.2%, respectively) and accurate (percent recovery values ranged from 92.5 to 110%). AFM1 could be detected with the immunosensor developed in both processed and unprocessed milk of different animal species without any dilution. The excellent analytical characteristics and the small instrument size make the proposed sensor suitable for accurate low-cost AFM1 determination in milk samples at the point-of-

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need.

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Keywords: Aflatoxin M1; Milk; White Light Reflectance Spectroscopy; Immunosensor

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1. Introduction

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Aflatoxin M1 (AFM1) is the hydroxylated metabolite of Aflatoxin B1 (AFB1), the most

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carcinogenic and toxic aflatoxin produced by fungus [1]. It is estimated that 0.3-6.2% of AFB1 delivered through the food is metabolized to AFM1 and excreted in the milk

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of animals (including human) that have ingested food contaminated with AFB1 [2, 3]. The exact percentage of the conversion depends on the concentration of AFB1 in foods

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and animal feedstuffs, as well as on the health of the animals, the milking process, and the environmental conditions [4]. AFM1 is, as its parent compound AFB1, a very

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mutagenic and carcinogenic compound and therefore since 2002 it has been categorized by the International Agency for Research on Cancer as group I carcinogen to humans

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[5]. Due to its high toxicity, the levels of AFM1 in milk are strictly regulated since the ingestion of even low concentrations pose a significant risk to human health, particularly for infants who are the major consumers of milk. Thus, in European Union the maximum allowable limit for AFM1 in infant milk is 0.025 ng/mL and in milk for adult consumption is 0.05 ng/mL [6], whereas, in USA the limit is 0.5 ng/mL for all

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milk types [2]. Any milk should be checked for the presence of AFM1 preferably at the point of its collection (e.g., the farms) and the measurements should be confirmed by the dairy industry central quality assurance laboratories after transfer to milk processing facilities [1]. A plethora of methods have been developed so far for the determination of

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AFM1levels in milk [7], including thin-layer chromatography [8] and high performance liquid chromatography coupled to a mass spectrometry [9 -12] or fluorescence detector

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[13, 14]. The latter are also the reference methods employed by the analytical labs for

the determination of AFM1 in milk samples. These techniques provide high sensitivity

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and accuracy, but involve time consuming sample preparation [15], expensive

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equipment and should be performed by well-trained personnel [7]. Immunochemical

[19-21]

and

chemiluminescence

immunoassays

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or

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fluorescence

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methods, like enzyme-linked immunosorbent assays (ELISA) [1, 6, 16-18],

immunochromatographic strips [23, 24] are also employed for the determination of

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AFM1 in routine analysis. Standard microtiter plate immunoassays offer the required detection sensitivity with simpler sample preparation procedures and instrumentation

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of lower cost compared to conventional chromatographic instrumental methods; however, most of them are time consuming and not suitable for on-site analysis [25].

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On the other hand, immunochromatographic strips are suitable for on-site determinations but they offer only semi-quantitative results and are, therefore, used

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mainly for screening purposes. Thus, there is a demand for analytical methods that could provide quantitative

results at the point-of-need [26, 27]. To satisfy this demand, several biosensors based on electrochemical [28-32] or optical transducers [33-37] have been reported for the determination of AFM1 in milk samples. Optical biosensors and specifically those

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offering label-free detection, have considerable advantages over other sensing principles since they combine increased miniaturization with less interference from the sample matrix [38]. Nonetheless, the optical label-free sensors developed, so far, for the determination of AFM1 [33, 34] do not fulfill the detection sensitivity requirements set by the regulatory EU authorities. An alternative approach for determination of

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AFM1 in milk, proposed in the literature, relies on the implementation of Fourier transform infrared (FT-IR) spectroscopy combined with Principal Component Analysis

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(PCA) [39]. This method, although rapid, requires a rather cumbersome sample

treatment prior to their analysis including sonication so as to homogenize the fat

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globules and avoid interference in the measurements by scattering of light from fat

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particles, thus complicating its application for on-site determinations.

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In this report, we present a rapid and easy-to-use method for the label-free

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determination of AFM1 in milk samples implementing White Light Reflectance Spectroscopy (WLRS). The WLRS sensing system consists of two main components:

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the USB powered & controlled measurement set-up and the biochip [39]. The measurement set-up is a compact unit that encompasses all the necessary electronic and

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optical parts for the implementation of the optical measurements: the reflection probe, the light source, the spectrometer, and the readout and control electronics. The

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reflection probe is a bundle of six optical fibers arranged at the periphery of a central seventh fiber; the six peripheral fibers guide the light from the source vertically to the

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biochip while the central one collects the reflected light. The interference reflectance spectrum is created when the light incidents vertically a surface that is composed by a reflective surface (Si) and a transparent layer (SiO2). The light is partially reflected at the SiO2/environment interface, passes through the transparent SiO2 layer and reflected again at the SiO2/Si interface; this way the SiO2 layer acts as interference spacer.

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Biomolecular reactions that take place on the SiO2 surface cause a red shift of the reflectance spectrum due to increase of the biomolecular adlayer thickness. This shift can be monitored in real-time by a miniaturized spectrometer with high spectral resolution and converted to effective biomolecular layer thickness by the implementation of dedicated algorithms [41-43]. For the determination of AFM1 with

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the WLRS platform, a competitive immunoassay format was followed which included

functionalization of the chip through immobilization of an AFM1-protein conjugate.

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The assay was performed by running mixtures of anti-AFM1 specific antibody with the calibrators or the samples over the biochip, followed by reaction with biotinylated

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secondary antibody and streptavidin in order to increase the biomolecular adlayer

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thickness. The assay developed was evaluated with respect to its analytical

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characteristics as well as through the determination of AFM1 in milk samples of

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different origin, both unprocessed and processed ones (pasteurized and homogenized). The solution developed addresses all the requirements for application at the point-of-

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need determination of AFM1 levels, e.g., short assay time and low analysis and instrumentation cost, with analytical sensitivity that well surpasses the requirements set

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by EU and USA regulation authorities.

2. Materials and methods 2.1 Materials

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AFM1 and AFM1 conjugate with bovine serum albumin (AFM1-BSA), aflatoxin

B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), goat polyclonal anti-rabbit IgG antibody and streptavidin-peroxidase conjugate were purchased from Sigma-Aldrich (St. Louis, MO). The polyclonal rabbit anti-AFM1 antibody was purchased from AntiProt (Puchheim, Germany). Bovine serum albumin

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(BSA) was obtained from Acros Organics (Geel, Belgium). 3-Sulfo-succinimidyl-6[biotinamido]hexanoate (biotin-LC-NHS) and ImmunoPure Streptavidin were from Thermo Fisher Scientific (Waltham, MO). Highly pure methanol (CHROMASOLV® for HPLC, ≥99.9%), (3-aminopropyl) triethoxysilane (APTES) and 2’,2-azino-bis(3ethylbenzothiazoline-6-sulphonic acid) (ABTS), as well as a commercially available

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ELISA kit for the determination of AFM1 in milk (High Sensitivity Aflatoxin M1 ELISA Kit) were also from Sigma-Aldrich. All other reagents were from Merck

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(Darmstadt, Germany). The water used throughout the study was doubly-distilled.

Eight-well polystyrene ELISA strips were purchased from Greiner Bio-One GmbH

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(Frickenhausen, Germany). Four-inch Si wafers (100) were purchased from Si-Mat

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(Kaufering, Germany). A silicon dioxide layer with an average thickness of 1100 nm

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was grown on the wafers by wet oxidation for 3 h at 1100 °C. After that, the wafers are

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diced to produce chips with dimensions 5 mm×15 mm. Spotting of the chips with the AFM1-BSA conjugate was performed using the BioOdyssey Calligrapher MiniArrayer

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(Bio-Rad Laboratories Inc., Hercules, CA).

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2.2. WLRS instrumentation

A picture of the WLRS platform used in this study is provided in Fig. 1a.The

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optical module of the measurement set-up consists of three core elements: a hybrid visible-near infrared light source that guarantee low power consumption and extremely

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long and stable operation (manufactured by ThetaMetrisis SA), a miniaturized USB controlled spectrometer (Ocean Optics) that covers a spectral range from 410 to 720 nm, and a proprietary designed reflection probe consisting of fibers with 200-μm diameter. The biochip is the Si/SiO2 chip covered by a custom designed microfluidic cell (Jobst Technologies GmbH) providing the fluidic connections to the solutions and

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the micropump (Fig. 1b). For the implementation of the assay and the facile monitoring of the biomolecular interactions the biochip is inserted in an opaque docking station that provides for automatic alignment of the bioreactive zone to the reflection probe and at the same time allows for operation under ambient light conditions which is important for use of the device at the point-of-need. A miniaturized USB powered and

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controlled peristaltic micropump (Jobst Technologies GmbH) was employed for the

continuous delivery of the reagents to the biochip surface. The reflected spectrum is

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recorded continuously (integration time was 15 ms; 1 spectrum per second) from the

spectrometer and processed by the dedicated software (ThetaMetrisis SA)

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implementing the interference equation and the Levenberg-Marquart algorithm to

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transform in real-time the observed spectral shift to effective biomolecular adlayer

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thickness increase (Fig. 1c).

2.3. Biotinylation of secondary antibody

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The goat anti-rabbit IgG antibody was extensively dialyzed against a 0.25 M carbonate buffer, pH 9.1, containing 0.9% (w/v) NaCl solution and its concentration

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was adjusted to 1 mg/mL in the same buffer. Sulfo-NHS-LC-biotin was dissolved in DMSO to a final concentration of 100 mg/mL and an appropriate volume of this

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solution was added dropwise to the antibody solution, so as the weight ratio of sulfoNHS-LC-biotin to antibody to be 2:1in the final reaction mixture. The reaction mixture

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was left 2 hours at room temperature (RT) and then extensively dialyzed against 0.1 M NaHCO3 solution, pH 8.5, 0.9% (w/v) NaCl, 0.05% (w/v) NaN3. The biotinylated antibody concentration was determined photometrically at 280 nm.

2.4. Preparation of calibrators and milk samples

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Αn AFM1 stock solution with concentration of 100 μg/mL was prepared in highly pure methanol and stored at 4 οC. From this solution, AFM1calibrators with concentrations ranging from 0.002 to 2.0ng/mL were prepared in 0.05 M Tris-HCl buffer, pH 7.8, containing 0.9% (w/v) NaCl, 0.5% (w/v) BSA, and 0.05% (w/v) bovine gamma-globulins (assay buffer). Raw cow, goat and ewe’s milk samples were collected

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from different small farms located at North and Central Greece and stored aliquoted at -20 oC. Pasteurized goat and cow milk from different milk companies (DELTA FOODS

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S.A., Olympos Larisa Milk Company S.A., EVOL S.A.) were purchased by local stores. Human breast milk samples were provided by anonymous donors after informed

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consent.

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2.5. Surface chemical activation and biofunctionalization

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The chips were cleaned and hydrophylized by a 30-s O2 plasma treatment at 10 mTorr in a Reactive Ion Etching reactor. Then, they were immersed for 20 min in a 2%

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(v/v) aqueous APTES solution, followed by gentle washing with distilled water, and drying under a nitrogen stream. After that, the chips were cured at 120 οC for 20 min

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and kept at RT in a desiccator until use. For the biofunctionalization, a 3x5 mm2 area at the center of the APTES-modified chips was spotted with a 50 μg/mL AFM1-BSA

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conjugate solution in 0.05 M carbonate buffer, pH 9.2 (coating buffer). After spotting, the chips were incubated overnight at RT under controlled humidity conditions (75%)

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and then, were immersed in a 1% (w/v) BSA solution in 0.1 M NaHCO3, pH 8.5 (blocking solution), for 2 h at RT. Following that, the surfaces were rinsed with 0.01 M Tris-HCl buffer, pH 8.25, 0.9% (w/v) NaCl (washing solution) and distilled water, dried under a nitrogen stream and used for the assay.

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2.6. AFM1 biosensor assay Prior to the assay, the biochips were assembled with the microfluidic cell and placed in the docking station. At first, the biochip was equilibrated with assay buffer, and then 1:1 (v/v) mixtures of AFM1calibrators or milk samples with a 1 μg/mL antiAFM1 antibody solution in assay buffer were flowed over the biochip for 10 min at a

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rate of 50 μL/min, followed by washing with assay buffer for 5 min. Then, a 10 μg/mL biotinylated goat anti-rabbit IgG antibody solution in assay buffer was run for 5 min

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followed by a 5μg/mL streptavidin solution in assay buffer for another 5 min at the

same flow rate. Finally, the biochip was washed with assay buffer and regenerated by

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passing a 0.5% (w/v) SDS solution, adjusted at pH 1.3 with 0.1 M HCl, for 4 min. After

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that, the surface was equilibrated with assay buffer prior to the next run. A schematic

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of the assay procedure is illustrated in Fig. 1d. The calibration curve was created by

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plotting in linear/log scale the effective biomolecular layer thickness (Sx) corresponding to different calibrators expressed as percentage of the zero calibrator

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signal (maximum signal; S0) against the respective AFM1 concentration in the calibrators. The linear/log format was applied in order to linearize the calibration curve

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over the range of calibrators used.

3. Results and discussion 3.1. Assay optimization

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For the development of the AFM1 immunoassay on the WLRS biosensing platform

several parameters have been optimized based on both the absolute maximum signal (zero calibrator signal) and the assay sensitivity as expressed by the percent signal drop in presence of AFM1 with respect to zero calibrator signal. Firstly, the concentration of AFM1-BSA conjugate used for coating and the concentration of the anti-AFM1

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antibody were optimized. The primary immunoreaction was followed by a 5-min reaction with a 10 μg/mL biotinylated goat anti-rabbit IgG antibody solution and 5-min reaction with a 5 μg/mL streptavidin solution. As shown in Fig. 2a, maximum zero calibrator values were obtained when AFM1-BSA conjugate concentration equal to or higher than 50 μg/mL is employed for all the antibody concentrations tested (1 to 4

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μg/mL). Thus, this AFM1-BSA conjugate concentration was selected for further

experimentation. Regarding the selection of anti-AFM1 antibody concentration, the

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absolute zero calibrator signal increased as the antibody concentration increased from

0.5 to 4 μg/mL (Fig. 2b). Nonetheless, in presence of a calibrator containing 0.1 ng/mL AFM1, the percent signal with respect to zero calibrator signal was approximately 55%

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for antibody concentrations ranging between 0.5 and 1 μg/mL and increased from 64

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to 80% as the anti-AFM1 concentration increased from 2 to 4 μg/mL. Thus, in order to combine the higher possible zero calibrator signal with the higher assay sensitivity (as

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expressed by the percent signal drop in presence of AFM1), a 1 μg/mL anti-AFM1

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antibody concentration was selected for further optimization of the assay conditions. Using the selected AFM1-BSA and anti-AFM1 antibody concentrations, all the

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other assay parameters were optimized. Regarding assay time, the duration of the primary immunoreaction, i.e., the reaction of anti-AFM1 antibody with the immobilized

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AFM1-BSA conjugate, as well as the duration of the reaction of the secondary antibody with the anti-AFM1 antibody bound onto the biochip were optimized with respect to

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zero calibrator signal. Concerning the duration of the primary immunoreaction, the zero calibrator signal increased with the reaction duration and maximum signal values were obtained for duration equal to or higher than 60 min (see Fig. S1a, Supporting information). Nevertheless, in order to select the primary immunoreaction duration, the effect of reaction time to assay sensitivity was determined using calibrators containing

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0.02 and 0.1 ng/mL AFM1. As shown in Fig. S1b of Supporting information, the increase of primary immunoreaction duration from 10 to 60 min affected adversely the sensitivity of the calibration curve. Based on these results, a 10-min reaction duration was adopted for the primary immunoreaction since it provided for an adequate zero calibrator signal and the highest possible sensitivity of the calibration curve.

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Regarding the reaction of the secondary anti-rabbit IgG antibody with the immunoadsorbed onto biochip anti-AFM1 antibodies, a 5-min reaction was adopted in

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the final protocol since almost 80% of the maximum plateau signal was obtained for

this assay duration (See Fig. S1c of Supporting information). Similarly, the reaction

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time selected for the last assay step, i.e., that of streptavidin reaction with the

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immunoadsorbed biotinylated anti-rabbit IgG antibody molecules was set to 5 min

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which was more than enough in order to receive the maximum plateau signal values for

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this particular step. Representative real-time graphs of the sensor response when running AFM1 calibrators in buffer (0-2.0 ng/mL) with the finally selected conditions

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are provided in Fig. 3. As shown, the reaction with the secondary antibody resulted in approximately 2-fold increase of the signal with respect to that obtained during the

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primary immunoreaction. This was expected since the polyclonal secondary antibody can bind to multiple sites (epitopes) of the anti-AFM1 antibody. Similarly, the

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additional 2 to 3-fold increase of the signal achieved after reaction of the immunoadsorbed biotinylated secondary antibody with streptavidin is attributed to the

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existence of multiple biotin moieties on this antibody. To access the non-specific binding, the response from a chip coated with BSA upon running the zero calibrator is also provided in Fig. 3 (blank curve). As shown, no noticeable response was observed along the whole assay cycle.

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The effect of mixing and incubation of the calibrators or the samples with the antibody solution, prior to the mixture introduction in the fluidic cell, to both the signal and the sensitivity of the calibration curve was also determined. For this purpose, calibrator solutions containing 0, 0.02, and 0.1 ng/mL AFM1 were mixed at 1:1 volume ratio with the anti-AFM1 antibody solution and incubated for 10, 30 and 60 min prior

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to their introduction in the fluidic cell. The signals obtained were compared with those

received when the mixture was immediately introduced after mixing in the reaction cell.

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The results provided in Fig. S3 of Supporting information, show clearly that the mixing and incubation of calibrators with the antibody solution did not affected the signals

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obtained neither the assay sensitivity.

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3.2. Analytical characteristics of the developed immunosensor

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A typical AFM1 calibration curve obtained following the developed 25-min assay is provided in Fig. 4. The linear regression equation was y = -35.83(±0.33) log(x) +

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20.1(±0.42) and the correlation coefficient was r2=0.9997. The assay limit of detection (LOD) The assay limit of detection (LOD) was determined as the concentration

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corresponding to signal equal to mean value of 20 replicate measurements of zero calibrator -3SD and it was found to be 0.006 ng/mL. Similarly, the quantification limit

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was determined as the concentration corresponding to mean value -6SD of 20 replicate measurements of zero calibrator and was 0.012 ng/mL, which is at least 3-times lower

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than the maximum allowable limit of AFM1 in milk set by the European legislation (0.05ng/mL) for baby milk. The assay dynamic range extended up to 2.0 ng/mL. Compared with the competitive ELISA developed using the same immunoreagents, the WLSR sensor had 6-times inferior LOD in milk (0.001 ng/mL AFM1 for the ELISA, see Fig. S2 of Supporting information), but the analysis time was 6-times shorter since

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ELISA took 2 h to be completed. In addition, the milk should be diluted at least 10times prior to the analysis with the ELISA developed to avoid matrix effects. Furthermore the reproducibility and accuracy of the developed immunosensor were also evaluated. Reproducibility was determined by running three control samples prepared in full-fat pasteurized cow’s milk by spiking different concentrations of AFM1

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to cover the assay dynamic range. The intra-assay coefficients of variation (CV) were obtained from 4 repetitive measurements of each control within the same day, while the

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inter-assay CVs were obtained from 4 measurements carried out in 4 different days in

a period of 20 days and their values ranged from 2.1 to 6.3% and 3.5 to 8.2%,

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respectively. In order to investigate the accuracy of the method, recovery experiments

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were performed by spiking raw cow and goat milk samples, which did not contain

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detectable amounts of AFM1 as it was found by assaying them with a commercially

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available ELISA kit, with known amounts of AFM1. Thus, percent recovery was calculated as the ratio of the added amount determined to the actual amount added in

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each sample. The results provided in Table 1 indicate the high accuracy of the developed immunosensor, since the recovery values ranged from 92.5 to 110%.

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The specificity of anti-AFM1 antibody used in the study towards the other

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aflatoxins, namely aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2), was also evaluated. For this purpose, solutions of each one of the potential cross-reactants with concentration ranging from 0.1 to 1000 ng/mL were

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prepared (by 10-fold serial dilution) in assay buffer, and run as calibrators (Fig. S3 of Supporting information). Percent cross-reactivity (%CR) was calculated as follows: %CR = (IC50 AFM1/IC50 cross-reactant)x100, where IC50 AFM1 is the concentration of AFM1 corresponding to 50% inhibition and IC50 cross-reactant is the concentration of the tested compound which provided 50% inhibition of the respective zero calibrator 14

signal. The cross-reactivities determined were approximately 1% for AFB1, 0.2% for AFB2 and AFG1 and 0.1% for AFG2. Considering the low cross-reactivity values determined and taking into account that these mycotoxins are not expected to be found in milk samples [44], no effect in AFM1 determination from cross-reactants of similar

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molecular structure with the proposed sensor is expected.

3.3. Sample matrix effect

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To evaluate the matrix effect on the assay performance, undiluted milk as well as

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different milk dilutions with assay buffer ranging from 2 to 100 times were tested using

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a commercial full-fat cow’s milk that did not contained detectable concentrations of

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AFM1 (as determined by a commercially available ELISA kit). It was found that the presence of milk, even 100-times diluted, had a significant effect on the signal recorded

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in real-time due to light absorption or scattering from milk. This effect was increased

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as the milk dilution decreased and became maximal when undiluted milk flew over the chip surface covering the signal changes due to the specific reaction (Fig. S5 of

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Supporting information). However, as shown in Fig. S5, by introducing a 5-min washing with assay buffer after the primary immunoreaction step, it was possible to monitor in real-time the reaction with the biotinylated secondary antibody and the

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streptavidin. Most importantly, it was found that the overall signal values determined after the assay completion for calibrators in undiluted milk were identical to those received with calibrators prepared in assay buffer. As shown in Fig. S6, identical calibration curves were obtained with AFM1 calibrators prepared either in assay buffer

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or in full-fat cow’s milk and thus, AFM1 calibrators prepared in assay buffer could be used for milk analysis. The effect of milk origin on the assay was also investigated. Skim cow milk powder, full-fat pasteurized cow and goat milk purchased from local stores, full-fat raw cow, goat and ewe milk obtained from different farms, as well as human breast milk

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were analyzed with the developed immunosensor. As shown in Fig. 5, the biosensor responses for all the above milk types are practically the same. Consequently, the

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developed assay permits the analysis of milk samples regardless of their origin. It is

worth mentioning that no special pre-treatment of the milk samples, like centrifugation

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to remove the lipids, sonication or addition of specific solvents was required for the

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analysis of all the milk samples with the WLRS biosensor. This is an important

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advantage of the developed biosensor compared with other sensors, electrochemical or

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optical, reported in the literature, which require milk pre-treatment prior to analysis.

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3.4. Regeneration and stability of the biosensor The possibility of regeneration and re-use of the biochip was also investigated since

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could further reduce the analysis cost. At first, several solutions were tested to find the most appropriate for regeneration of the chip. The solutions examined were: a

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commercially available IgG elution buffer for immunoaffinity chromatography, 0.05 M HCl, 0.1 Μ HCl, 0.1 M NaOH, 0.1 M HCl, 0.5% (w/v) SDS, pH 1.3. In order to evaluate

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the ability of those solutions to remove the immunoadsorbed anti-AFM1 antibodies, they were run over the biochip for 5 min after the completion of the assay followed by reaction with biotinylated anti-rabbit IgG antibody and streptavidin. As shown in Fig. S7 of Supporting information, the 0.5% (w/v) SDS solution, pH 1.3, provided almost complete removal of the bound antibody molecules. Having selected this solution for

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biosensor regeneration, the regeneration time was also tested. It was found that a stable percent signal ascribed to antibody molecules remaining on the surface after the regeneration step was achieved for duration of the regeneration step equal to or greater than 4 minutes and remained constant for consecutive assay/regeneration cycles, while for shorter times the percentage of remaining molecules was gradually increased.

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Therefore, a 4-min regeneration step was selected. In order to investigate the stability of the biofunctionalized with the AFM1-BSA conjugate biochips against regeneration

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with the selected solution, 30 regeneration/assay cycles were run on the same biochip

and the results are presented in Fig. 6. As shown, the values obtained for the first 25

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regeneration/assay cycles fall within the mean value ± 2SD range, proving the high

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stability of the AFM1-protein conjugate modified biochips against regeneration. The stability of measurements obtained with biochips prepared in a single batch

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and kept at 4 oC in presence of desiccator was also determined. For this purpose, zero calibrators were run in duplicate using different biochips within a period of three

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months. It was found that the biochips provided signals within the mean value ± 2SD

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range, with a coefficient of variation lower than 10%.

3.5. Comparison with other optical immunosensors

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The analytical performance of the developed WLRS biosensor with respect to

AFM1 determination in milk as compared to that of other optical biosensors are

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summarized in Table 2. The LOD achieved with the WLRS biosensor was significantly superior compared to all optical label-free immunosensors reported in the literature. In particular, the LOD achieved is approximately 280-times lower than that reported for a silicon based microring resonator (SiON MRR) biosensor [34] and 500-times compared to the respective value reported for a sensor based on asymmetric Mach-Zehnder

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interferometer (aMZI) [33], whereas the assay duration was in all cases 25 min. Two SPR biosensors employing labels have been also reported regarding the determination of AFM1. In the first of them, an antibody labelled with gold nanoparticles was employed [36], leading to LOD that was 3-times higher (18 pg/mL) than that of the developed WLRS sensors. The second SPR biosensor was based on long range surface

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plasmon-enhanced fluorescence spectroscopy (LRSPFS) and demonstrated a LOD

comparable to that of the WLRS sensor, though two times longer analysis time (53 min)

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was required [37]. A multiplex planar waveguide fluorescence immunosensor

(MPWFI) was also implemented for the determination of AFM1 [35]. This

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immunosensor had 9-times higher LOD (55 pg/mL) compared to the WLRS sensor

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developed, while the analysis duration was comparable. It should be mentioned, that all

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the reported biosensors required treatment of the milk samples prior to analysis for the

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removal of the fat layer, including centrifugation [35-37], and/or sonication and extraction [35]. On the contrary, using the developed WLRS biosensor, milk analysis

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was feasible without special pre-treatment of samples. In addition the cost of the biochip is considerably lower compared to the technologies employed in reports

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mentioned in Table 2 which is an additional advantage for the targeted application at the field. Finally, compared to the optical method based on Fourier transform infrared

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spectroscopy (FT-IR) [39], the proposed sensor had an approximately 3-times lower LOD (20 pg/mL), while the time required for the analysis was difficult to compare since

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the data acquisition with the FT-IR is rapid but their processing time might take some time. Overall, the developed WLRS immunosensor is suitable for the AFM1 determination in milk samples at the point-of-need due to the direct analysis of whole milk, the short analysis time, the small size of the instrumentation along with the lowpower consumption and the high detection sensitivity and reliability.

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4. Conclusion and future outlook The WLRS biosensor developed for the determination of AFM1 in milk samples is characterized by high detection sensitivity, short analysis time, and low cost of consumables (biochip, analytes). The sensor could detect AFM1 in whole milk at

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concentrations as low as 6 pg/mL that is well below the maximum allowable limit in milk for infant or adult consumption set by EU as well as by other regulatory

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authorities. Milk from different origins (cow, goat, sheep and human) could be analyzed without any pretreatment. In addition, the same biochip could be regenerated and reused

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for up to 25 times, thus providing for further suppression of the analysis cost. Based on

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these features and by further miniaturization of the instrumentation employed, the

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developed sensor could be used for the rapid and sensitive detection of AFM1 at the

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point-of-need.

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Acknowledgments

Mrs Dimitra Tsounidi and Dr Grigorios Zisis were financially supported by the program

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of Industrial Scholarships of Stavros Niarchos Foundation.

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[32] E. Larou, I. Yiakoumettis, G. Kaltsas, A. Petropoulos, P. Skandamis S. Kintzios, High throughput cellular biosensor for the ultra-sensitive, ultra-rapid detection of aflatoxin M1, Food Control 29 (2013) 208-212.

23

[33] T. Chalyan, R. Guider, L. Pasquardini, M. Zanetti, F. Falke, E. Schreuder, R.G. Heideman, C. Pederzolli, L. Pavesi, Asymmetric Mach-Zehnder interferometer based biosensors for Aflatoxin M1 detection, Biosensor 6 (2016) 1. [34] T. Chalyan, L. Pasquardini, D. Gandolfi, R. Guider, A. Samusenko, M. Zanetti, G. Pucker, C. Pederzolli, L. Pavesi, Aptamer- and Fab'- functionalized microring

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resonators for Aflatoxin M1 detection. IEEE J. Select. Topic Quantum Electron. 23 (2017) art. no 5900208.

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[35] H.L. Guo, X.H. Zhou, Y. Zhang, B.D. Song, J.X. Zhang, H.C. Shi, Highly sensitive and simultaneous detection of melamine and aflatoxin M1 in milk products by

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multiplexed planar waveguide fluorescence immunosensor (MPWFI), Food

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Chem. 197 (2016) 359-366.

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Emmenegger, J. Dostalek, K.H. Feller, Sensitive and rapid detection of aflatoxin M1 in milk utilizing enhanced SPR and p(HEMA) brushes. Biosens. Bioelectron.

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fluorescence spectroscopy for the detection of aflatoxin M1 in milk, Biosens.Bioelectron. 24 (2009) 2264-2267.

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[38] K. Narsaiah, S.N. Jha, R. Bhardwaj, R. Sharma, R. Kumar, Optical biosensors for food quality and safety assurance- A review, J. Food Sci. Technol. 49 (2012) 383-

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[40] G. Koukouvinos, P. Petrou, D. Goustouridis, K. Misiakos, S. Kakabakos, I. Raptis, Development and Bioanalytical Applications of a White Light Reflectance Spectroscopy Label-Free Sensing Platform, Biosensor 7 (2017) 46. [41] G. Koukouvinos, P. Petrou, K. Misiakos, D. Drygiannakis, I. Raptis, G. Stefanitsis, S. Martini, D.Nikita, D.Goustouridis, I.Moser, G. Jobst, S. Kakabakos,

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Simultaneous determination of CRP and D-dimer in human blood plasma

samples with White Light Reflectance Spectroscopy. Biosens. Bioelectron.84

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(2016) 89-96.

[42] G. Koukouvinos, P.S. Petrou, K. Misiakos, D. Drygiannakis, I. Raptis, D.

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Goustouridis, S.E.Kakabakos, A label-free flow-through immunosensor for

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determination of total- and free-PSA in human serum samples based on white-

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light reflectance spectroscopy, Sensor Actuator B 209 (2015) 1041-1048.

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[43] G. Koukouvinos, Z.Tsialla, P.S. Petrou, K. Misiakos, D.Goustouridis, A. UclesMoreno, A.R.Fernandez-Alba, I. Raptis, S.E. Kakabakos, Fast simultaneous

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detection of three pesticides by a White Light Reflectance Spectroscopy sensing platform, Sensor Actuator B 238 (2017) 1214-1223.

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[44] P.T. Scaglioni, T. Becker-Algeri, D. Drunkler, E. Badiale-Furlong, Aflatoxin B1

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and M1 in milk, Anal. Chim. Acta 829 (2014) 68-74.

25

Author biography

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Mrs Dimitra Tsounidi received her B.Sc. Degree in Chemistry in 2015 and M.S. Degree in “Analytical Chemistry and Nanotechnology” in 2017 from the Department of Chemistry of University of Patras. Since 2017, she is a PhD candidate of the Department of Chemistry of University of Patras and a recipient of an Industrial Scholarship of Stavros Niarchos Foundation. Her PhD thesis focus on the development of optical sensor for multiplexed detection of analytes.

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Dr Georgios Koukouvinos received his B.Sc. Degree in Chemistry in 2008, his M.S. Degree in Biochemistry in 2010 and his Ph.D. in Chemistry in 2015 from the University of Athens. Since 2012, he has been working as a DNA expert at the Forensic Science Division of the Hellenic Police. He is also acting since 2015 as a post-doctoral fellow at the Immunoassay/Immunosensors Lab of the Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, of NCSR “Demokritos”. His research interests include DNA STRs analysis, DNA and protein microarrays, microfluidic systems, chemically modified surfaces and biosensors for bioanalytical applications. He is the author/co-author of 12 publications in international Journals and more than 20 presentations in conferences.

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Dr Panagiota Petrou obtained her B.Sc. and Ph.D. in Chemistry from the University of Athens in 1993 and 2000 respectively. From 2000 to 2001, she was a Post-doctoral fellow in the Institute of Microsystems Technology of Albert-Ludwig University of Freiburg. At the end of 2002, she joined the Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, of NCSR “Demokritos”, where currently holds a Senior Researcher position. Her activities include development of advanced bioanalytical microsystems, protein and DNA arrays, modification of surfaces with self-assembled monolayers, etc. She has participated in numerous European and national projects. She is the author/co-author of 95 papers in international Journals and of more than 150 conference communications. She holds 15 Greek and international patents.

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Dr Konstantinos Misiakos, received his B.Sc. in Electrical Engineering from the National Technical University of Athens, 1978, and the M.Sc. from Clemson University, Clemson, SC, USA in 1984. He obtained his Ph.D. in semiconductor device physics from the University of Florida, Gainesville, FL, USA in 1987. He served as a visiting assistant professor in Electrical Engineering at the University of Florida from August 1987 until May 1989. Since 1989, he is with the Institute of Microelectronics, NCSR “Demokritos”, where he now holds the position of Director of Research. His research interests include optical biosensors, monolithic silicon optocouplers for biosensing, solar cells and radiation detectors. Dr. Misiakos is the author or co-author of >125 publications in refereed international journals, 60 communications in international conferences, three book chapters and holds five international patents on biosensors. He coordinated three European projects: EUBRITE III Project “BOEMIS” (1997-2000), EU-IST Project “BIOMIC” (2001-2004) and the EU-IST project “NEMOSLAB” (2006-2009), all in biosensors. 26

Dr Grigoriοs Zisis obtained his B.Sc. degree in Material Science from the University of Patras in 2011. In January 2016, he obtained his Ph.D. from the Optoelectronics Research Center (ORC) of the University of Southampton for his work on laser-assisted domain engineering, waveguide fabrication, and micro-structuring of lithium niobate. Subsequently, he joined Thetametrisis S.A. as a senior optical engineer, and a year afterwards, he started his industrial postdoctoral fellowship (a corporation between NCSR Demokritos and Thetametris S.A.), studying on a radical optical approach for the precise characterization of 2D and photonic materials.

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Dr Dimitris Goustouridis received the B.Sc. in 1992 from the Physics Depsrtment of the University of Patras. In 2002 he received the Ph.D. degree in microelectronics from the Department of Applied Sciences of National Technical University of Athens for his work on capacitive type pressure sensors. From 2000 until 2010 he was working as research associate at the institute of Microelectronics of NCSR “Demokritos”. His current position is Assistance Professor in Department of Electronic Engineering of Piraeus University of Applied Science. His interests include silicon micromachining, capacitive pressure sensors, optical sensors, chemical and biological sensing devices and measurements set-up for sensors characterization. He is/was Key Researcher in several research projects in the areas of micro-fabrication and bio(chem)sensors funded by EU, national funding agencies and industries. He is author of more than 60 publications in international journals and holder of 4 patents. In parallel in 2008 began the commercial exploitation of his research results in the area of optical metrology by co-founding ThetaMetrisis, a spin-off company of Institute of Microelectronics of NCSR “Demokritos”.

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Dr Ioannis Raptis received his B.Sc. degree in Physics (1989) and his Ph.D. on e-beam lithography (1996) from the University of Athens. The experimental part of his Ph.D. thesis was carried out at IESS-CNR (Rome, Italy). The software part of his thesis became a commercial product by Sigma-C GmbH. Since 2003 he works at NCSR-D as researcher, and since 2013 as a Director of Research on the implementation of technologies and electronic/photonic devices in the micro/nano scale for bio/chemical sensing applications. He is/was Key Researcher and Coordinator of several EU (FP6, FP7) and national (GSRT) funded research projects. He is program and steering committee member in several international conferences and serves as associate editor in journals published by Elsevier, IEEE and Nature. He is author of >140 articles in peer-reviewed international journals and holder of 5 patents. In 2008 he co-founded ThetaMetrisis, and he is member of the BoD.

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Dr Sotirios Kakabakos obtained his B.Sc. in Pharmacy, University of Athens, in 1980 and his Ph.D. in Pharmacy, University of Patras in 1989. In 1990-91 he was a postdoc fellow in the Clinical Biochemistry Department of the Medical School at the University of Toronto. In 1992 he joined the Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, of NCSR “Demokritos”, where he holds the position of the Director of Research, on the development and evaluation of advanced immunoassay technologies focusing mainly on optical immunosensors. He was scientific responsible of the institute for several European and national projects. He is author/co-author of 115 papers in peer-reviewed international journals and of more than 200 communications in international conferences. He also holds 26 national, international and European patents.

27

FIGURE CAPTIONS Fig. 1. (a) Image of the WLRS biosensing platform with the docking station (inside which the biochip is placed), the optical set-up and the miniaturized USB powered and controlled peristaltic micropump. (b) Image of the Si/SiO2 chip with its microfluidic

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cell on top of a 1-euro coin. (c) Real-time reaction monitoring as presented in the tablet screen. (d) Schematic of the AFM1 assay procedure taking place onto the biochip

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surface and monitored by WLRS platform. The arrangement of the six peripheral fibers (1a-1f) which send the incident light to the surface, as well as of the central fiber (2)

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that collects the reflected light, in the reflection probe is also illustrated on the left side.

c

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b

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d

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a

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FIGURE 1

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Fig. 2. (a) Zero calibrator signal values obtained for biochips spotted with AFM1-BSA solutions with concentrations ranging from 10 to 200 μg/mL for anti-AFM1 antibody concentrations of 1.0 (black squares), 2.0 (red circles), 3.0 (green upward triangles), or 4.0 μg/mL (blue downward triangles). (b) Sensor responses corresponding to zero

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calibrator (blue columns) and a calibrator containing 0.1 ng/mL AFM1 (red columns) obtained from chips spotted with a 50 μg/mL AFM1-BSA solution for anti-AFM1

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antibody concentrations ranging from 0.5 to 4 μg/mL. In all cases, after the primary immunoreaction, a 10 μg/mL biotinylated goat anti-rabbit IgG antibody solution was run for 5 min followed by a 5 μg/mL streptavidin solution for another 5 min. The flow

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rate throughout the experiment was 50 μL/min. Each point is the mean of 3

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measurements ± SD.

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FIGURE 2

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2.0 1.5 1.0

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Effective biomolecular adlayer thickness (nm)

2.5

anti-AFM1 Ab concentration 1.0 2.0 3.0 4.0

0.5

0

50

100

2.5 2.0 1.5 1.0 0.5 0.0

150

AFM1-BSA (ug/mL)

200

0.5

1

2

3

4

Anti-AFM1 antibody concentration (ug/mL)

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0.0

b Effective biomolecular adlayer thickness (nm)

a

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Fig. 3. Real-time signal recordings corresponding to AFM1 calibrators (0 – 2.0 ng/mL) prepared in assay buffer. The arrows show the sequence of solutions run over the biochip: assay buffer: start to arrow 1; mixture of zero calibrator with anti-AFM1 antibody: arrow 1 to 2; biotinylated anti-rabbit IgG antibody: arrow 2 to 3; streptavidin: arrow 3 to 4; assay buffer: arrow 4 to end.

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FIGURE 3

4

1.2

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1.0 0.8

AFM1 (ng/mL) 0 0.02 0.05

0.6

2

1

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0.4

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3

0.2 0.0 5

10

15

0.1 0.5

2.0 blank

20

25

Time (min)

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0

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Effective biomolecular layer thickness (nm)

1.4

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Fig. 4. Typical AFM1calibration curve. Each point is the mean of 3 measurements ± SD.

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FIGURE 4

30

100

(Sx/S0)x100

80 60 40

0

0.01

0.1

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20

1

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AFM1 (ng/mL)

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Fig. 5. Sensor responses obtained for zero calibrators prepared in assay buffer (i), skim

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cow milk powder (ii), full-fat pasteurized cow milk (iii), full-fat pasteurized goat milk

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(iv), raw cow milk (v), raw goat milk (vi), raw ewe milk (vii) or human breast milk

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(viii). Each point is the mean of 3 measurements ± SD.

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FIGURE 5

Effective biomolecular layer thickness (nm)

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1.2 1.0 0.8 0.6 0.4 0.2 0.0

(i)

(ii)

(iii) (iv)

(v)

(vi) (vii) (viii)

Milk samples

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Fig. 6. Zero calibrator values obtained after repetitive assay/regeneration cycles. Solid red line corresponds to mean value of the 30 measurements; dashed green lines correspond to mean ± 2SD.

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FIGURE 6

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1.0 0.9

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0.8

5

10

15

20

Regeneration cycle

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30

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0

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0.7

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Effective biomolecular adlayer thickness (nm)

1.1

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Table 1.Recovery of AFM1 added to milk samples Amount determined (ng/mL)

Recovery %

0.05

0.053

106

0.4

0.37

92.5

1.5

1.4

93.0

0.05

0.052

0.2

0.22

1

0.96

cow milk

105 110

96.0

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goat milk

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Amount added (ng/mL)

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Sample

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Table 2. Comparison of the performance of WLRS sensor against other optical sensors for the determination of AFM1 in milk samples

(pg/mL)

Analysis time (min)

Sample type

-

6.0

25 min

milk

SiON MRR

-

1640

25 min

buffer

[34]

aMZI

-

3000

25 min

buffer

[33]

SPR

Gold nanoparticles

18

55 min

milk

[36]

LRSPFS

Fluorescence

6.0

53 min

milk

[37]

MPWFI

Fluorescence

55

17 min

milk

[35]

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Ref. #

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proposed WLRS sensor

LOD

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Label

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A

Sensor type

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