Absorbance based on LED and photodetector

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Physical and chemical properties for OTA

OTA is a white, crystalline powder that is very unstable in the presence of light but fairly heat resistant, is a weak organic acid with a pKa value of 7.1 and its molecular weight is 403.8 g/mol [13]. With a crystalline structure varying from colorless to white. OTA possess an intense green fluorescence under UV light in acid medium and blue fluorescence in alkaline conditions [15]. A neutral or acid pH, OTA is soluble in polar organic solvents (alcohols, ketones, chloroform), slightly soluble in water and insoluble in petroleum ethers and saturated hydrocarbons. At alkaline pH, this molecule is soluble in aqueous sodium bicarbonate solution (0.1 M, pH 7.4) and in alkaline solutions.
Its melting point is 90 °C when it is crystallized from benzene and 169 °C when it is crystallized in xylene [44]. The particularity of OTA is its high stability. OTA possesses a resistance to acidity and high temperatures. Thus, once foodstuffs are contaminated, it is very difficult to totally remove this molecule [13, 45].
OTA is optically active, its rotational coefficient is 𝛼𝐷21=−46.8° . The UV absorption spectrum of OTA varies depending on the pH and polarity of the solvent. OTA has an absorption maximum at 333 nm with a molar extinction coefficient of 5500/mol×cm in methanol. It absorbs at 338 nm with a coefficient of 5600. OTA has an absorption maximum at 378 nm with an extinction coefficient of 14700. Ochratoxin A is also fluorescent; it has an emission maximum at 467 nm in 96% ethanol and 428 nm in absolute ethanol after excitation at 340 nm.
Moreover, after excitation at 365 nm, OTA has green fluorescence in an acidic medium and blue fluorescence in alkaline medium; This property is used for the detection and determination of OTA [13, 44]. OTA is relatively stable to temperature; it is however completely degraded by sodium hypochlorite [46]. These properties are used in the laboratory, for the decontamination of equipment.

Toxicological profile

The toxicological status of OTA has been examined and it was the subject of a complete monograph by the IARC (International Agency for Research on Cancer) in 1993 [47]. Several studies were carried out in order to show the implication of OTA in diseases with effects such as nephrotoxic, hepatotoxic, neurotoxic, teratogenic and immunotoxic on several species of animals [8], and can cause kidney and liver tumors in mice and rats. However, its toxicity varies depending on the sex, the species and the cellular type of the tested animals [16].
The genotoxic status of OTA is still controversial, due to contradictory results obtained in various microbial and mammalian tests. Ochratoxins have been related to human and animal diseases in literature especially since the early 1970s. Most reports indicate that Ochratoxin plagued especially the North Eastern European countries and Africa [48, 49].


In studies with broiler chicks, liver damage was present in concert with nephrotoxicity. Lymphocyte infiltration occurred in the liver along with lymphocytosis in lymphoid organs. Necrotic changes in periportal cells were observed in rats given an LD50 (20 mg/kg body weight) dose of Ochratoxin A and apparently prevented fatty degeneration of the liver caused by aflatoxins when the two toxins were given simultaneously to broiler chickens [50].

ImmunoAffinity columns

Antibody-based immunoaffinity columns clean-up combined with HPLC/fluorescence detection has become very common in mycotoxin analysis of foodstuffs, feedstuffs and biological fluids due to a large number of advantages over other commonly used clean-up procedures. IAC tools are based on the use of anti-mycotoxin antibody into solid support such as agarose gel. Principle of the cleanup is showed in the figure 7 and described below:
(1) Filtered extract is loaded onto the column containing specific antibodies for the mycotoxin of interest.
(2) Antibodies selectively bind the mycotoxin from the crude extract.
(3) Impurities are removed from the column by washing with water or PBS while the toxin is immobilized by antibodies in the column.
(4) Mycotoxin is eluted from the column for its detection by a dedicated fluorometer, HPLC or by other methods. OTA could be eluted with methanol [77].

Molecularly Imprinted columns

Attempts to replace the bio recognition element of IAC by a less expensive and more stable biomimetic counterpart have been described recently. A molecularly imprinted polymer (MIP) able to recognize Ochratoxin A was prepared [70].
Molecular imprinted technique (MIT) has been investigated to be an efficient and powerful technique for sample clean up and pre-concentration of mycotoxins in applications to different matrices successfully. MIP is a synthetic material with an artificially generated three-dimensional network that is able to bind to a target molecule, specifically.
It has the advantages to be inexpensive, chemically, thermally stable and compatible with all solvents. MIP specifically designed for OTA has already been generated using either OTA or a structural analog as template and successfully applied as SPE sorbent (MIP-SPE) for sample pretreatment. In comparison with IAC, MIP-SPE exhibits reusability, simple operation and longer storage time and is further considered as an alternative to IAC [84]. MIP currently seem to be very promising and cheaper alternative to SPE and IAC sorbents for clean-up and pre-concentration of OTA [70] .
Molecular imprinting is a process where the target molecule (or a derivative thereof) acts as a template around which interacting and cross-linking monomers are arranged and copolymerized to form a cast-like shell, scheme in the figure 8. The use of MIP for Ochratoxin A [85] for extraction and analysis [86] in ginger [84], coffee, grape juice and urine [87] and wine [88] has been documented.

High performance liquid chromatography

HPLC coupled with UV, a diode array detector (DAD) or a fluorescence detector (FD) is the most widely used technique for the identification of mycotoxins in food by its characteristics such us accuracy, repeatability, reproducibility and have reasonably low levels of detection. Compounds eluted from the column pass through a detector of some sort (usually fluorescence or ultraviolet depending on the physical and chemical attributes of the analyte of interest) and the detector helps to quantify the specific compounds in the original sample injected onto the column. It is sometimes necessary or an advantage, to use pre-column or post-column derivatization to assist sensitive detection of the mycotoxin. For many mycotoxins, the time for analysis following injection onto the column is less than 20 minutes.
These methods have been adopted as official or standard methods by the AOAC International or the European Standardization Committee. In particular, methods for Ochratoxin A in barley (2000.03), Ochratoxin A in roasted coffee (2000.09), Ochratoxin A in wine and beer (2001.01) and Ochratoxin A in green coffee (2004.10). Additionally, HPLC/immunoaffinity column methods have been validated for the measurement of Ochratoxin A in cocoa powder. HPLC-FD is highly sensitive, selective and repeatable, so specific labeling reagents have been developed and are commercially available for the derivatization of non-fluorescent mycotoxins to form fluorescent derivatives [3]. The presence of OTA in wine has been determined using clean up with commercially available IA columns and separation with RP-HPLC C-18 column, the sensitive FD method allowed estimation of OTA in 0.01 ng/mL concentration. The researchers employed direct injection of the grape must sample in a HPLC–FD system without prior clean-up procedure, the LOD and LOQ were 0.22 and 0.77 μg/L, respectively [67].
OTA contamination in dried figs was studied using HPLC–FD extraction with methanol and orthophosphoric acid following clean up by an IAC. The LOD for OTA was 0.12 g/Kg. The mean concentration of OTA determined using HPLC was 0.39 ng/mL of plasma. The combination of HPLC method with clean-up step by IAC was used for the detection of OTA in green and roasted coffee beans as well as in the coffee brew. HPLC based OTA detection in dry-cured meat products has been described that comprised no clean-up step [67]. The main advantage is its identification power and possibility to perform determination of multiple analytes from different chemical groups in the same run. The advantages and disadvantages of the most common methods to detect OTA are showed in the table 7.

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Flow-based indirect competitive aptasensor

First, the non-specific sites on the SPCE surface were blocked with 100 μL of BSA 2% for 1 h at room temperature. Then 4 μL of OTA modified MBs diluted in binding buffer was injected onto the SPCE surface. The second step consisted of passing 150 nM of biotinylated aptamer with free OTA in buffer solution or real sample. Therefore, OTA present in food samples competed with the immobilized OTA on the MBs. After, a solution of avidin–ALP was bound to the biotinylated aptamer. The electrochemical detection was carried out in the similar way as described for the flow-based direct competitive aptasensor.

Amperometric detection of OTA

The measurement of the activity of ALP, the enzyme that labeled the OTA or the aptamer was carried out by amperometry; the amperometric measurements were performed using the data acquisition card (PMD1208FS), which connected the potentiostat Polarostat type PRGE, Tacussel electronique with the computer for the acquisition of the signal.
The substrate ALP (1-NP) was injected on the aptasensor in the flow cell connected to the potentiostat. After incubation of the substrate for 6 minutes, a potential of 200 mV was applied. This potential corresponds to the oxidation potential of the product 1-naphthol 1-iminoquinone. The signal obtained is proportional to the enzymatic activity of ALP. It is therefore proportional to the amount of OTA in the case of the direct competitive aptasensor and inversely proportional to the amount of OTA in the case of indirect competitive assay.

Table of contents :

List of figures
List of tables
Chapitre I
1. Introduction
1.1 Définition du problème
1.2 Objectifs de la recherche
1.2.1 Général
1.2.2 En particulier
Chapitre II
2.1 Définition, la structure et le profil toxicologique de l’OTA
2.2 Contamination et règlement
2.3 Méthodes de détection dans les denrées alimentaires contaminées par l’OTA.
2.3.1 Biocapteurs
2.3.2 La détection optique La fluorescence de l’OTA Optoélectronique Colorimétrie
Chapitre III
Partie A: Absorbance basée sur l’émetteur et les photos détecteurs
3.1 Photo détecteurs pour détecter les pesticides
3.1.1 Détermination spectrophotométrique de l’activité des enzymes et des constantes d’inhibition
3.1.2 Détermination de l’activité acétylcholinestérase (AChE).
3.1.3 Détermination de la constante d’inhibition (Ki)
3.1.4 Détermination optique des activités enzymatiques et des constantes d’inhibition
3.2 LED-UV et photo détecteurs pour détecter OTA
Partie B: Fluorescence avec capteur CMOS.
3.3 ArduCAM pour détecter la fluorescence
3.4 CMOS pour détecter la fluorescence
3.5 Téléphone mobile pour détecter la fluorescence
3.6 Image de fluorescence
3.7 Fluorescence avec HPLC (selon l’acronyme anglais)
3.8 Méthodes d’extraction
3.8.1 Extraction de l’OTA du cacao en utilisant des colonnes MIP
3.8.2 Extraction avec des solutions à 1% NaHCO3 dans l’eau
3.8.3 Extraction avec l’acétonitrile
3.8.4 Extraction de l’OTA à partir du vin et de la bière échantillon Colonnes IAC (selon l’acronyme anglais) Colonnes MIP (selon l’acronyme anglais)
3.9 Les systèmes d’écoulement
3.10 L’evaluation de la performance du dispositif de fluorescence dans des conditions particulières
Partie C: Traitement de l’image
Chapitre IV
Partie A: Absorbance basée sur l’émetteur et les photo détecteurs
4.1 La détection des pesticides à l’aide d’un phototransistor et d’une diode
4.2 La détection de l’OTA en utilisant un photo détecteur et UV-LED
Partie B: Fluorescence avec capteur CMOS.
4.3 Calibration de l’OTA en utilisant ArduCAM
4.3.1 Des échantillons d’OTA préparés dans l’éthanol (EtOH)
4.3.2 Des échantillons d’OTA préparés dans le méthanol (MeOH)
4.4 Détection d’OTA en utilisant un capteur CMOS
4.4.1 Extraction avec des colonnes IAC pour le vin et la bière
4.4.2 Extraction avec des colonnes MIP pour le vin et la bière
4.4.3 Extraction de l’OTA du cacao avec des colonnes MIP
4.5 Téléphone mobile comme détecteur d’OTA
4.6 Les systèmes d’écoulement
4.7 L’évaluation de la performance du dispositif de fluorescence dans des conditions particulières
4.7.1 Effet du solvant sur l’intensité de fluorescence de l’OTA
4.7.2 Effet de la concentration en sel du tampon et l’intensité de fluorescence de l’OTA 36
4.7.3 Effet du pH sur l’intensité de fluorescence de l’OTA
Partie C: Traitement de l’image
Chapitre V
Chapter I Background of the study
1. Introduction
1.1 Problem definition
1.2 Research Objectives
1.2.1 General
1.2.2 Particulars
1.3 Thesis structure
Chapter II Review
2.1 Definition
2.1.1 Structure
2.2 Physical and chemical properties for OTA
2.3. Toxicological profile
2.3.1 Hepatotoxic
2.3.2 Nephrotoxicity
2.3.3 Neurotoxicity
2.3.4 Teratogenicity
2.3.5 Immunotoxicity
2.3.6 Carcinogenesis
2.4 Contaminated food
2.5 Regulation and Legislation
2.6 Pesticides
2.7 Methods of detection in foodstuffs contaminated by OTA
2.7.1 Methods of extraction ImmunoAffinity columns Molecularly Imprinted columns
2.7.2 Biosensors
2.7.3 Chromatography methods High performance liquid chromatography
2.8 Optical detection
2.8.1 Fluorescence of OTA
2.8.2 Optoelectronics
2.8.3 Colorimetry
Chapter III Methodology
PART A: Absorbance based on LED and photodetector
3.1 Photodetectors to detect pesticide
3.1.1 Reagents and materials
3.1.2 Optical design
3.1.3 Basic principle
3.1.4 Methodology Spectrophotometric determination of enzyme activities and inhibition constants Determination of acetylcholinesterase activity (AChE). Determination of inhibition constants (Ki) Optical determination of enzyme activities and inhibition constants
3.2 UV-LED and photodetectors to detect OTA
PART B: Fluorescence with CMOS sensor
3.3 Material and software
3.4 ArduCAM as fluorescence set-up
3.5 CMOS sensor in serial port camera module for detect OTA
3.6 Smartphone as detector of OTA
3.7 Fluorescence image
3.8 Fluorescence with HPLC
3.9 Methods of extraction
3.9.1 Reagents and materials
3.9.2 Equipment and instruments
3.9.3 Extraction of OTA from cocoa using MIP columns Extraction based on 1% NaHCO3 in water Extraction based on acetonitrile: water mixture (2%NaCl), classical method
3.9.4 Extraction of OTA from wine and beer sample IAC columns MIP columns
3.10 Flow systems
3.10.1 Flow system for Aptamer columns
3.11 Evaluation of the fluorescence device performance under specific conditions
3.12 APP designed
PART C: Image processing
Chapter IV Results
PART A: Absorbance based on LED and photodetector
4.1 Detection of pesticides using a phototransistor and a LED
4.2 Detection of OTA using a photodetector and UV-LED
PART B: Fluorescence with CMOS sensor
4.3 Calibration of OTA using ArduCAM fluorescence set up
4.3.1 Samples of OTA prepared in Ethanol (EtOH)
4.3.2 Samples of OTA prepared in Methanol (MeOH)
4.4 Detection of OTA using CMOS sensor in serial port camera module
4.4.1 Extraction with IAC columns for wine and beer
4.4.2 Extraction with MIP for wine and beer samples
4.4.3 Extraction of OTA from cocoa using MIP columns
4.5 Smartphone as detector of OTA
4.6 Flow system
4.6.1 Flow system for Aptamer columns
4.7 Evaluation of the fluorescence device performance under specific conditions
4.7.1 Effect of solvent of fluorescence intensity of OTA
4.7.2 Effect of salt composition of fluorescence intensity of OTA
4.7.3 Effect of pH on fluorescence intensity of OTA
4.8 Employing the APP
PART C: Image processing
Chapter V Conclusions


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