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Aptamers for the detection of OTA
The first aptamer towards OTA was published by Cruz-Aguado and Penner in 2008.128 The aptamer which they isolated contains 36 nucleotides with the sequence GAT-CGG-GTG-TGG-GTG-GCG-TAA-AGG-GAG-CAT-CGG-ACA, and displays an affinity Kd of 49 nM towards OTA. They also showed that the aptamer does not bind with chemically similar molecules like warfarin and N-acetyl-L-phenylalanine and has a strongly reduced affinity towards ochratoxin B. Upon binding with OTA this aptamer takes the structure G-quadruplex, as depicted in Figure 6.82 More recently, Barthelmebs et al. isolated two new 30mer aptamers against OTA with dissociation constants of 96 nM and 130 nM.83 The specificity of the aptamers was also tested towards ochratoxin B and phenylalanine. Despite this alternative, the more commonly used aptamer in literature is the first one found by Cruz-Aguado and Penner.
Aptamer-based biosensors for the detection of OTA
There is a large number of different aptamer based methods for the detection of OTA with electrochemical or optical detection methods. An overview of the methods and their sensitivities is given in Table 2. The different detection methods show a wide range of limit of detection (LOD). Depending on the underlying method the fluorescent and luminescent methods show high sensitivities with very low LODs (fM – pM range). Electrochemical methods also show relatively high sensitivities (pM – nM range), while the colorimetric methods are less sensitive (nM range). The response range of sensors is the range in which they show a linear response with the concentration of the target, and is therefore also an important characteristic of sensors.
Challenges for biosensors
The performance of biosensors regarding sensitivity, selectivity, speed, stability, complexity, and processing depends strongly on the nature of the chosen bioreceptor and transducer. The substrate for the bioreceptor plays a major role, as its intrinsic properties define the possible measurement methods, which in turn also determine the biosensors speed, ease-of-use and possible miniaturization for portable devices. The need of high stability and reproducibility demands a reliable and well-controlled chemistry. This ensures dependable probe immobilization but also institutes the possibility of applying (antifouling) layers to avoid non-specific adsorption from targets such as proteins and bacteria. A reliable probe immobilization is also crucial for the dependability of the biosensor. The biosensors specificity and selectivity are mainly defined by the probes which are used. Another point to consider is that the probes are biological components and their production can be complex and their shelf life has to be considered before choosing a biosensor system.
Strategy proposed for detecting the interaction aptamer-OTA
The goal of this thesis is to study the interaction of pathogens with aptamers on a stable and reproducible biochip architecture based on an hydrogenated amorphous silicon carbon alloy (a-Si1-XCX:H) deposited on an aluminium back-reflector for reliable and sensitive detection of pathogens by fluorescence. The silicon surface enables the grafting of acid-terminated organic monolayers with robust Si-C bounds. The acid-terminated layers are excellent candidates for a reliable immobilization of amine-terminated probes by covalent peptide bonds. The silicon substrate is not only the basis for a reliable immobilization but also provides a platform for analysis and quantification by infrared spectroscopy, as well as analysis with fluorescence for an increased sensitivity. On this architecture we introduce the interaction of the toxin ochratoxin A (OTA) with its 36mer DNA-aptamer as a model system.
The well-controlled multi-step modification process which we carry out on silicon is displayed in Figure 7, and will be explained in more detail in chapter 2. Hydrogen terminated silicon surfaces (1) are used as a basis for the grafting of an acid-terminated organic monolayer by photochemical hydrosilylation (2). This results in a very stable monolayer attached by covalent Si-C bonds. The stable acid-terminated layer is then the basis for the next step (3), where the amino-terminated aptamers are immobilized on the surface by a two-step amidation process. The two-step amidation consists first of an activation reaction of the surface with the carbodiimide EDC in presence of NHS. This is followed by a subsequent aminolysis reaction, leading to aptamers immobilized on the organic monolayer by stable peptide bonds. This immobilization strategy provides the substrate with a high chemical stability with respect to ageing and long exposure in physiological media. Once the aptamers are immobilized on the surface the interaction aptamer-OTA is carried out (4).
Direct detection of the interaction aptamer-OTA by ATR-FTIR
Carrying out the surface modifications on crystalline silicon (111) allows studying of all steps by Fourier-transform infrared spectroscopy in attenuated total reflexion geometry (ATR-FTIR). This enables the study and quantification of the surface functionalization, the probe immobilization and the interaction of OTA with its aptamer without any labelling.
Indirect detection of the interaction aptamer-OTA by fluorescence
The chemistry is transferred to the biochip architecture. While the chemistry remains largely the same (HF-etching, hydrosilylation, 2-step amidation), it is now conducted on a thin layer of an amorphous silicon-carbon alloy (a-Si0.85C0.15:H) on an aluminium back reflector. The role of the back reflector is to increase the fluorescence sensitivity by constructive interference at the a-Si1-XCX:H layer.20 The local and defined deposition of different aptamers and oligonucleotides is carried out with a robotic spotter, giving it its biochip (multiplex) capabilities. The biochips are based on an indirect detection of the interaction aptamer-OTA by fluorescence. Two different methods of detection on surfaces are proposed in literature, “signal OFF” and “signal ON”. In the “signal OFF” detection method (Figure 8, left) short complementary oligonucleotides labelled with fluorophores are hybridized to the aptamers. Successful association with OTA removes the complementary strands, resulting in a decrease of fluorescence intensity. For the “signal ON” method (Figure 8 right), the attached aptamers have a fluorophore at their extremity, while the short complementary oligonucleotide has a quencher, which suppresses fluorescence of nearby fluorophores. Successful association of OTA will remove the quenchers and therefore increase the fluorescence signal.
Table of contents :
RESUME
GENERAL INTRODUCTION
1 STATE OF THE ART & STRATEGY
1.1 Introduction
1.2 Pathogens
1.2.1 Definition and types
1.2.2 Ochratoxin
1.2.3 Methods for detecting pathogens
1.3 Aptamer-based biosensors
1.3.1 Aptamers as probes in biosensors
1.3.2 Aptamers for the detection of OTA
1.3.3 Aptamer-based biosensors for the detection of OTA
1.3.4 Challenges for biosensors
1.4 Strategy proposed for detecting the interaction aptamer-OTA
1.4.1 Direct detection of the interaction aptamer-OTA by ATR-FTIR
1.4.2 Indirect detection of the interaction aptamer-OTA by fluorescence
2 SURFACE FUNCTIONALIZATION OF OXIDE FREE SILICON SURFACES
2.1 Introduction
2.2 State of the Art
2.2.1 Functionalization of silicon surfaces
2.2.2 Two-step amidation of acid terminated surfaces
2.3 Materials and Methods
2.3.1 Chemicals and substrates
2.3.2 Hydrogenated silicon surfaces
2.3.3 Photochemical hydrosilylation of undecylenic acid
2.3.4 Activation with EDC/NHS of acid-terminated surfaces
2.3.5 Aminolysis of PEG 750 on activated silicon surfaces
2.3.6 Adsorption test of E. coli Katushka K12 on PEG-surfaces
2.4 Results and discussion
2.4.1 Hydrogenation of silicon surfaces
2.4.2 Grafting of undecylenic acid on silicon surfaces by hydrosilylation
2.4.3 Activation with EDC/NHS of acid-terminated surfaces
2.4.4 Aminolysis of an activated silicon surface with PEG 750
2.4.5 Adsorption test of E. coli Katushka K12 on PEG-surfaces
2.5 Conclusion
3 CHARACTERIZATION OF OCHRATOXIN A IN PHYSIOLOGICAL BUFFER SOLUTIONS
3.1 Introduction
3.2 Materials and methods
3.2.1 Chemicals and substrates
3.2.2 Preparation of OTA-solutions for in-situ IR spectroscopy
3.2.3 Preparation of OTA-solutions for UV-vis spectroscopy
3.3 Results and discussion
3.3.1 The relation of OTA-structure with IR response and UV-vis absorption
3.3.2 Calibration of OTA IR-intensity and UV-vis absorption to OTA-concentration in solution
3.4 Conclusion
4 INTERACTION APTAMER – OTA
4.1 Introduction
4.2 Materials and methods
4.2.1 Chemicals, substrates and devices
4.2.2 Aminolysis with aptamers
4.2.3 Interaction of OTA and warfarin with aptamers
4.2.4 Regeneration of OTA from its aptamers
4.3 Results and discussion
4.3.1 Aminolysis of activated silicon surfaces with aptamers
4.3.2 Interaction of OTA with its aptamer by IR on crystalline silicon surfaces
4.3.3 Regeneration of aptamers
4.3.4 Non-specific interaction of aptamers with warfarin
4.4 Conclusion
5 FLUORESCENT BIOCHIPS FOR OTA DETECTION
5.1 Introduction
5.2 Materials and Methods
5.2.1 Chemicals and substrates
5.2.2 Transfer of protocols from crystalline silicon
5.2.3 Preparation of solutions for spotting
5.2.4 Blocking of remaining active surface sites
5.2.5 Hybridization with complementary oligonucleotides
5.2.6 Association with OTA
5.3 Results and discussion
5.3.1 PECVD-calibration
5.3.2 Fluorescent assays
5.4 Conclusion
6 GENERAL CONCLUSION AND OUTLOOK
ANNEX
A.I. ATR-FTIR spectroscopy
A.II. Scanning electron microscopy
A.III. Thermal vapor deposition
A.IV. Plasma enhanced chemical vapor deposition
A.V. Robotic spotter
A.VI. Fluorescence imaging
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