The presence of harmful chemicals and microorganisms in food is often addressed ad food contamination, which can cause consumer illness at different levels of severity. Potential hazardous residues in food include several different substances: natural and environmental contaminants (e.g. toxins, heavy metal ions), agrochemicals pollutants like pesticides, drugs, growth promoters, packaging components, etc. The detection of contaminants is then of utterly importance in food safety and environmental analysis; thus, it requires highly sensitive and easy-to-use analytical procedures to be developed. Conventional analytical methods used for this kind of analysis include separation techniques (e.g. high-performance liquid chromatography, tandem mass spectrometry), which often provide sensitive and selective results. Despite the advantages of these techniques, the high costs, the expensive instrumentation, the technical skills needed for users and the complex pretreatment processes are pushing scientists to find out rapid, low cost, highly sensitive and simple alternative analytical methods. For this purpose, biosensors can act as an option for solving the problems mentioned before or become a helpful tool at least. Biosensors development can be classified as an interdisciplinary field and one of the most active research areas in analytical chemistry. As well as other analytical methods, biosensors performance is evaluated by considering their detection limit, their sensitivity, selectivity and reproducibility in terms of linear and dynamic range and response to interfering substances. The most used receptors in biosensing applications are probably the antibodies, which are able to bind target molecules with high selectivity and sensitivity, but their use is characterized by some limitations. As one of the drawbacks in developing contaminants biosensors is the synthesis of antibodies for these highly toxic targets, the use of biomimetic receptors has recently become an interesting alternative. This kind of probes includes biological “bricks” assembled in vitro or synthetic molecules assembled to mimic the recognition capabilities of antibodies. The advances in nanotechnology have led to the discovery and the employment of a great number of new materials in nanoscale dimensions (comprised between 1 and 100 nm, even if for biological application dimensions can raise up to 500 nm and rarely up to 700 nm). Because the common biological systems (such as proteins, viruses, membranes, etc.) are nanostructured and their interactions take place at nanometric scale, nanomaterials become ideal candidates for the development of advanced biosensing devices. Nanostructures present several advantages in analytical applications and can be mainly used as transducers (due to their unique optical, chemical, electrical, and catalytic properties) or as a component of the recognition element of a biosensing device (due to the high surface-to-volume ratio that increases the number of bioreceptors attached to the sensing surface). This thesis presents different strategies for the development of electrochemical biosensors based on nanostructured sensing platforms and biomimetic probe molecules for the determination of a pattern of contaminants (e.g. pesticides, toxins, allergens) related to food and environmental analysis. The dissertation is subdivided in ten chapters. Chapter 1. The definition of biosensors is provided, highlighting the classification and advantages of electrochemical ones. Moreover, a short description of aptamers, Affibodies® and molecularly imprinted polymers as bioreceptors is presented. A particular attention has been posed in the description of different aptamer assay formats and the aptasensing approaches based on screen-printed electrochemical transducers. Chapter 2. The role of biosensors in contaminants detection for food analysis is described. A classification of the contaminants analyzed in this work is also given, divided by their chemical classes, underlining their hazardous potential. Chapter 3. The electrochemical techniques (cyclic voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy) used in this work are introduced, describing their basic principles. The experimental set-up, including the graphite screen-printed cells used as transducers in this thesis, is described. Chapter 4. An overview of the conductive nanostructured materials for sensing platforms development is presented. In particular, a short description of the conductive polymers (i.e. polyaniline, poly(aniline-co-anthranilic acid), poly-L-lysine) and the gold nanoparticles used in this work is given, also providing their electrodeposition protocols and electrochemical characterization. Chapter 5. The application of a sensing platform based on gold nanoparticles and polyaniline-modified graphite screen-printed electrodes for dopamine detection is reported. Dopamine was chosen as a model analyte due to its easiness of determination by being oxidized at an electrodic surface. The analytical usefulness of the sensor was also demonstrated by analyzing spiked commercial serum samples. Chapter 6. Profenofos pesticide is detected by means of an aptasensor based on a competitive format, which employs a gold/polyaniline-modified transducer as sensor platform and an enzyme-linked label for a dual amplification of the signal. The nanostructured electrodes were modified with a mixed monolayer of a thiol-tethered DNA probe and 6-mercapto-1-hexanol. A biotinylated DNA aptamer was incubated with the pesticide and then dropped onto the sensing surface: the aptamer sequences which did not bind the analyte were free to hybridize with the immobilized DNA probe. The binding was traced with the addition of streptavidin-alkaline phosphatase enzyme conjugate: the enzymatic substrate 1-naphthyl phosphate was converted into the electroactive product 1-naphthol, which was finally oxidized and detected by differential pulse voltammetry. The bindings of the aptamer with the analyte and the DNA probe were also preliminary assessed by melting temperatures study. Chapter 7. Aflatoxin B1 mycotoxin is detected by means of an enzyme-linked oligonucleotide array based on a competitive format. The developed assay makes use of a sensing platform composed of poly(aniline-co-anthranilic acid)-modified electrodes; a conjugate between aflatoxin B1 and bovine serum albumin was immobilized by amide coupling between the carboxylic groups of the copolymer and the amine groups of the protein. Each phase involved in the assembly of the aptasensor was characterized and evaluated by means of cyclic voltammetry and electrochemical impedance spectroscopy techniques. The competition was achieved between free and immobilized AFB1 molecules for the binding with a biotinylated DNA aptamer and the affinity reaction was traced by streptavidin-alkaline phosphatase in the same way as previously described. Preliminary experiments in maize flour samples spiked with AFB1 were also conducted. Chapter 8. Deoxynivalenol mycotoxin is detected by means of an aptasensor based on a competitive format. The sensing strategy is somehow similar to that employed for the pesticide detection, as it shares with the aforementioned assay both the nanostructured platform and the enzymatic labeling. However, in this case, a thiol-tethered DNA aptamer was immobilized on the electrodic surface, while the competition occurs in solution between deoxynivalenol molecules and a biotinylated complementary DNA sequence. The enzyme and its substrate were then used for the electrochemical detection by differential pulse voltammetry. Apart from being one of the first electrochemical aptasensors reported for deoxynivalenol detection, the novelty of the work consists in the investigation of the molecular interaction between the aptamer and the mycotoxin by a docking study, which allows to verify if the aptamer region binding with the complementary oligonucleotide sequence chosen for the competitive assay includes the interaction sites between the mycotoxin and the DNA aptamer, while also determining the preferred orientation assumed by DON in the binding event.7 Chapter 9. β-Lactoglobulin milk allergen is detected by means of a switch-on assay employing a gold/poly-L-lysine-modified transducer as the sensing platform. The nanostructured electrodes were modified with a mixed monolayer of a thiol-tethered DNA aptamer, bearing the electroactive methylene blue moiety to the free 3’-end, and 6-mercapto-1-hexanol. Upon the binding with the analyte, the aptamer changed its conformation, making the labeled end to be closer to the electrodic surface and to be more easily oxidized. The electrochemical folding-based aptasensor allowed unambiguous identification of the protein, while no significant non-specific signals were detected in case of negative controls. Chapter 10. Concluding remarks are reported.
Development of biomimetic nanostructured sensors in food and environmental applications / Giulia Selvolini. - (2021).
Development of biomimetic nanostructured sensors in food and environmental applications
Giulia Selvolini
2021
Abstract
The presence of harmful chemicals and microorganisms in food is often addressed ad food contamination, which can cause consumer illness at different levels of severity. Potential hazardous residues in food include several different substances: natural and environmental contaminants (e.g. toxins, heavy metal ions), agrochemicals pollutants like pesticides, drugs, growth promoters, packaging components, etc. The detection of contaminants is then of utterly importance in food safety and environmental analysis; thus, it requires highly sensitive and easy-to-use analytical procedures to be developed. Conventional analytical methods used for this kind of analysis include separation techniques (e.g. high-performance liquid chromatography, tandem mass spectrometry), which often provide sensitive and selective results. Despite the advantages of these techniques, the high costs, the expensive instrumentation, the technical skills needed for users and the complex pretreatment processes are pushing scientists to find out rapid, low cost, highly sensitive and simple alternative analytical methods. For this purpose, biosensors can act as an option for solving the problems mentioned before or become a helpful tool at least. Biosensors development can be classified as an interdisciplinary field and one of the most active research areas in analytical chemistry. As well as other analytical methods, biosensors performance is evaluated by considering their detection limit, their sensitivity, selectivity and reproducibility in terms of linear and dynamic range and response to interfering substances. The most used receptors in biosensing applications are probably the antibodies, which are able to bind target molecules with high selectivity and sensitivity, but their use is characterized by some limitations. As one of the drawbacks in developing contaminants biosensors is the synthesis of antibodies for these highly toxic targets, the use of biomimetic receptors has recently become an interesting alternative. This kind of probes includes biological “bricks” assembled in vitro or synthetic molecules assembled to mimic the recognition capabilities of antibodies. The advances in nanotechnology have led to the discovery and the employment of a great number of new materials in nanoscale dimensions (comprised between 1 and 100 nm, even if for biological application dimensions can raise up to 500 nm and rarely up to 700 nm). Because the common biological systems (such as proteins, viruses, membranes, etc.) are nanostructured and their interactions take place at nanometric scale, nanomaterials become ideal candidates for the development of advanced biosensing devices. Nanostructures present several advantages in analytical applications and can be mainly used as transducers (due to their unique optical, chemical, electrical, and catalytic properties) or as a component of the recognition element of a biosensing device (due to the high surface-to-volume ratio that increases the number of bioreceptors attached to the sensing surface). This thesis presents different strategies for the development of electrochemical biosensors based on nanostructured sensing platforms and biomimetic probe molecules for the determination of a pattern of contaminants (e.g. pesticides, toxins, allergens) related to food and environmental analysis. The dissertation is subdivided in ten chapters. Chapter 1. The definition of biosensors is provided, highlighting the classification and advantages of electrochemical ones. Moreover, a short description of aptamers, Affibodies® and molecularly imprinted polymers as bioreceptors is presented. A particular attention has been posed in the description of different aptamer assay formats and the aptasensing approaches based on screen-printed electrochemical transducers. Chapter 2. The role of biosensors in contaminants detection for food analysis is described. A classification of the contaminants analyzed in this work is also given, divided by their chemical classes, underlining their hazardous potential. Chapter 3. The electrochemical techniques (cyclic voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy) used in this work are introduced, describing their basic principles. The experimental set-up, including the graphite screen-printed cells used as transducers in this thesis, is described. Chapter 4. An overview of the conductive nanostructured materials for sensing platforms development is presented. In particular, a short description of the conductive polymers (i.e. polyaniline, poly(aniline-co-anthranilic acid), poly-L-lysine) and the gold nanoparticles used in this work is given, also providing their electrodeposition protocols and electrochemical characterization. Chapter 5. The application of a sensing platform based on gold nanoparticles and polyaniline-modified graphite screen-printed electrodes for dopamine detection is reported. Dopamine was chosen as a model analyte due to its easiness of determination by being oxidized at an electrodic surface. The analytical usefulness of the sensor was also demonstrated by analyzing spiked commercial serum samples. Chapter 6. Profenofos pesticide is detected by means of an aptasensor based on a competitive format, which employs a gold/polyaniline-modified transducer as sensor platform and an enzyme-linked label for a dual amplification of the signal. The nanostructured electrodes were modified with a mixed monolayer of a thiol-tethered DNA probe and 6-mercapto-1-hexanol. A biotinylated DNA aptamer was incubated with the pesticide and then dropped onto the sensing surface: the aptamer sequences which did not bind the analyte were free to hybridize with the immobilized DNA probe. The binding was traced with the addition of streptavidin-alkaline phosphatase enzyme conjugate: the enzymatic substrate 1-naphthyl phosphate was converted into the electroactive product 1-naphthol, which was finally oxidized and detected by differential pulse voltammetry. The bindings of the aptamer with the analyte and the DNA probe were also preliminary assessed by melting temperatures study. Chapter 7. Aflatoxin B1 mycotoxin is detected by means of an enzyme-linked oligonucleotide array based on a competitive format. The developed assay makes use of a sensing platform composed of poly(aniline-co-anthranilic acid)-modified electrodes; a conjugate between aflatoxin B1 and bovine serum albumin was immobilized by amide coupling between the carboxylic groups of the copolymer and the amine groups of the protein. Each phase involved in the assembly of the aptasensor was characterized and evaluated by means of cyclic voltammetry and electrochemical impedance spectroscopy techniques. The competition was achieved between free and immobilized AFB1 molecules for the binding with a biotinylated DNA aptamer and the affinity reaction was traced by streptavidin-alkaline phosphatase in the same way as previously described. Preliminary experiments in maize flour samples spiked with AFB1 were also conducted. Chapter 8. Deoxynivalenol mycotoxin is detected by means of an aptasensor based on a competitive format. The sensing strategy is somehow similar to that employed for the pesticide detection, as it shares with the aforementioned assay both the nanostructured platform and the enzymatic labeling. However, in this case, a thiol-tethered DNA aptamer was immobilized on the electrodic surface, while the competition occurs in solution between deoxynivalenol molecules and a biotinylated complementary DNA sequence. The enzyme and its substrate were then used for the electrochemical detection by differential pulse voltammetry. Apart from being one of the first electrochemical aptasensors reported for deoxynivalenol detection, the novelty of the work consists in the investigation of the molecular interaction between the aptamer and the mycotoxin by a docking study, which allows to verify if the aptamer region binding with the complementary oligonucleotide sequence chosen for the competitive assay includes the interaction sites between the mycotoxin and the DNA aptamer, while also determining the preferred orientation assumed by DON in the binding event.7 Chapter 9. β-Lactoglobulin milk allergen is detected by means of a switch-on assay employing a gold/poly-L-lysine-modified transducer as the sensing platform. The nanostructured electrodes were modified with a mixed monolayer of a thiol-tethered DNA aptamer, bearing the electroactive methylene blue moiety to the free 3’-end, and 6-mercapto-1-hexanol. Upon the binding with the analyte, the aptamer changed its conformation, making the labeled end to be closer to the electrodic surface and to be more easily oxidized. The electrochemical folding-based aptasensor allowed unambiguous identification of the protein, while no significant non-specific signals were detected in case of negative controls. Chapter 10. Concluding remarks are reported.File | Dimensione | Formato | |
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