Resumen de Aptamer biosensors engineering based on reflectometric interference spectroscopy in nanoporous anodic alumina
In the last few decades, advances in biomedical applications have generated the need to investigate new analysis methods.Biosensors are analytical tools capable of detecting any type of biomolecule in an easy and fast way. Therefore, the design and manufacture of a good biosensor provides an extremely useful tool in a large number of biomedical applications. The first commercialized biosensors were blood glucose sensors, which were a breakthrough in the treatment of diabetic patients.1) Inmobilization of an aptamer in the surface of the NAA.2) Study the best cross-linking methods to modify the surface on NAA to be able to inmobilize the aptamer in the surface of NAA.3) Characterize in real-time by means of RIfS the modification process of NAA until the inmobilization of NAA.4) Sensing in real time the target molecule under study.Historically, the most widely used supporting nanomaterial in the manufacture of biosensors has been gold. However, the developmentof more complex analysis methods has allowed the introduction of new nanomaterials in the fabrication of biosensors. Within these naomaterials, nanoporous materials have meant a great advance in the design of biosensors, since they allow obtaining a sensor with a large surface area. Furthermore, nanoporous materials allow the introduction of new and highly precise analysis techniques.Many optical sensing techniques have been developed in porous materials, including the Reflectometric Interference Spectroscopy (RIfS) which has been reported as a very useful method to use in NAA based biosensors. A hinghly effective fluid system has been developed in NAA by using the RIfS technique (NAA-RIfS system). In this procedure, by means of RIfS, the change in the signal is detected when a fluid with different molecules is introduced into the system.In this PhD dessertation it is presented thte study of the fabrication of an aptamer biosensor based on NAA-RIfS system. The main objectives are:Immobilization of streptavidin on the alumina surface was first studied. Streptavidin is a protein with an amino group on one end and a carboxylic group on the other. Amino groups and carboxyl groups can be linked together in a stable covalent bond. Therefore, by inserting an amino group or a carboxyl group on the surface of the alumina, a NAA substrate is obtained on which streptavidin can be easily immobilized. In this case we use the molecule (3-Aminopropyl)triethoxysilane (APTES) that forms a self-assembled monolayer (SAM) on the surface of the alumina and generates a free amino group able to bind streptavidin.In the following years, research in biomedicine continued to advance and the need to investigate new types of biosensors became clear. In the first years, biosensors based on the detection of chemical reactions that take place when the biosensor is put in contact with the sample under study were studied. One of the most advanced biosensors is the glucose oxidase sensor, which in contact with a glucose molecule oxidizes it and produces a detectable signal.Later, antibody-based sensors began to be developed, whose operation is based on the detection of the specific binding of an antibody with its antigen. These sensors are the most used for their high versatility and specificity.Today, the need for more sensitive and accurate analysis has extended the research to newer types of sensors based on newer molecules. Among others, aptamer-based biosensors emerged. Aptamers are DNA molecules that are synthetically manufactured to bind very specifically to the molecule under study. Therefore, they provide the ability to cheaply and easily manufacture biosensors with high affinity and sensitivity.The design and manufacture of a good biosensor, in addition to the specific sensing molecules, must include a suitable support material. In this sense, the introduction of nanomaterials in the manufacture of biosensors has allowed the manufacture of highly efficient sensors due to their particular characteristics.
Historically, the most widely used supporting nanomaterial in the manufacture of biosensors has been gold. However, the developmentof more complex analysis methods has allowed the introduction of new nanomaterials in the fabrication of biosensors. Within these naomaterials, nanoporous materials have meant a great advance in the design of biosensors, since they allow obtaining a sensor with a large surface area. Furthermore, nanoporous materials allow the introduction of new and highly precise analysis techniques.
Nanoporous silicon has been the most widely used nanoporous material this field, but currently nanoporous alumina (NAA) has been reported as a powerful alternative. Nanoporous alumina offers some advantages over silicon since the manufacturing process is simpler and its structure can be easily tuned. Furthermore, the structure of the alumina is stable while the structure of the silicon changes its properties over time.
The detection method of the signal that occurs when the biosensor comes into contact with the molecule to be studied is also a crucial element in obtaining the best results. The most studied methods have been based on generating an electrical response when a chemical reaction occurs when biosensor is in contact with the specific molecule (electrochemical biosensors). This type of biosensors are not effective in all circumstances, since there are interaction mechanisms between molecules that do not generate a detectable electrical response, or the response that is generated does not allow ussee the evolution of the reaction that occurs. In the latest advances in biosensor research, optical sensors have been reported as a very good alternative,since they allow the detection of interactions between molecules which would not be possible with other methods. Optical sensors are based on generating a change in the optical properties of the biosensor surface when it detects the presence of the molecule under study. They allow not only the detection of the molecule , but also allow registering in real time the evolution of the recognition process to be studied in real time.
Many optical sensing techniques have been developed in porous materials, including the Reflectometric Interference Spectroscopy (RIfS) which has been reported as a very useful method to use in NAA based biosensors. A hinghly effective fluid system has been developed in NAA by using the RIfS technique (NAA-RIfS system). In this procedure, by means of RIfS, the change in the signal is detected when a fluid with different molecules is introduced into the system.
In this PhD dessertation it is presented thte study of the fabrication of an aptamer biosensor based on NAA-RIfS system. The main objectives are: 1) Inmobilization of an aptamer in the surface of the NAA .2) Study the best cross-linking methods to modify the surface on NAA to be able to inmobilize the aptamer in the surface of NAA .3) Characterize in real-time by means of RIfS the modification process of NAA until the inmobilization of NAA .4) Sensing in real time the target molecule under study.
As it is mentioned above, the main objective of this thesis is the immobilization of the aptamer on the surface of the alumina and subsequent sensing of a protein. In this case, since Thrombin Binding Aptamer (TBA) it is one of the most studied aptamers and its operating conditions are well known, we have chosen to study the process using the thronbin binding aptamer and the thrombin protein as models.To carry out the immobilization of the aptamer on the surface of the alumina, the streptabidin-biotin binding complex has been used, since it is one of the simplest and strongest molecule binding methods.
Immobilization of streptavidin on the alumina surface was first studied. Streptavidin is a protein with an amino group on one end and a carboxylic group on the other. Amino groups and carboxyl groups can be linked together in a stable covalent bond. Therefore, by inserting an amino group or a carboxyl group on the surface of the alumina, a NAA substrate is obtained on which streptavidin can be easily immobilized. In this case we use the molecule (3-Aminopropyl)triethoxysilane (APTES) that forms a self-assembled monolayer (SAM) on the surface of the alumina and generates a free amino group able to bind streptavidin.
In this thesis, we have studied different types of streptavidin binding methods with APTES and the ability of these methods to work in the fluid medium of the NAA-RIfS system (Glutaraldehyde (GTA), Carbodiimide Cross-linker (EDC/NHS) and Biotin-Sulfo-NHS reagent).We have also studied the system's ability to detect streptavidin binding processes with APTES, as well as streptavidin binding event with biotin and with a biotinylated molecule (biotinylated thrombin).
After verifying that the RIfS system was capable of detecting and monitoring the different binding processes (APTES - streptavidin - biotin) in real time, other important parameters for the manufacture of the sensor were studied.Knowing that the detection of biotinylated thrombin is possible, we studied how it affects the pore size of alumina throughout the process. For this, studies on 50 and 60 nm NAA substrates experiments were carried out, keeping the concentrations of all the molecules constant (APTES, streptavidin and biotinylated thrombin). The best results were obtained with alumina substrates with60 nm pores, therefore all the following experiments were performed on alumina substrates with 60nm pores.Once the ideal pore size for working with this system was known, the behavior of the system was studied using different concentrations of streptavidin, the results suggest that the most suitable is 1 μg / ml.The ability of the system to detect and quantify different concentrations of biotinylated thrombin was also studied, and it was observed that the system is capable of detecting thromnin concentrations above 10 μg / ml.
Achieving the results described above, the biosensor was designed and manufactured with the aptamer (TBA) immobilized on the surface of the alumina. For this, the same previously mentioned binding mechanism (streptavidin-biotin) was used, but in this case, streptavidinwas linked to APTES by means of the Biotin-Sulfo-NHS cross-linker. Streptavidin was immobilized on the surface of the alumina and the biotinylated aptamer (TBA) was then attached to it. The entire process of immobilization of the aptamer (Biotin-Sulfo-NHS - streptavidin - biotinylated aptamer) was characterized in real time by means of RIfS. Then the alumina substrates with the aptamer immobilized on its surface were used to detect different concentrations of thrombin. With the obtained data, a regression curve was made from which the characteristics of the biosensor were studied. Finally, studies of the specificity of the biosensor were made.
The conclusions of the work are included in the final part of this thesis. On the one hand, we can conclude that the streptavidin-biotin complex is suitable for the immobilization of biotinylated thrombin and the RIfS system is capable of detecting it in real time. This suggests that, by means of this binding complex and using the NAA-RIfS system, any type of biotinylated molecules could be immobilized and monitored.The proper pore size for all immobilized molecules to fit inside the pores has been confirmed to be around 60nm. While the appropriate streptavidin concentration when using EDC / NHS as a cross-linker is 1 μg / ml.Furthermore, we have shown that the binding of different thrombin concentrations can be monitored in real time. The system has been shown to be able to detect and quantify such concentrations avobe 10 μg / ml.
On the other hand, it has been shown that it is possible to immobilize and detect the TBA aptamer on the surface of NAA by means of the streptavidin-biotin binding complex using Biotin-Sulfo-NHS as a cross-linking method obtaining high reproducibility between experiments. In addition, the capability of the system to detect different concentrations of thrombinin the μM range was proved. Furthermore, the limit of detection and the sensitivity were estimated as 7.2 nM 45.5 nm/μM respectively. In addition, high specificity of the biosensor was proved.
Summarizing, all these results prove that the flow cell NAA-RIfS system can works not only as a characterization method of the modification procedure of the biosensor surfaces, but also as an accurate and specific method for biosensing.These results also reveal that this system can provide new analytic devices with different biotinylated receptors. These biosensor devices make possible the detection of a wide range of molecules among others, proteins, toxins and viruses.
