Novel Strategies For Biosensors Based On Smart Surface Switches

Controlled study of microscopic fields is one of the most important challenges to scientific and technological development. Scientists use chemical and biological methods to build complex molecular-level devices from the basic building blocks of atomic or molecular microstructure. This strategy, known as "bottom-up" design allows for devices with specific structure or function to be built. Of particular interest has been the development of molecular switches, which could serve as the precursor to the preparation of more complicated molecular devices. Molecular switches are molecules which are capable of changing their behavior in a controlled fashion based on external stimuli such as light, electricity, heat, magnetic fields, or pH. By taking advantage of such switching behavior it should be possible to realize the transmission of information.Switching behavior requires an external stimuli to cause either an electronic transition or a structural rearrangement. The three most important stimuli are light (absorption of photons), electricity (addition or removal of electrons) and chemical energy (binding of protons, metal ions, or other special molecules). The most common photochemical switches are related to light-induced isomerization and light-induced oxidation and reduction reactions. Electrochemical methods can also be used to produce redox type switching behavior. Moreover, electrochemical techniques are a useful way to monitor the operation of molecular machinery, with the electrode serving as the connection to the macroscopic world. As an example, surface electric switches can be used to control protein adsorption and separation. By combining molecular design, organic synthesis, and polymer chemistry, it is possible to introduce hydrophilic or hydrophobic functional groups or antigen-antibody complexes to the surface resulting in a reversible switch.This thesis is based on the use of a low-density self-assembled monolayer and temperature-sensitive polymer to build a biocompatible surface. By using these innovative methods, controlled in situ determination and monitoring for biological molecules could be carried on. The intelligent modification of surface properties allows for the detection of a wide range of biological species. The focus of this work has been the development of a sensitive and reversible switch for the detection of specific biomolecules. By using a low-density self-assembled monolayer (SAM) to establish a hydrophobic/hydrophilic switch it has been possible to achieve the rapid and controlled detection of avidin and streptavidin. Reversible control of the surface properties has been achieved with various methods, including: photoillumination, potential effects, and thermal driving. By using temperature-sensitive poly (N-isopropylacrylamide) (PNIPAAm) materials, an immune switch sensor for Anti-BSA antibody and FITC-BSA could be created. The switch is stable in biological environments, does not interfere with the antibody/antigen detection, and can be reused more than 30 times. In the first chapter we review the research on surface molecular switches and related technical progress in the field of biosensors. Polymers and self-assembly are two important ways to build molecular surface switches. Using self-assembled monolayers could supply a thermodynamically stable and ordered membrane. The structure of SAMs can easily be varied by the introduction of a wide range of functional groups. Temperature-sensitive materials are also found to be good candidates and have the useful ability to respond to external temperature stimuli. In recent years PNIPAAm has attracted interest in the study of temperature-sensitive polymer materials because of its potential application in drug delivery, immunoassays, and life science research.The second chapter describes using SAMs to constructsmart hydrophobic/hydrophilic reversible surfaces as a new method for biosensor development. The inclusion complex (IC) between 16-mercaptohexadecanoic acid (MHA) andα-,β- orγ-cyclodextrin acted as the space-filling group of the SAM. The unwrapping procedure, the removal of n-CD from the SAM, was accomplished by dissociating the bond n-CD—MHA with ethanol. The surface coverage for LD-SAM-n referring to high-density SAM shows an obvious decrease from 100% to 61.2%, 45.3% and 29.2%, respectively. The thus-prepared low density-SAM shows reversible conformational reorientation under negative and positive potential; this induces changes in wettability, which can be observed by means of contact angle measurements. The removal of n-CD was monitored by quartz crystal microbalance (QCM), nuclear magnetic resonance (NMR), impedance (IMP), and MALDI-TOF-MS.The third chapter describes the application of our LD-SAM surface to the controlled assembly of two kinds of fluorescent-labeled avidin and streptavidin. Selective protein adsorption was demonstrated on the above-mentioned switchable surface. The assembly of the two proteins on MHA-SAM-n was performed in PBS buffer (pH 7.4) at an applied potential of 0.3 V and -0.3 V (vs.SCE), respectively. From either QCM or fluorescence spectra (FL) data, distinctly different assembly behaviors for the two proteins were observed at two controlled potentials. For example, the emission intensity for avidin-LD-SAM-1 assembled at negative potential for 30 min was about 4.9 times that for assembly at positive potential. The dissimilarity of the surface loading for these two proteins is believed to be mainly due to the charge status difference originating from their isoelectric points. We believe that it might lead to versatile applications, e.g. control of protein adsorption/release in a functionalized capillary or microfluidics channel, or design of intelligent protein chips. We have also attempted to apply this system to a homemade microfluidic chip.The fourth chapter works on establishing the PNIPAAm-antibody surface that could supply the most effective dissociation of the antigen and regeneration of antibody into reuse of the immunosensors. As a model antibody-antigen system, bovine serum albumin (BSA) and the corresponding antibody (anti-BSA) were chosen. A reversible PNIPAAm-antibody (anti-BSA) conjugates surface was established by triggered control of external temperature. This took advantage of the thermally tunable conformational changes for the PNIPAAm-conjugated antibody surface, and could be used for switchable antigen association and dissociation. The temperature controlling strategy could realized the regeneration of the immunosensor on which immobilized anti-BSA antibodies retain the activity and specificity necessary to carry out more than 30 reproducible assays for BSA. The dissociation reaches 89%, which can compare with the general recovery methods. The controlled binding and unbinding were monitored by quartz crystal microbalance (QCM), confocal fluorescence, native electrophoresis, laser induced fluorescence, and electrochemical impedance.The fifth chapter describes a highly enantioselective and sensitive immunosensor for the detection of chiral amino acids based on capacitive measurement. The sensor was prepared by first binding mercaptoacetic acid to the surface of a gold electrode, followed by modification with tyramine utilizing carbodiimide activation. Stereoselective binding of an anti-D-amino acid antibody to the hapten-modified sensor surface resulted in capacitance changes that were detected with high sensitivity by a potentiostatic step method. Using capacitance measurement, detection limits of 5 pg of antibody/mL were attained. The exquisite stereoselectivity of the antibody was also utilized in a competitive setup to quantitatively determine the concentration of D-phenylalanine in nonracemic samples. Trace impurities of D-phenylalanine as low as 0.001% could be detected. This method should be useful not only for the enantioselective detection of amino acids as described here but also for investigating other targets (e.g., drugs) using appropriate antibodies.The sixth chapter is the summary of this thesis and the future prospects for related research.This thesis reports on the development of several different surface molecular switches which can be used as biosensors. Using self-assembled monolayers, hydrophobic/hydrophilic surface switches capable of responding to external temperature and electrical stimuli were prepared. These switches could supply a smart and biocompatible interface to determine and analyze proteins and antigen-antibody immune systems, which may open a new paradigm for the design of functional biocomposite films.