Preparation Of Hemoglobin Imprinted Surface Plasmon Resonance Biosensors

Abstract

Proteins hold many pivotal structural, functional and organization features and closely associate with the other macromolecules such as lipids and carbohydrates to generate larger and more complex units in cellular machinery. Researchers have been focused on discovering fundamental properties of proteins, and realized that they are also one of the major indicators and predictors in disease stages, as well as recent studies pointed out that their structure, concentration, and even, orientation are crucial for facilitating cellular machinery. Due to their multiple roles in cell functionality and structure, one of the most attractive arena to investigate the significance of proteins is to detect small biological units for diagnosing diseases. For instance, hemoglobin --an iron carrying protein in red blood cells-- transports oxygen and carbon dioxide around the human body and also maintains the acid-base balance in the blood. In clinical practice, hemoglobin concentrations are closely correlated with several diseases and health status, including thalassemia, anemia, leukemia, heart disease, and excessive ii loss of blood. Sensitive and accurate detection platforms will have potentially create new avenues to monitor the concentrations of such vital protein marker, hemoglobin, in the early detection and highly reliable prediction of disease. Surface plasmon resonance technology has been considerably utilized to detect protein biomarkers, eukaryotic cells, bacteria, and viruses for diagnosis purposes. This sensing platform provides excellent optical modality to measure the changes in refractive index at the close vicinity of the metal surface. Compared with other sensitive biosensing modalities, the surface plasmon resonance biosensors holds multiple advantageous, including real‐time and label-free analysis, high dual sensing modality (surface and bulk sensitivity), short assay time, independent of small changes in temperature and surface oscillations, low-cost assay, and multiplexing. Besides these prominent features, the surface plasmon resonance technology cannot only potentially be integrated with different surface sensitive tools, and it also enables versatile surface modifications that can easily be tailored to multiplexed detection. Molecular imprinting method, one of a fascinating surface modification techniques, utilizes molecules as templates to create cavities for recognition of targets in the polymeric matrix. This method provides a broad range of versatility to imprint targets with different molecular size, three dimensional structure, and physicochemical properties. In contrast to the complex and time-consuming laboratory surface modification methods, this method offers a rapid, sensitive, inexpensive, easy-to-use, and selective approach for the diagnosis, screening and monitoring disorders. Owing to high selectivity, physical and chemical robustness, high stability, low-cost and reusability of this method, molecularly imprinted polymers have become very attractive and been applied in many fields, especially biosensors, diagnosis, and environmental monitoring. In this study, a molecularly imprinted surface plasmon resonance biosensor was designed to detect hemoglobin as a model protein marker. First, hemoglobin:acrylamide pre-complex was prepared with template and monomer iii mixture, and the cross-linker (methylenebis acrylamide) was applied to the pre-complex mixture to form a final mixture for polymerization. Followed by addition an initiator and activator (ammonium persulfate and tetramethyl ethylenediamine) pair to the final mixture, the monomer mixture was then used to decorate to the surface plasmon resonance biosensor surfaces. By employing spin coating technique, the monomer solution was uniformly distributed on the surface plasmon resonance biosensor surfaces. The polymerization was carried out under by photo-polymerization method. At the end of the polymerization, the unreacted monomers and impurities were removed and dried at room temperature. The hemoglobin imprinted surface plasmon resonance biosensor was characterized by Fourier transform infrared spectroscopy-attenuated total reflectance, atomic force microscope, an ellipsometer, and contact angle measurements. The hemoglobin imprinted surface plasmon resonance biosensor was tested for real-time detection of hemoglobin from hemoglobin solutions that have different hemoglobin concentrations. The selectivity and reusability performance of the hemoglobin imprinted surface plasmon resonance biosensor was also investigated. In addition, the microfluidic-integrated surface plasmon resonance biosensors were also prepared for real-time hemoglobin detection by using different layers that are polymethyl methacrylate, double sided adhesive and gold coating substrate. After the different modification steps, the microfluidic-integrated surface plasmon resonance biosensors interacted with different hemoglobin concentration solutions. Finally, the equilibrium and adsorption isotherm models of interactions between hemoglobin solutions and two different surface plasmon resonance biosensors were determined.