Mikroalbümin Tayini için SPR Temelli Nanosensörlerin Hazırlanması

Abstract

Human serum albumin (HSA) is the most abundant protein in the blood. Enlargement of the pores in the glomeruli and remarkable amount of plasma proteins’ leakage into urine is caused by the kidney damages. A certain amount of leaked HSA (in spot urine 0-20 mg/L) is defined as microalbumin. Since the increase in the amount of microalbumin is a sign of some important diseases, early identification is very important for the diagnosis. Devices which convert biological response to electrical signals are defined as ‘’Biosensors’’. Biosensors are analytical devices that contain biological recognition elements and physicochemical transducers in their bodies. Molecular imprinted polymers was unified with the transducers through major developments in the field of biosensors and interactions between analyte and MIP can be converted into a processable signals. In this sense, surface plasmon resonance (SPR) based optical devices have a great potential. In this study, it was aimed to prepare SPR based nanosensor using molecular imprinting technique for microalbumin detection in urine. In the first step, NMethacryloyl-(L)-Leucine Methyl Ester (MALM) monomer having capability of interaction with HSA to create cavities was synthesized by reacting of L-Leucine methyl ester with methacrloyl Chloride. To define optimum ratio between template HSA molecule and MALM monomer, HSA was mixed with different ratios of MALM monomer and optimum ratio was determined by using UV-visible spectrophotometer instrument. Nanosensor preparation studies were realized according to the optimized ratio conditions. Nanosensors are prepared in two ways by micro-contact method with HSA imprinted [PEDMALM-HSA] (MIP) and non imprinted [PEDMALM] (NIP) nanosensor preparation and by attaching HSA imprinted [PEDMALM-HSA] (MIP) and non imprinted [PEDMALM] (NIP) nanoparticles to the chip gold surface respectively. For this specified purpose, HSA imprinted poly(N-Methacryloyl-(L)-Leucine Methyl Ester-Human Serum Albumin) [PEDMALM-HSA] (MIP) nanofilm was synthesized onto the allyl mercaptan modified gold chip surface by micro-contact method. Mixture prepared from specific proportions of MALM monomer, crosslinker ethylene dimethacrylate (EDMA) and initiator 2,2'-azobis (isobutyronitrile) (AIBN) was poured (20 μL) onto the SPR gold sensor surface and after then HSA immobilized glass slides was inserted onto the poured mixture by pressing to form stamped structure. Special cavities for the target HSA protein was formed when the mixture put under the UV light for 4 hours. For control experiments nonimprinted nanofilm preparation was performed in the same conditions without adding target HSA protein. SPR biosensors prepared by HSA imprinted [PEDMALM-HSA] (MIP) and non imprinted [PEDMALM-HSA] (NIP) nanofilms were characterized by FTIR-ATR, Atomic force microscope (AFM), Contact angle (CA), Ellipsometer measurements. Surface rougness measurements were obtained by Atomic force microscopy (AFM) method and images showed that roughness of HSA imprinted [PEDMALMHSA] (MIP) and non imprinted [PEDMALM] (NIP) nanofilms was estimated as 3.64 and 2.47 nm respectively. Rougness difference between HSA imprinted [PEDMALM-HSA] and non imprinted [PEDMALM-HSA] (MIP) nanofilms show that imprinting process of HSA molecules was performed successfully. Film thickness of HSA imprinted [PEDMALM-HSA] (MIP) and non imprinted [PEDMALM] (NIP) nanofilms was estimated by ellipsometry measurements and recorded as 95±4.9 and 89.9±4.9 nm for imprinted and non imprinted nanofilms respectively. Wettability studies was performed by contact angle measurements. Increasing contact angle values for unmodified gold chip surface (80°±0.73), HSA imprinted [PEDMALM-HSA] (MIP) (55.2°±2.24) and HSA non imprinted [PEDMALM] (NIP) (61.5±4.63°) nanofilms show that imprinting process enabled hydrophilicity increment. In the second part of the study to work in a a targeted concentration range [PEDMALM-HSA] (MIP) nanoparticles were synthesized by a two-phase miniemulsion polymerization method. The first aqueous phase PHASE I was prepared by dissolving of PVA (95 mg), SDS (15 mg) and NaHCO3 (12 mg) in 5.0 mL deionized water. The second phase PHASE II was prepared by dissolving of PVA (50 mg) and SDS (50 mg) in 100 mL of deionized water. Prepared MALM:HSA pre-complex (0.15; 0.015 mmol) was dissolved in a crosslinker, ethylene glycol dimethacrylate (EDMA; 4.8 mmol) to form oil phase. The oil phase was slowly added to the first aqueous phase. In order to obtain mini-emulsion, the mixture was homogenized at 25 000 rpm by a homogenizer. After homogenization, mixture was added to the PHASE II. Then, initiators, sodium bisulfite (125 mg) and ammonium persulfate (125 mg), were added into the solution. Polymerization was continued for 24 h at 40°C. Besides this, for control experiment, the non-imprinted [PEDMALM] (NIP) nanoparticles were synthesized by applying same procedure with imprinted nanoparticles except addition of template HSA molecules. Size distrubution of the prepared nanoparticles was characterized by zeta size and scanning electron microscopy measurements. Averaged size distrubution value of the HSA imprinted [PEDMALM-HSA] (MIP) and nonimprinted [PEDMALM] (NIP) nanoparticles was estimated as 63 and 55 nm respectively. Because of low polydispersity index values for imprinted and nonimprinted nanoparticles (PDI:0.138; PDI:0.113) homogeneous average nanoparticle size distrubition was attained. Average size of HSA imprinted [PEDMALM-HSA] (MIP) nanoparticles was determined as 60-65 nm by Scanning electron microscopy measurements. Target protein HSA imprinted [PEDMALM-HSA] (MIP) and non imprinted [PEDMALM] (NIP) nanoparticles were attached to the chip surface by dropping 5 µl of the prepared particles diluted 100 times with distilled water onto the chip surface which was already cleaned with ethyl alcohol and dried in a vacuum oven and put under the UV light for 2 hours. Prepared HSA imprinted [PEDMALM-HSA] (MIP) and non imprinted [PEDMALM] (NIP) nanoparticles attached SPR nanosensors were characterized by FTIR-ATR, atomic force microscope (AFM) method, contact angle (CA), ellipsometer measurements. Surface rougness measurements were obtained by Atomic force microscopy (AFM) and images showed that roughness values of HSA imprinted [PEDMALM-HSA] (MIP) and non imprinted [PEDMALM] (NIP) nanoparticles were estimated as 1.61 and 1.28 nm respectively. Rougness difference between HSA imprinted [PEDMALM-HSA] (MIP) and non imprinted [PEDMALM] (NIP) nanoparticles attached nanoparticles show that imprinting process of HSA molecules was performed successfully. Thickness of HSA imprinted [PEDMALMHSA] (MIP) and non imprinted [PEDMALM] (NIP) nanoparticles attached biosensor chips was estimated by ellipsometry measurements and recorded as 118.4 ±0.3 and 116 ± 0.3 nm for imprinted and non imprinted nanofilms respectively. Wettability studies was performed by contact angle measurements. Increasing contact angle values for unmodified gold chip surface (80°±0,73), HSA imprinted [PEDMALM-HSA] (MIP) (67.3°±2.24) and HSA non imprinted [PEDMALM] (NIP) (84.5°±4.63) biosensors show that imprinting process enabled hydrophilicity increment. Detection of HSA by micro-contact method was performed in the 1,5-300 nM concentration range and by nanoparticles in the 0,15-500 nM concentration range for HSA solutions prepared with 50 mM pH= 7.4 phosphate buffer solution. Desorption studies were carried out by using 0.05 M NaCI solution. Adsorption analyses for the HSA spiked artificial urine and plasma samples which was prepared in pH=7.4 phosphate buffer were examined. Selectivity studies of [PEDMALM-HSA] (MIP) and [PEDMALM] (NIP) SPR biosensors for HSA determination were investigated by using different protein samples such as hemoglobine and transferrine. Higher selectivity factor was estimated for the [PEDMALM-HSA] (MIP) nanosensor which was prepared by micro-contact method than nanoparticles attached [PEDMALM-HSA] (MIP) nanosensor as k’=5.02, k’=4.25 for Hb and HTR respectively. Limit of detection value for the nanoparticles was estimated lower (LOD: 0.7 pM) than the nanofilms prepared by micro-contact method (LOD: 8.8 pM). As a result nanoparticles attached biosensors more sensitive than the nanofilm attached ones. Repeatability experiments of [PEDMALM-HSA] nanosensor prepared by microcontact method and nanoparticles attached nanosensor was performed five times (5 times) by using HSA solutions (300 nM and 500 nM respectively) and reflectance values were measured. Stability studies were performed for the 3 times in the three months periods with four repeatibity. To determine the kinetic and adsorption models of interactions between [PEDMALM-HSA] (MIP) nanofilm attached SPR nanosensor and HSA solution, four different adsorption models named Scatchard, Langmuir, Freundlich and Langmuir-Freundlich (LF) were employed. Langmuir adsorption model was found most applicable model for this affinity system and results showed that affinity regions on the surface of [PEDMALM-HSA] (MIP) SPR nanosensor for the HSA template were homogeneously distributed and have monolayer structure. To determine the kinetic and adsorption models of interactions between [PEDMALM-HSA] (MIP) nanoparticles attached SPR nanosensor and HSA solution, four different adsorption models named Scatchard, Langmuir, Freundlich and Langmuir-Freundlich (LF) were employed. Langmuir adsorption model was found most applicable model for the low concentratins of HSA while Freundlich adsorption model fits for the high HSA concentrations in this affinity system and results showed that affinity regions on the surface of [PEDMALM-HSA] (MIP) SPR nanoensor for the HSA template were homogeneously distributed and have monolayer structure for the low concentrations. As a result, albumin level are used as a Biomarker for the 0-20 mg/L (0-300 nM) concentration range. Nanosensor prepared by the micro-contact method was estimated more selective for the HSA determination than the nanosensor prepared by the nanoparticle attached method. Although the nanosensor prepared by the micro-contact method can be used successfully for the estimation of HSA in the 1.5-300 nM concentration level, it can not detect precisely 0-300 nM HSA concentration level. So it has been required to use nanoparticles to detect HSA with lower concentration limits. Selective and sensitive nanosensor prepared by micro-contact method can be used for HSA detection by real time measurement, no need labeling, low cost and ease of miniaturization when compared with the other methods and nanoparticles can be used for the detection of lower HSA concentrations. This study will contribute to the literatüre by comparing the advantages of the nanofilm attached nanosensor prepared by micro-contact method and nanoparticles attached nanosensor.