Proceedings of the 6th International Conference on Nanostructures (ICNS6) 7-10 March 2016, Kish Island, Iran
Experimental investigation and simulation of surface plasmon resonance biosensor with human serum albumin for small drugs detection A. Nikniazia, S. Ahmadi Kandjani a*, V. Siahpousha, M.R. Rashidib,d, H. Naghsharac, F. Fathid, F. Ranjbarib a
Research institute for applied physics and astronomy, University of Tabriz, Tabriz, Iran b Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran c Department of physics, University of Tabriz, Tabriz, Iran d Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical Sciences, Tabriz, Iran *
[email protected]
Abstract: Since the first application of the surface plasmon resonance (SPR) phenomenon for sensing, this method has made great strides both in terms of instrumentation development and applications in the areas of biochemistry, biology, and medical sciences. Today SPR sensor technology has been commercialized and SPR biosensors have become a central tool for characterizing and quantifying biomolecular and drugs interactions. In this project we present simulation of SPR biosensor for combination of 55 nm of Cr/Au that configuration by Kretschmann structure for showing the changing of resonance parameters for deposition of MPA as perpetration layer and human albumin as Ligand. Also experimental investigation of these structure and both results comparison reported.
Keywords: Surface plasmon resonance; biosensor; human serum albumin Introduction Protein - antibody interactions are one of important research topics for pharmaceutical industry and protein research. Antibody-based biotechnology drugs have been shown to be effective treatments to some obstinate diseases, like Bevacizumab for cancer therapy [1-2] and Adalimumab for rheumatoid arthritis [3-4]. Human serum albumin (HSA) is of key interest in distribution pharmacology, because it is abundant amount in serum and reversibly binds most drugs [5]. HSA main functions is to carry fatty acids and maintain blood colloidal osmotic pressure, among that it is an important carrier for many hormones and drugs, especially hydrophobic ones. Although drug binding to HSA decreases the accessibility of free drug in circulation, it increases drug half-life, which makes it extremely important for clinical care. Early drug discovery, the plasma protein binding is important to determine because it is used to evaluate drug dosing needs and clearance from the body. Surface plasmon resonance (SPR) is a simple and direct optical sensing technique that based on free electrons resonating at a metal surface, which are excited with visible or near infrared light. This phenomenon can be observed when an excitation source of energy (in our case, p-polarized light) is directed towards a metal whose conduction band electrons can be approximated as a free electron plasma. Under certain resonance conductions, collective resonating oscillation of free electrons may exist on the metal surface, producing a charge density wave propagating along the plasma surface. These charge fluctuations, called the surface plasmons, are accompanied
by a mixed transversal and longitudinal electromagnetic field which has its maximum on the metal surface and decays exponentially away from the metal–dielectric interface. There is an absorption maximum as a function of the angle of the incident light, and the SPR phenomenon is highly dependent on the dielectric constant near the metal surface. This explains their sensitivity to surface properties of the adjacent dielectric medium and any changes near the surface, such as antibody binding, change the angle of the absorption maximum, and reflect the amount of molecules close to surface [6]. Instead of measuring reflectance, attempts have been made to use phase shift in measuring SPR angle and SPR wavelength. Other possibilities to measure the magnitude of magnetic field on SPR sensors include magnetoresistive magnetometers, Hall devices, and magneto-optic imagers [7]. Thus, the present study investigates experimentally the optical characteristics of the Kretschmann configuration to analyze the SPR sensitivity to HAS immobilization, and it numerical simulation for these sensors with the use of the transmission matrix method.
Materials and method Transmission Matrix Method (TMM) The reflection of light from a single interface between two media is described by the Fresnel equations. However, when there are multiple interfaces, such as in the figure, the reflections themselves are also partially transmitted and then partially reflected. Depending on the exact path length, these reflections can interfere destructively or
Proceedings of the 6th International Conference on Nanostructures (ICNS6) 7-10 March 2016, Kish Island, Iran
constructively. The overall reflection of a layer structure is the sum of an infinite number of reflections, which is cumbersome to calculate. The Transfer Matrix technique is a compact, analytically exact method of treating with plane wave propagation in infinite layered planar media. If the field is known at the beginning of a layer, the field at the end of the layer can be derived from a simple matrix operation. A stack of layers can then be represented as a system matrix, which is the product of the individual layer matrices. The final step of the method involves converting the system matrix back into reflection and transmission coefficients [8]. 𝒏𝟎
𝒏𝟏
𝒏𝟐
𝒏𝟑
𝒏𝑵+𝟏
𝒅𝟏
𝒅𝟐
𝒅𝟑
𝒅𝑵+𝟏
𝜃0
𝒅𝟎
Fig. 1. A schematic of multi-layer thin film structure
To study the reflection and the transmission of electromagnetic radiation through a multilayer with the TMM, we consider a one dimensional structure consisting of different refractive indices coupled to a homogeneous medium characterized by refractive index 𝑛0 at the interface. Fig. 2 shows a schematic of a multi-layer structure where the 𝑖-th layer of thickness 𝑑𝑖 has a complex refractive index, 𝑛̃𝑖 = 𝑛𝑖 + 𝑗𝑘𝑖 , and the interference matrix of the 𝑖-th layer for p-polarized light can be expressed by 𝑀𝑖 =
(𝑛̃ 1 [ 𝑖 2𝑛̃𝑖 +1 (𝑛 ̃𝑖
+ 𝑛̃𝑖+1 )𝑒 −𝑗𝜑𝑖 − 𝑛̃𝑖+1 )𝑒 −𝑗𝜑𝑖
(𝑛̃𝑖 − 𝑛̃𝑖+1 )𝑒 𝑗𝜑𝑖 ] (𝑛̃𝑖 + 𝑛̃𝑖+1 )𝑒 𝑗𝜑𝑖
(4)
Where 𝑛̃𝑖 = 𝑛𝑖 𝑠𝑒𝑐𝜃𝑖 and 𝜑𝑖 = 𝑛𝑖 𝑘0 𝑑1 𝑐𝑜𝑠𝜃𝑖 [9]. We applied the 632.8 nm wavelength incident light to an angular modulation SPR. The optical properties of materials used in the present simulation are listed in Table 1. Table 1. Optical properties of materials at 632.8 𝑛𝑚 wavelength of light and 27.2℃ [10-11]
Material
Refractive index
Thickness
BK7 Prism Titanium Gold MPA HSA Air
1.62 2.7038+3.7683i 0.18012+3.4398i 1.364 1.367 1
Semi-infinite 5 𝑛𝑚 50 𝑛𝑚 10 𝑛𝑚 100 𝑛𝑚 Semi-infinite
Immobilization of HSA Phosphate-buffered saline (PBS), sulfuric acid (H2SO4), hydrogen peroxide solution (H2O2), mercaptopropionic acid (MPA), N-Hydroxysuccinimide (NHS), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and human serum albumin (HAS) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Formation of Self-assembly monolayer of MPA on gold surface: First the gold surface on prism was washed in a piranha solution (1:3 mixture of 30% H2O2–H2SO4 conc) for 3 min for rinsing any attachments on gold surface. Then rinse the gold film clean with deionized water and drying it with N2 .After that, the gold films were immersed in absolute ethanol solution consisting of MPA in different concentrations 10.0 mM during different immersion periods 24 h. After immobilizaion of MPA the gold surface were rinsed several times with ethanol and water. The SPR angle and angle-shifting of attached layer was monitored in our SPR instrument. Activation with NHS, EDC and immobilization of HAS: A defined fraction of the carboxy groups was activated to form reactive N-hydroxysuccinimide esters using a solution of 0.2 M N-ethyl-N'-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.05 M N-hydroxysuccinimide (NHS) in water for 1h. Then HSA at 1 𝜇𝑔/𝑚𝐿 was diluted to 15 µg/mL in PBS buffer (pH 5.2) and then gold surface immersed in this solution for immobilizing HSA on sensor chip using amine coupling. Then angle-shifting of binding albumin protein on activated carboxyl groups in MPA was monitored in SPR instrument. Experimental Measurement Technique For manufacturing conventional Kretschmann multilayer structures, 5 nm titanium film was first sputtered on the surface of BK7 prism to resolve the adhesion problem of gold film to the glass substrate and then 50nm gold films deposited by physical evaporation method. Then we used MPA as preparation layer and after activation of MPA, HSA is immobilized on gold surface. Beam Splitter
He-Ne Laser 𝜆 = 632.8 𝑛𝑚
Homemade Rotation Stage
ND filter Polarizer PC Lens
Digital Oscilloscope
Photo Detector
Photo Detector
Fig. 2. An experimental setup of angular modulation SPR sensor
Proceedings of the 6th International Conference on Nanostructures (ICNS6) 7-10 March 2016, Kish Island, Iran
Fig. 3. Simulation results for HAS immobilization process.
Fig. 2 illustrates the experimental apparatus for a SPR sensor. Incident light of 632.8 nm from 5 mW He-Ne lasers was TM polarized while passing the p-polarizer, and then excited the surface plasmons by approaching the gold film surface of the SPR sensor. To vary the incidence angle, we used a homemade rotation stage and a two-axis stepping motor drive controller with 0.08° at each step. In addition, we used several filters and lenses to achieve greater sensitivity. A neutral density (ND) filter was used to reduce the power of the incident light because very high intensity light cannot be detected by the photodetector. A plano-convex lens was used to collimate light to a spot on the SPR surface. The intensity of reflected light after the SPR sensor was measured with a 4.8 mm Semi-Lens silicon pin photodetector (PD438C EverLight). Using a beam splitter, we measured the reflectance of incident light.
Results and Discussion In order to Table 1. information, simulation results for this process that explain in HAS immobilization part by TMM method, shows in Fig. 3. and the experimental result for this process for MPA deposition on gold surface as shown in Fig. 4. As show in both figures the SPR angle shifted for changing of layer on gold surface (Sensor surface) depend on thickness of layer and mass of molecules.
Conclusions As show in Fig. 3 and Fig. 4 we can conclude that SPR angle displacement depends on surface change has good agreement in our experimental and simulation results. So by this methods we can predict that MPA layer thickness that deposited on gold surface is 10 nm. Finally we can utilize this method to measured layer thickness of material deposited on sensor chip.
Fig. 4. Experimental results for MPA deposition on gold surface.
References [1] N. Ferrara, K. J. Hillan, W. Novotny, “Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy”, Biochemical and Biophysical Research Communications, 333 (2005) 2. [2] F. Petrelli, A. Coinu, M. Cabiddu, K. Borgonovo, V. Lonati, M. Ghilardi, S. Barni, “Prognostic factors for survival with bevacizumab-based therapy in colorectal cancer patients: a systematic review and pooled analysis of 11,585 patients”, Medical Oncology, 2015, DOI: 10.1007/s12032-014-0456-z. [3] F. Navarro-Sarabia, R. Ariza-Ariza, B. HernandezCruz, I. Villanueva, “Adalimumab for treating rheumatoid arthritis”, The Cochrane Library, 2005, DOI: 10.1002/14651858.CD005113.pub2 [4] R. Landewe, M. Stergaard, E. C. Keystone, S. Florentinus, S. Liu, D. van der Heijde, “Analysis of Integrated Radiographic Data From Two Long-Term, Open-Label Extension Studies of Adalimumab for the Treatment of Rheumatoid Arthritis”, Arthritis Care & Research, 67 (2015) 2. [5] T. Peters, “All about albumin”. Elsevier: Academic Press, 1996, ISBN: 978-0-12-552110-9 [6] J. Homola, S. S. Yee and G. Gauglitz, “Surface plasmon resonance sensors: review”, Sensors and Actuators B, 54 (1999) 3–15.Ref_4,11 [7] H. R. Gwon, S. H. Lee, “Spectral and Angular Responses of Surface Plasmon Resonance Based on the Kretschmann Prism Configuration”, Materials Transactions, 56 (2010) 6. [8] S. A. Maier, “Plasmonics: Fundamentals and Applications”, Springer, 2007, ISBN: 0-387-33150-6 [9] B. E. A. Saleh, M. C. Teich, “Fundamental of photonics”, John Wiley and sons, 2007, ISBN: 978-0471-35832-9 [10] J. Marvin, J. Weber: “Handbook of Optical Materials”, CRC Press, 2003 [11] http://refractiveindex.info, Johnson and Christy 1972.
