Label-Free Analysis of Cell Membrane Proteins via Evanescent Field Excited Surface-Enhanced Raman Scattering
Yu TianWeiqing XuKongshuo MaLili CongYanting ShenXiao HanChongyang LiangLijia LiangGuohua QiYongdong JinShuping Xu
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Challenges in studying the structures and functions of cell membrane proteins lie in their lipophilicity, which makes them hard to be stabilized, crystallized, and expressed by E. coli. Herein, we propose an evanescent field excited surface-enhanced Raman scattering (EF-SERS) strategy for label-free analysis of membrane proteins in situ. Extracted cell membranes tightly wrapped the metal nanoparticles by an extruder, which ensures the SERS signals of the membrane proteins precisely benefit from the localized surface plasmons (LSPs). The leaky mode of a waveguide was employed to improve the plasmon excitation coupling. Thus, the LSPs and waveguide modes together enable the achievement of high-quality SERS profiles of membrane proteins. By spectral analysis, the structural changes of membrane proteins can be deeply understood at the molecular level. This method has broader applicability in establishing the Ramanomics of membrane proteins and unraveling the exact changes of membrane proteins during physiological processes.Keywords:
Cell membrane
Waveguide
Surface-plasmon-resonance (SPR) sensors are widely used in biological, chemical, medical, and environmental sensing. SPR sensors supporting two surface-plasmon modes can differentiate surface binding interactions from bulk index changes at a single sensing location. We present a new approach to dual-mode SPR sensing that offers improved differentiation between surface and bulk effects. By using an angular interrogation, both long- and short-range surface plasmons are simultaneously excited at the same location and wavelength but at different angles. Initial experiments indicate that angular interrogation offers at least a factor of 3.6 improvement in surface and bulk cross-sensitivity compared to wavelength-interrogated dual-mode SPR sensors.
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The most expedient unit of the human body is its cell. Encapsulated within the cell are many infinitesimal entities and molecules which are protected by a cell membrane. The proteins that are associated with this lipid based bilayer cell membrane are known as membrane proteins and are considered to play a significant role. These membrane proteins exhibit their effect in cellular activities inside and outside of the cell. According to the scientists in pharmaceutical organizations, these membrane proteins perform key task in drug interactions. In this study, a technique is presented that is based on various computationally intelligent methods used for the prediction of membrane protein without the experimental use of mass spectrometry. Statistical moments were used to extract features and furthermore a Multilayer Neural Network was trained using backpropagation for the prediction of membrane proteins. Results show that the proposed technique performs better than existing methodologies.
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Starting with a comprehensive review of both surface plasmon resonance (SPR) based and localized surface plasmon resonance (LSPR) based sensors, this thesis reports the studies on the development of a novel sensitive gold nanorod (GNR) based label-free LSPR optical fibre biosensor, and the development of a novel robust method for effectively modifying the surface of cetyl-trimethyl ammonium bromide (CTAB) capped GNRs and their LSPR biosensing applications.
A novel GNR-based LSPR optical fibre sensor was fabricated and evaluated in this work. The sensor probe was prepared by covalently immobilizing GNRs, synthesized using a seed-mediated growth method, on the decladed surface of a piece of multimode optical fibre. In order to operate the LSPR sensor as a reflective sensor, a silver mirror was also coated at one distal end of the sensor probe by a dip coating method. In the refractive index sensitivity test, it was found that the longitudinal plasmon band (LPB) of GNRs is highly sensitive to the refractive index change close to the GNRs surface, and the sensitivity of the LSPR optical fibre sensor increases with the increase of the aspect ratio of GNRs. The results showed that the GNR-based LSPR optical fibre sensors prepared in this work have linear and high refract index sensitivities. For sensors based on GNRs with aspect ratios of 2.6, 3.1, 3.7 and 4.3, their refractive index sensitivities were found to be 269, 401, 506 and 766 nm/RIU (RIU = refractive index unit), respectively, in the refractive index range from 1.34 to 1.41. In order to evaluate the biosensing performance, the GNR-based LSPR optical fibre sensor with aspect ratio of 4.1 and a 2 cm sensing length was further functionalized with human IgG to detect the specific target — anti-human IgG, and a detection limit of 1.6 nM was observed using a wavelength-based interrogation approach.
In another study, in order to overcome the drawbacks of the CTAB-capped GNRs found in biosensing and biomedical applications, a simple yet robust pH-mediated method for effectively modifying the surface of CTAB-capped GNRs synthesized by the seed-mediated growth method was developed. This method allows the complete replacement of the CTAB molecules attached on the GNRs surface with the 11-mercaptoundecaonic acid (MUA) molecules to take place in a total aqueous environment by controlling the pH of the MUA aqueous solution, thus avoiding the irreversible aggregation of GNRs during the complex surface modification process observed in the previous reported methods. The success of the complete replacement of CTAB with MUA was confirmed by the surface elemental analysis using an X-ray photoelectron spectroscopy (XPS), and the MUA-modified GNRs created in this work demonstrated a high stability up to 4 months at least when stored in a buffer solution at pH 9 at 4°C. The MUA-modified GNRs with an aspect ratio of 3.9 were furthered developed as a solution-phase-based label-free LSPR biosensor by functionalizing the GNRs with human IgG. A detection limit as low as 0.4 nM for detecting anti-human IgG was achieved by this sensor.
The achievements of this work are concluded and the directions of future work are also pointed out.
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In this work, two of surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR)-based optical fibre sensors have been successfully developed and cross-compared. With one SPR sensor being coated with a thin layer of gold film and the other gold-nanorods (GNRs), forming a LSPR sensor, both sensors are subjected to various refractive index changes. As a result their sensitivities are measured in the form of resonance wavelength shift as a function of refractive index variation. The results demonstrate that the thin-film coated SPR sensor has much higher sensitivity than that of GNRs coated LSPR sensor but with worse linearity.
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We have systematically investigated the localized surface plasmon resonance (LSPR) of the silver nanoparticles by using electron energy loss spectroscopy (EELS) and optical simulation based on boundary element methods with respect to the diameter and the impact parameter variations. The both peaks of EELS and optical curve were occurred from 3.2 to 3.8 eV. Interestingly, we found two types of plasmon modes. At the impact parameter from 0 to R, the plasmon showed the properties as bulk plasmon, while at the greater value than R it showed the surface plasmon mode. This result showed the EELS simulation was better to observe a high-order of LSPR spectra than optical simulation. High-order was originated from a higher multipolar mode and weak interaction in surface plasmon phenomenon. As shown above, the EELS measurement can detect a high-order mode of LSPR than the optical measurement.
Localized surface plasmon
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A surface plasmon resonance (SPR) sensor system for the determination of sucrose concentration was constructed with a gold thin film sensing chip. The properties of gold thin film are critical factors in exciting surface plasmon resonance phenomena. Therefore in the present paper, the fabrication conditions of gold thin film were investigated to optimize the SPR phenomena. The optimum thickness was obtained as with resonance angle and good surface roughness limitation. about . The linear resonance angle shifts and rapid response were observed from the sucrose concentrations ranged from 0 to 40wt%.
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This paper presents a strategy for the signal enhancement of surface plasmon resonance biosensors using colloidal gold nanoparticles and a silica layer. We describe the method for the deposition of a silica-stabilized gold nanoparticle layer on a gold film, namely an enhanced surface plasmon resonance chip. This chip shows significant changes in its surface plasmon resonance signals when biomolecules are attached to its surface as compared to a normal gold surface. These characteristics are closely related to the surface plasmon resonance effect as determined using prostate-specific antigen. The detection limit of the enhanced surface plasmon resonance chip is determined to be 0.01 ng/mL for a prostate-specific antigen immunoassay. The use of an enhanced surface plasmon resonance chip makes it possible to enhance signals 1000-fold compared to the signals obtained by conventional surface plasmon resonance sensing. The enhancement of the surface plasmon resonance spectral shift results from the coupling of the surface and particle plasmons through the application of a silica-stabilized gold nanoparticle layer on the gold surface.
Localized surface plasmon
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We have developed a method to measure the amounts of cell surface-expressed membrane proteins with bioluminescence. Dinoflagellate luciferase was expressed on the surface of a mammalian cell as a chimeric fusion protein with a membrane protein of interest. Using a membrane-impermeable substrate to quantify the membrane-displayed luciferase, the expression of the membrane protein on the cell surface was determined. By inclusion of a quenching step for the luminescent activity of luciferase on the cell surface, we were able to monitor the membrane protein expression kinetics by measuring the luminescence recovery from the cell surface after quenching. The reported methods provide a convenient way to monitor the kinetics of expression and transport of membrane proteins to the cell surface. It is applicable to the high-throughput analysis of drugs or drug candidates concerning their effects on membrane protein expression.
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