BING Nai-ci, TIAN Zhen, QIAO Wei, ZHU Xiang-rong, ZHANG Ye, SHEN Jiao-wen, CHEN Qin, ZHOU Yu-lin(School of Urban Construction and Environmental Engineering, Shanghai Second Polytechnic University, Shanghai 201209, P. R. China)

Application of Total Internal Reflection Fluorescence in Protein-Material Interactions Fields

BING Nai-ci, TIAN Zhen, QIAO Wei, ZHU Xiang-rong, ZHANG Ye, SHEN Jiao-wen, CHEN Qin, ZHOU Yu-lin
(School of Urban Construction and Environmental Engineering, Shanghai Second Polytechnic University, Shanghai 201209, P. R. China)

Protein-surface interactions play a significant role in drug delivery, biosensor technology, affinity or ion exchange chromatography and artificial materials under a biological environment. Many elaborate techniques have been applied for the investigation of protein density, structure or orientation at the interfaces. One particularly useful technique for studying protein surface-associated processes at the molecular level is total internal reflection fluorescence (TIRF), which is fast, non-destructive, sensitive and versatile technique. In this paper, the principles and techniques of TIRF were described and the broad range of TIRF and TIRF-electrochemical systems for detection and control of biomolecular interaction including protein-protein, protein-DNA, DNA-DNA, protein-membrane is summarized. These studies are providing enhanced understanding of protein-surface interaction. Several recent developments in TIRF from protein-material fields are likely to find future application in other biophysics and biochemistry.

total internal reflection fluorescence; protein-material; interactions; reviews

0 Introduction

Protein-surface interactions play a significant role in drug delivery[1,2]biosensor technology[3], affinity or ion exchange chromatography[4]and artificial materials under a biological environment[5]. The investigation of protein density, structure or orientation at the interfaces, dynamic adsorption and competitive adsorption at interfaces can provide the important information for understanding of specific interactions of protein molecules in biological systems, reproducing biological principles, modifying mimic structure in artificial systems, and computer modeling of structure/function relationships, etc. Many high-sensitivity optical measurement techniques have been used to investigate the protein-surface interactions including Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), time of flight-secondary ions mass spectroscopy (ToF-SIMS), Surface plasmon resonance (SPR), surface enhanced Raman spectroscopy (SERS), ellipsometry, quartz crystal microbalance and total internal reflection fluorescence (TIRF). Among them, TIRF has become a powerful and widely used technique for determination of surface-associated processes of proteins at molecular level[6].

TIRF is based on the surface-associated evanescent field that is created when light is internally reflected at a planar interface between two transparent materials with different refractive indices. Fluorescent molecules in the medium with the lower refractive index that are on or near the interface are selectively excited by the evanescent illumination[7]. Since 1990s, with the appearance of new objective lens and high-sensitive detectors, TIRF techniques have been fully developed. TIRF is super-sensitive, real-time, low volume, in situ, which is well-suited for single molecule detection, analysis of biomolecular interactions and studies of the mechanisms of biomolecular events.

In this paper, we described the principles and techniques of TIRF and summarized the broad range of TIRF and TIRF-electrochemical systems for detection and control of biomolecular interaction including protein-protein, receptor-ligand, protein-DNA, DNA-DNA, protein-membrane. These studies are providing enhanced understanding of protein-surface interaction. Several recent developments of TIRF in protein-material fields are likely to find future application in other biophysics and biochemistry.

1 Principle

1.1 TIR and Evanescent field[9-13]

When a light beam propagates through optically dense medium, such as glass (refractive index n1), and encounters an interface with optically less dense medium, such as water or aqueous solution (refractive index n2), it would undergo total internal refraction for all angles of incidence θ greater than a critical angle θc= arcsin(n2/n1) according to the Snell’s law (see Fig.1).

Fig.1 Illustration of total internal reflection(θ1: incident angle, θ2: refractive angle, θc: critical angle)

Although being fully reflected, the incident beam establishes an evanescent electromagnetic wave that extends beyond the interface and penetrates into the lower refractive index medium and decays exponentially with the distance from the interface. Typically, the penetration depth of evanescent field is in the range of half the wavelength of the light. According to the relation d = (λ/4π) (n12sin2θ – n22)-1/2(where λ corresponds to the wavelength of light), penetration depths (d) can be adjusted between about 70 nm and 300 nm (see Fig.2).

Fig.2 The relationship between penetration depths and incident angle[11]

1.2 Fluorophore molecules imaging

Owing to the excitation of the fluorescent molecules on or near the interface, the interface has an important influence on the transmission pattern of fluorophore molecules and fluorophore distribution exists fine structure. Illumination of a 3D fluorophore distribution C(x, y, z) by an exponentially decaying evanescent wave with a decay constant k= 1/d leads to the product C(x, y, z) ·exp (-k·z). Detecting (integrating) the fluorescent light with a microscope objective lens leads to an integral expression depending on the decay parameter k.[14]

One-dimensional geometry to determine cellsubstrate distances of the whole x, y plane by assuming top-hat functions for the fluorophore distribution in the z direction[15]. The same binary dependency along z has been approximated to spherical objects[14,16-17]. Figure.3 shows the comparison of axial distribution models of fluorescent molecules[16].

Fig.3 Comparison of axial distribution models of fluorescent molecules

2 Techniques

There are two main types of TIR optics in total internal reflection fluorescence microscopy (TIRFM): prism-type and objective-type. With the introduction of novel techniques, the equipment previously used for variable-angle TIRFM is recently replaced with a miniaturized illumination device, and objective-type TIRFM has been combined with two-photon microscopy using either wide-field detection or sample scanning, and the problem that different polarizations of incident light can excite different patterns of fluorophores has been overcome by using a prism combination that permits excitation by two orthogonal beams[18].

Table1 shows the differences and applications of the two types of TRIFM.

Tab.1 Difference and applications of two types of TRIFM

3 Applications

The broad range of TIRF and TIRF-electrochemical systems has been applied to the detection and the control of protein materials interaction.

Chen[21]developed a new method for quantitative determination of serum albumin in aqueous solution by the coupling technique of total internal reflection synchronous fluorescence (TIRSF) at the solid/liquid interface. The combination of BSA and TPPS adsorbed onto the glass surface produced a synchronous fluorescence signal. And the detection limit of 0.94 μg/mL. Tang[22]also studied the interaction and adsorption of BSA and TPPS at toluene-water interface successfully by TIRSF, and provided a new method for the determination of the critical micelle concentration, apparent adsorption equilibrium constant and maximum amount of adsorption at the liquid-liquid interface.

Guo[23]used TIRF to monitor the process that protein immobilized via DNA conjugation by utilizing laminarflow in a microfluidic device. Both the specificity and sensitivity of the method were high.

Fig.4 TIRF images at the inter face of the laminar flows[23]

Mashanov[24]studied the behavior of individual protein molecules within living mammalian cells by TIRFM. Wang[25]developed a sensitive single-molecule imaging method for quantification of protein by TIRFM with adsorption equilibrium (seen from Fig.5). Wang[6]applied TIRFM to the observation of the dynamic interaction between circularly permuted green fluorescent protein (cpGFP) and trypsin at the single-molecule level. Daly[26]had studied the adsorption of polyethylene glycol-modified (PEGylated) chicken egg lysozyme to silica. The combination of conventional TIRF exchange experiments with the pH-sensitive fluorophore TIRF approach to monitor that PEGylated lysozyme layer changes the shape of the adsorption isotherm and alters the preferred orientation of lysozyme on the surface.

Zhan Y, Gao S B, Xue P, et al[27]used TIRF technique to observe the aggregation of membrane protein (Reticulocalbin 2) surrounding STIM1 clusters by TIRF.

Fig.5 TIRFM image of GFP molecules bound to anti-GFP antibodies on glass surface ( Some spots have double intensity either because the antibody binds)

4 Outlook

With the development of life-science, the technical improvements of TIRF and the commercialization of TIRFM, TIRF will have a more widely application to understand protein–material interactions by putting together experimental results with simulation data and biophysical theories. The combination of TIRF with nanometer techniques, fluorescence resonance energy transfer and shallow angle fluorescence microscopy etc. will give a boost to the related research and motivate future investigations, at the same time, more breakthroughs are expected.

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全內(nèi)反射熒光光譜在蛋白質(zhì)材料相互作用領(lǐng)域的應(yīng)用

邴乃慈，田 震，喬 煒，祝向榮，張 燁，沈嬌雯，陳 欽，周玉林
(上海第二工業(yè)大學(xué)城市建設(shè)與環(huán)境工程學(xué)院, 上海201209)

蛋白質(zhì)表面相互作用對(duì)藥物釋放、生物傳感器、親和色譜和離子色譜以及仿生材料在生物環(huán)境中均起到關(guān)鍵作用。許多技術(shù)可用來(lái)研究不同材料界面間的蛋白質(zhì)密度、結(jié)構(gòu)和取向。全內(nèi)反射技術(shù)由于具有快速、無(wú)損、靈敏以及靈活等特點(diǎn)，在研究分子水平蛋白質(zhì)界面耦合過(guò)程發(fā)揮著重要的作用。描述了全內(nèi)反射的原理、技術(shù)以及在檢測(cè)和控制生物分子間作用等方面的應(yīng)用，主要包括蛋白質(zhì)—蛋白質(zhì)，蛋白質(zhì)-DNA，DNA-DNA以及蛋白質(zhì)—膜等界面體系。這些研究有助于進(jìn)一步加強(qiáng)對(duì)蛋白質(zhì)表面相互作用的理解。近年來(lái)全內(nèi)反射技術(shù)在蛋白質(zhì)領(lǐng)域的應(yīng)用，為其在生物物理和生物化學(xué)方面的應(yīng)用提供了依據(jù)和可能。

全內(nèi)反射熒光光譜；蛋白質(zhì)—材料；相互作用；綜述

O657

A

1001-4543(2011)03-0214-05

2011-01-06;

2011-04-15

邴乃慈（1979－），女，遼寧人，博士，主要研究方向?yàn)榄h(huán)境友好功能材料，電子郵箱ncbing@eed.sspu.cn。

上海市教委創(chuàng)新項(xiàng)目（No.09YZ446）