Fluorescence, protein adsorption

Protein adsorption has been studied with a variety of techniques such as ellipsome-try [107,108], ESCA [109], surface forces measurements [102], total internal reflection fluorescence (TIRE) [103,110], electron microscopy [111], and electrokinetic measurement of latex particles [112,113] and capillaries [114], The TIRE technique has recently been adapted to observe surface diffusion [106] and orientation [IIS] in adsorbed layers. These experiments point toward the significant influence of the protein-surface interaction on the adsorption characteristics [105,108,110]. A very important interaction is due to the hydrophobic interaction between parts of the protein and polymeric surfaces [18], although often electrostatic interactions are also influential [ 116]. Protein desorption can be affected by altering the pH [117] or by the introduction of a complexing agent [118]. 

When cells are suspended in a biological fluid or culture medium, both serum proteins and cells interact with the surface substrate. Serum protein adsorption behavior on SAMs has been examined with various analytical methods, including SPR [58-61], ellipsometry [13, 62, 63], and quartz QCM [64—66]. These methods allow in situ, highly sensitive detection of protein adsorption without any fluorescence or radioisotope labeling. SPR and QCM are compatible with SAMs that comprise alkanethiols. In our laboratory, we employed SPR to monitor protein adsorption on SAMs. 

As a technique for selective surface illumination at liquid/solid interfaces, TIRF was first introduced by Hirschfeld(1) in 1965. Other important early applications were pioneered by Harrick and Loeb(2) in 1973 for detecting fluorescence from a surface coated with dansyl-labeled bovine serum allbumin, by Kronick and Little(3) in 1975 for measuring the equilibrium constant between soluble fluorescent-labeled antibodies and surface-immobilized antigens, and by Watkins and Robertson(4) in 1977 for measuring kinetics of protein adsorption following a concentration jump. Previous rcvicws(5 7) contain additional references to some important early work. Section 7.5 presents a literature review of recent work. 

S. A. Rockhold, R. D. Quinn, R. A. VanWegenen, J. D. Andrade, and M. Reichert, Total internal reflection fluorescence (TIRF) as a quantitative probe of protein adsorption,... 

In TIRF protein adsorption experiments, it is desirable to correlate the intensity of excited fluorescence with excess protein concentration at the interface. Such an adsorbed layer is often in equilibrium with bulk-nonadsorbed protein molecules which are also situated inside the evanescent volume and thus contributing to the overall fluorescence. Various calibration schemes were proposed, using external nonadsorbing standards40,154 , internal standard in a form of protein solution together with a type of evanescent energy distribution calculation 154), and independent calibration of protein surface excess 155). Once the collected fluorescence intensity is correlated with the amount of adsorbed protein, TIRF can be applied in the study of various interactions between surface and protein. 

In conclusion, TIRF promises to be exceedingly useful in the study of protein-substrate interactions. It gives in situ, possibly remote, real-time information about protein adsorption-desorption parameters, conformational changes upon adsorption and hopefully, nanosecond time-resolved fluorescence lifetime information about adsorbed proteins 156). 

In protein separations, adsorption of protein molecules to the channel wall is always a problem. For instance, adsorption of green fluorescent protein (GFP) to the walls of plastic polycarbonate microchannels is problematic [597]. To avoid protein adsorption, the microchannels normally need to be treated with coatings, such as (acryloylaminopropyl)trimethylammonium chloride (BCQ), [806,809,812] or methacrylocyloxyethylphosphorylcholine (MPC) [1032]. In... 

TIRF Protein adsorption Modified proteins are required, fluorescence-based techitique... 

Interfacial fluorescence provides an excellent means of studying protein adsorption. The maximum energy available for excitation is localized within a few hundred angstroms of the surface where most of the protein is concen-... 

Interfacial protein fluorescence is an in situ method that can provide real time data with a resolution of 0.1 s. This technique is a major advantage in that the protein adsorption-desorption dynamics may be determined without resorting to sample manipulation prior to analysis. Figure 9 illustrates adsorption-desorption dynamics for both BSA and 7-globulin at bulk equimolar concentrations of 6.671xM/L. The 7-globulin required 40 min to reach... 

VanWagenen, R. A., Rockhold, S., Andrade, J. D., Probing Protein Adsorption Total Internal Reflection Intrinsic Fluorescence , in Interfacia Phenomena and Applications, Cooper, S. L., Pappas, N. A. (eds.) New York Wiley, 1982, p. 351. 

The application of total internal reflection fluorescence spectroscopy (TIRF) by this laboratory to the study of protein adsorption at solid-liquid interfaces is reviewed. TIRF has been used to determine adsorption isotherms and adsorption rates from single-and multi-component protein solutions. Initial adsorption rates of BSA can be explained qualitatively by the properties of the adsorbing surface. Most recently, a TIRF study using monoclonal antibodies to probe the conformation of adsorbed sperm whale myoglobin (Mb) elucidated two aspects of the Mb adsorption process 1) Mb adsorbs in a non-random manner. 2) Conformational changes of adsorbed Mb, if they occur, are minor and confined to local regions of the molecule. Fluorescence energy transfer and proteolytic enzyme techniques, when coupled with TIRF, can characterize, respectively, the conformation and orientation of adsorbed Mb. 

Current understanding of protein adsorption has been synthesized from investigations employing a number of techniques. One of these techniques, total internal reflection fluorescence (TIRF), has emerged as a versatile tool for studying proteins at surfaces. The investigation of protein adsorption at solid-liquid interfaces in this laboratory using TIRF is reviewed. Several reviews of the TIRF technique have appeared recently (13-15). 

TIRF Apparatus. The TIRF apparatus used to study protein adsorption in our laboratory is shown schematically in Figure 2. Important features of the apparatus are described in the Figure caption. In a typical experiment, an aqueous buffer solution of fluorescently-labeled protein (medium 2 of Figure 1) is pumped through the flow cell. The protein diffuses to and adsorbs onto a polymer film (medium 1 of Figure 1) coated on the glass microscope slide that constitutes one wall of the flow cell. The adsorption of protein is monitored continuously as a function of time through the fluorescence induced by the evanescent field. 

Also, the measured fluorescence signal may not be proportional to the surface concentration of protein. This has been observed during adsorption experiments using fluorescently-labeled 7-globulins (17) and sperm whale myoglobin (Mb) (19). Therefore, the TIRF apparatus must be properly calibrated to determine whether fluorescence intensity is indeed proportional to surface concentration. Some early calibration techniques must be viewed with caution because they were not performed under the same conditions as the protein adsorption experiments (20,21). More recently, a reliable calibration procedure that uses a double labeling technique has been developed (17). 

The coupling of the CZE step to detection systems other than UV has required the development of separation conditions compatible to the detection system used. For instance, the presence of primary amines, such as DAB, in buffers needed to be avoided for compatibility with laser-induced fluorescence (LIE) of compounds derivatized with fluorogenic substrates through their amino groups [90]. Baseline resolution of eight peaks in approximately the same time was achieved by substituting DAB by morpholine and tricine by boric acid (to avoid potential traces of primary amines in the tricine buffer) and by adjusting the concentration of other buffer components to compensate for the increase in electrical current. In the same work, modifications were also required to achieve compatibility with MS detection where nonvolatile salts, urea, and amines should be usually avoided. A physically adsorbed polyethylenimine-coated capillary was used to overcome protein adsorption to the capillary walls in the absence of cationic additives and the use of an acetate buffer at pH 5.05 allowed the partial resolution of at least five bands of rhEPO. Other types of coated capillaries have been used for the analysis of EPO by CE-MS as detailed in Section 22.4.3.3 [30,37,42,62,96]. 

In a similar manner, non-, mono-, and diphosphorylated myosin light chain using CIEF, either with UV or LIF detection, was separated by Shiraishi et al. Neutral coated capillaries (eCAP Beckman Coulter) and HPMC in the ampholyte solution reduced electroosmotic flow (EOF) and protein adsorption. A detection limit of 1 pg fluorescently labeled myosin light chain/capiUary was achieved. 

Uses Hydrophobic coating providing water repellency, lubricity, and surf, resistivity to glass and vitreous surfs, (e.g., laboratory glassware, optical fibers, porcelain ware, fluorescent light bulbs) reduces blood protein adsorption in treatment of analytical and diagnostic equip. 

Karlsson, M. Carlsson, U. Protein adsorption orientation in the light of fluorescent probes mapping of the interaction between site-directly labeled human carboiuc anhydrase n and silica nanoparticles. Biophys. J., 2005, 55(5), 3536 3544. 

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