Fluorescence adsorbed layer

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]. 

It is known that not all reactions proceed in the same manner on all adsorbent layers because the material in the layer may promote or retard the reaction. Thus, Ganshirt [209] was able to show that caffeine and codeine phosphate could be detected on aluminium oxide by chlorination and treatment with benzidine, but that there was no reaction with the same reagent on silica gel. Again the detection of amino acids and peptides by ninhydrin is more sensitive on pure cellulose than it is on layers containing fluorescence indicators [210]. The NBP reagent (. v.) cannot be employed on Nano-Sil-Ci8-100-UV2S4 plates because the whole of the plate background becomes colored. 

Ifi fluorescence intensity k proportionality factor lo intensity of irradiating hght e molar absorption coefficient a amount of substance applied d thickness of adsorbent layer... 

Figure 37. Experimental results and least-squares fits of data (solid lines) for a Pt/C LSM covered with an electrodeposited layer of copper and an adsorbed layer of iodine. Topmost curve IL fluorescence middle curve copper Ka fluorescence bottom curve reflectivity.

The evidence accumulated in the literature suggests that the structure of surfactant adsorbed layers is, in some respects, analogous to that of surfactant micelles. Fluorescence probing techniques - e.g., pyrene and dinaphtylpropane (DNP) fluorescence probes are used to investigate the structure of adsorbed layer of a surfactant - give information on the polarity of the microenvironment in the adsorbed... 

Chandar, P., P. Somasundaran, and N. J. Turro (1987), "Fluorescence Probe Studies on the Structure of the Adsorbed Layer of Dodecyl Sulfate at the Alumine Water Interface", J. Coll. Interf. Sci. 117, 31-46. 

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. 

Figure 13. A schematic diagram showing three different approaches to introducing a fluorescent label into thin films to measure surface diffusion in the adsorbed layer.

The FRAP apparatus can also be used in a semi-quantitative manner to measure the surface concentration and subsequent competitive displacement of adsorbed labelled species, such as the fluorescent-labelled protein in the adsorbed layer of a/w or o/w thin films [10]. This can be achieved by focusing the low power 488 nm beam on the film and detection of the emitted fluorescence using the FRAP photon counting photomultiplier. The detected fluorescence signal is proportional to the amount of adsorbed protein at the interfaces of the thin film provided that the incident laser intensity is kept constant. Calculations have proved that the contributions from non-adsorbed protein molecules in the interlamellar region of the film are negligible [12],... 

An improvement in foam stability was observed as R was increased to >0.15 (Figure 17). This was accompanied by the onset of surface diffusion of a-la in the adsorbed protein layer. This is significantly different compared to our observations with /8-lg, where the onset and increase in surface diffusion was accompanied with a decrease in foam stability. Fluorescence and surface tension measurements confirmed that a-la was still present in the adsorbed layer of the film up to R = 2.5. Thus, the enhancement of foam stability to levels in excess of that observed with a-la alone supports the presence of a synergistic effect between the protein and surfactant in this mixed system (i.e., the combined effect of the two components exceeds the sum of their individual effects). It is important to note that Tween 20 alone does not form a stable foam at concentrations <40 jtM [22], It is possible that a-la, which is a small protein (Mr = 14,800), is capable of stabilizing thin films by a Marangoni type mechanism [2] once a-la/a-la interactions have been broken down by competitive adsorption of Tween 20. 

These data can be compared with those for a/w films shown in Figure 15. Such comparison suggests that there is substantially less protein at the interface in o/w thin films, indeed almost five times less. However, care needs to be exercised when equating surface concentration to fluorescence intensity. It is possible that the fluorophore is located in different environments in the two types of thin film and that the difference in fluorescence intensity is a fluorescence quantum yield effect. However, this is unlikely since the surface concentration, as judged by the surface fluorescence signal at which surface diffusion is first observed, in both a/w and o/w films is very similar at approximately 600 counts per channel. It is reasonable to assume that the structure of the adsorbed layer is similar at the point where surface diffusion is first observed. The presence of similar surface counts indicates that the quantum yield of fluorescence is similar at both o/w and a/w interfaces. Thus, this strongly supports the... 

Irregularities in the adsorbent layer can lead to significant error by causing poorly reproducible Rp values and irregularities in the size and shape of the spots. Thick areas of the plate may have less of the sample at or near the surface of the layer, causing a reduction in reflectance or fluorescence response when measurements are made on the same side of... 

Chandar, P., Somasundaran, P., and Turro, N.J. (1987). Fluorescence probe studies on the structure of the adsorbed layer of dodecyl sulfate at the alumina-water interface. J. 

Thin-layer chromatography is frequently employed for following the course of chemical reactions and for small-scale, preparative purposes. Often, it may be advantageous to employ a fluorescent adsorbent and to use a p-toluenesulfonic ester instead of the corresponding methanesulfonic ester, since p-toluenesulfonic esters can then be located by examination of a chromatoplate under ultraviolet light. 

Some essential discoveries concerning the organization of the adsorbed layer derive from the various spectroscopic measurements [38-46]. Here considerable experimental evidence is consistent with the postulate that ionic surfactants form localized aggregates on the solid surface. Microscopic properties like polarity and viscosity as well as aggregation number of such adsorbate microstructures for different regions in the adsorption isotherm of the sodium dedecyl sulfate/water/alumina system were determined by fluorescence decay (FDS) and electron spin resonance (ESR) spectroscopic methods. Two types of molecular probes incorporated in the solid-liquid interface under in situ equilibrium conditions... 

We consider three decay channels for D in addition to injection Fluorescence (rate constant k ), intramolecular radiationless decay (rate constant k ), and energy transfer quenching within the adsorbed layer (rate constant kg) ... 

Figure 25 depicts the angular dependence of the electric field and fluorescence yield for an adsorbate layer located at varying positions with respect to the diffracting planes. The phase and amplitude of this modulation (or so-called coherent position and coherent fraction) are a measure of the mean position and width of the distribution of atoms in the overlayer. The... 

Figure 33. Experimental results and least squares fit of data (solid lines) for a Pt/C LSM covered with an electrodeposited layer of copper and an adsorbed layer of iodine. Topmost curve /j. fluorescence. Middle curve copper K, fluorescence. Bottom curve reflectivity. (From Abruna, H. D., White, J. H., et al., J. Phys. Chem. 92, 7045 (1988), with permission.)...

Understanding of the structure of the adsorbed surfactant and polymer layers at a molecular level is helpful for improving various interfacial processes by manipulating the adsorbed layers for optimum configurational characteristics. Until recently, methods of surface characterization were limited to the measurement of macroscopic properties like adsorption density, zeta-potential and wettability. Such studies, while being helpful to provide an insight into the mechanisms, could not yield any direct information on the nanoscopic characteristics of the adsorbed species. Recently, a number of spectroscopic techniques such as fluorescence, electron spin resonance, infrared and Raman have been successfully applied to probe the microstructure of the adsorbed layers of surfactants and polymers at mineral-solution interfaces. 

Information on microviscosity is obtained by studying the excimer forming capabilities of suitable fluorescent probes. The excimer, which is a complex of a ground state and excited state monomer, has a characteristic emission frequency. The intramolecular excimer formation for example, of 1,3-dinaphthyl propane (DNP), is a sensitive function of the microviscosity of its neighborhood. This property, expressed as the ratio of the excimer and monomer yield (/e//m) for DNP, has been determined for dodecyl sulfonate solutions and its adsorbed layer for the various regions of the adsorption isotherm (Fig. 4.18) (Somasundaran et al., 1986). Comparing the ratios thus obtained to the /e//m values of DNP in mixtures of ethanol and glycerol of known viscosities, a microviscosity value of 90 to 120 cPs is obtained for the adsorbed layer in contrast to a value of 8 cPs for micelles. The constancy of microviscosity as reported by DNP is indicative of the existence of a condensed surfactant assembly (solloids) that holds the probe. 

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