Fluorescence adsorbed density

The peculiar maximum near 100 nM in the measured adsorption isotherms remains a puzzle. Although fluorescence quenching appears to be an unlikely explanation, detailed measurements of the fluorescence quantum yield as a function of adsorbed density have not been carried out and are warranted to address this issue. In any case, the anomalous behavior of the isotherms occurs at concentrations far higher than typically employed in biological applications of the probe arrays (1 pM to 10 nM) and is therefore not expected to be relevant to them. 

Fig.44 (left) Dry thickness (in nm) of a poly(2-hydroxyethyl methacrylate) (PHEMA) brush in an orthogonal gradient as a function of the PHEMA grafting density and molecular weight The scale represents the thickness of dry polymer (in nm). (right) Adsorbed amount of lysozyme as a fimction of the position on the orthogonal PHEMA gradient. The scale represents the fluorescence intensity (in a.u.). (Reproduced with permission from [164])... 

The dependence of 0 on e was studied in our laboratory in an attempt to clarify the competition between energy transfer and injection for dyes adsorbed from solution (14). The fluorescence quantum yield of rhodamine B adsorbed on Sn-doped In203 and on glass was determined as a function of (measured by the optical density of the adsorbed dye). A sharp difference was found between the behavior of 0 on glass and on the semiconductor as 0 0 increased by more than one order of magnitude on glass... 

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. 

The conversion from fluorescence intensity (counts/pixel) to adsorbed target density (molecules/jim or pmol/cm ) was accomplished by measuring the system gain of the optical scanner. The system gain was determined from the derivative of the axial dependence of the signal from a solution of fluorophore labeled oligonucleotide of known quantum yield and concentration. The measured adsorbed target densities are estimated to be accurate to 0.2 pmol/cm. ... 

When light traversing an optically dense medium approaches an interface with a more optically rare medium at an angle exceeding a critical value, Bent = sin (rerare/ dens), total internal reflection occurs and an evanescent wave of exponentially deca5ung intensity penetrates the rarer medium. This phenomenon is at the heart of certain spectroscopic methods used to probe biomolecules at interfaces (199). In total internal reflection fluorescence (TIRF) spectroscopy (200-202), the evanescent wave excites fluorescent probes attached to the biomolecules, and detection of the emission associated with their decay provides information on the density, composition, and conformation of adsorbed molecules. In fourier transform infrared attenuated total reflection (FTIR-ATIR) spectroscopy (203,204), the evanescent wave excites certain molecular vibrational degrees of freedom, and the detected loss in intensity due to these absorbances can provide quantitative data on density, composition, and conformation. 

Fluorescing spots can be evaluated directly on the adsorbent layer. The conditions for this are favourable The relation F 8 x f holds, where F = the fluorescence value, read from a suitable detector, 8 = radiation density of a fluorescent spot, regarded as a light source and / = spot area. The total fluorescence of a particular amount of substance is thus not significantly influenced by the spot shape. 

This model has been recently confirmed, since the existence of adsorbed intermediate [NiCitMo02]ads by the in situ surface Raman spectroscopic smdies has been detected [154]. However, it should be noted here that the whole mechanism has been based on the X-ray fluorescence analysis of the electrodeposited alloys, and from the content of Ni and Mo their partial current densities of electrodeposition were calculated and used for the analysis of the mechanism [117-120]. 

The structure of polymer adsorbed layers at the air-liquid interface is similar to that at the solid-liquid interface. Adsorption at air-liquid interface has been studied by ellipsometry (72), X-ray and neutron reflectivity (73,74), surface tension measurements (75), X-ray evanescent wave-induced fluorescence (76), and Langmuir trough techniques (73). Neutron reflectivity measurements indicate that in the case of homopolymers the segment density decreases as in good agreement with scaling predictions for homopolymers at the solid-liquid interface (73). 

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