Concentration single molecule experiments

In addition to proflavin and rhodamine, the photobleaching-resistant Cy3 and Cy5 fluorophores are also frequently used in single-molecule experiments and have been incorporated in the form of hydrazide derivatives into tRNAs via D residues (Pan et al., 2009) (Fig. 4.2). However, quantitative uptake of these hydrazide dyes requires modification of three reaction parameters higher concentrations of the hydrazide dyes (40 mM) than that required for proflavin or rhodamine (22 mM), pH 3.7 rather than pH 3.0, and 2 h reaction time instead of 45—90 min. The requirement of higher concentration is to promote formation of hydrazide adduct, while the slighdy elevated pH prevents hydrolysis of the adduct, which is acid labile. Thus, while the labeling method can be adapted to incorporate new fluorophores besides proflavin and rhodamine, it is prudent to systematically evaluate for the fluorophores under consideration for coupling efficiency as a function of dye concentration, pH, and reaction time. 

M in concentration. This is in the range required for single-molecule detection. These sensitivity levels have been obtained on colloidal clusters at near-infrared excitation. Figure 10.3 is a schematic representation of a single-molecule experiment performed in a gold or silver colloidal solution. The analyte is provided as a solution at concentrations smaller than 10-11 M, Table 10.1 lists the anti-Stokes/Stokes intensity ratios for crystal violet (CY) at 1174 cm-1 using 830-nm near-infrared radiation well away from the resonance absorption of CY with a power of 106 W/cm2 [34]. CV is attached to various colloidal clusters as indicated in the table. Raman cross sections of 10-16 cm2/molecule or an enhancement factor of 1014 can be inferred from the data. 

Sulfide is here represented by the bisulfide ion, the major free species at the oceanographic pH of 8.2. Relative to sulfate (0.01 M), equilibrium sulfide concentrations are on the order of 10 140 M in this system (261. It can be calculated that an imaginaty search through IO100 terrestrial oceans would be needed to yield even a single molecule of the bisulfide if it were under equilibrium control. A number of kinetic studies have shown that the sulfides are also short lived in oxic seawater. Oxidation experiments are complicated by catalytic and wall effects so that lifetimes have varied from minutes to days, but the latest values fall towards the upper end of the range (12.131. 

In the vast majority of cases reported where a streak camera has been used to measure fluorescence lifetimes, the measurements have been made from a single laser shot. Since a high fluorescence efficiency is necessary for single shot experiments, most of these studies have been concerned with measuring the lifetimes and quenching of organic dye molecules in solution. For example, Yu etal. [67] have made a study of the fluorescence lifetime of malachite green as a function of solvent viscosity and the lifetime and relative yield of erythrosin as a function of water concentration in a water—acetone mixture. The fluorescence lifetimes of these dyes are... 

Around the same time as this, Reinhoudt developed a calix[4]arene system with only two urea or thiourea functionalities attached on opposite faces 70-72 [167]. These less substituted systems display both inter- and intramolecular hydrogen bonding as a result of the calixarene adopting a pinched cone conformation (demonstrated by the use of NOESY NMR). In some spectra it is impossible for the connectivities to be made within a single molecule, so the only possibility left is that dimerisation occurs. As with the initial experiments of Rebek and Bohmer, the extent of hydrogen bonding was observed to be solvent dependent. Concentration dependant FTIR was also used, to observe the effects on the NH stretching vibrations, but no concentration dependence was observed. Of the three urea derivatives used, only 72 showed no evidence of dimerisation... 

It is important to stress that single-molecule enz3mie turnover experiments are distinctly different from conventional in vitro enz3miatic assays in that they are conducted under a nonequilibrium steady state condition with invariant substrate concentrations [32,33], which is the usual condition in a living cell. 

To achieve single-molecule fluorescence detection, there are three common experimental practices. First, experiments are done at low concentrations (10 9-l() 12 mol I ) to spatially separate molecules, so each of them can be studied without interference from surrounding molecules. Second, fluorescence signal detection is confined to a small volume (<10-151) to minimize background noises for single-molecule sensitivity. Third, biomolecules are often immobilized, so a single molecule can be studied over time. 

For the conditions of the discussed experiments it is 10 molecules/cm sec. Here Cg is the concentration (molecules/cm ) of the molecules to be sorbed, m is the mass of a single molecule, and k is Boltzmann s constant. With the localization of the activated complex on the surface, the theoretical value of the pre-exponential factor decreases. If the desorption realized from the whole surface is limited, the maximum theoretical value of the pre-exponential factor reaches 10 molecules/cm sec. 

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