Most spectroscopic techniques are based on the comparison of the signal which passed the sample with a reference signal. If the difference between the two signals becomes small because the concentration of the species generating the signal alteration is low, the resulting signal becomes more and more covered by noise. In contrary, fluorescence spectroscopy detects an absolute signal, i.e. the spectrum is not generated by the comparison with a reference. This is the reason why fluorescence techniques can be extremely sensitive.
Coupling fluorescence dyes selectively to certain functional groups at the surface of a polymer allows to correlate the fluorecence intensity with the concentration of the functional group. With this kind of derivatization the concentration of hydroxyl, carboxyl, carbonyl, and amino groups can be determined.
With state of the art equipment we are able to record the fluorescence spectrum of a femtomolar fluorescein solution. This value corresponds to a surface concentration in the order of 10-16 mol/cm2. In a practical experiment, this limit is shifted toward higher concentration mostly due to defraction of the exciting light in the sample and fluorescent contaminations. However, a realistic concentration limit of about 10-12 mol/cm2 is well in the sub-monolayer range.
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