I have taken a plot of the sensitivity of my probe towards a metal ion. It has been found that with an increase in the concentration of metal ion, fluorescence intensity keeps on quenching. From this data, how can I calculate the linear range and limit of detection?
The lower limit of detection is the concentration at which the observed change in intensity reaches a certain level of statistical significance. This is typically stated as being 3 standard deviations over the blank, but that is for an increasing signal. I I think in the case of the quenching assay, I might set the LLOD at the concentration at which the P-value for the t-test for the comparison with zero concentration reached 0.01. This requires multiple measurements for each concentration in order to calculate standard deviations.
This paper gives more sophisticated equations for LLOD calculation in a quenching assay (section 2.4), taking into account the linear standard curve.
https://www.mdpi.com/1420-3049/24/16/2927/pdf
The linear range can be measured simply by making a plot of analyte concentration versus fluorescence, using evenly-spaced analyte concentrations, and seeing at what concentration the data deviate from a straight line that is tangent to the low end of the concentration range.
To calculate LOD, he change in the quenching efficiency is plotted against the addition of different concentrations of metal ions. A linear decrease in the emission intensity will be observed. The limit of detection (LOD) is measured by using the formulae:
LOD = 3.3xσ/m
Where m is slope and σ is standard deviation.
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