Question

You may search online for additional information on protein microarray. Here we assume the microarray is based on a fluorescence-based sandwich assay. A layer of capture antibody is immobilized on the microarray surface. The detection antibody is labeled with a fluorophore and the detection is done measuring the fluorescence intensity of the microarray spot.

Assuming a single spot in the microarray has a surface area of 100 µm2, the surface area is completely covered with a single layer of capture antibody, the binding affinity constant between the antibody and antigen protein, K, is 108 M-1, each detection antibody is labeled with one single fluorophore, and the limit of fluorophore detection is approximately 10,000 fluorophores per microarray spot. Predict the lower limit of detection (LLOD) for the protein antigen in Molar concentration (M).

Answer 1

Inorder to predict the lower detection limit, there are few common methods as described in the literature.

1. Visual definition

2. Calculating the ratio of signal to noise

3. Calculating from the standard deviation of the blank

4. Finally, calculating from the calibration line at low concentrations

For a coventional flourescence the detection limit of 1000-fold sensitive can measure concentration down to 1fM or fetamolar. Hence, 1fM is 1x10^-15 M, therefore for a detection limit of 10,000 fluorophores per microarray spot would be 1x10^-14 M which is the lower limit of detection (LLOD) for this technique.