Methods and devices for performing high dynamic range immunoassays
A method for performing a high dynamic range immunoassay includes (a) measuring a first signal from a sandwich immunoassay to detect a target analyte in a fluidic sample in a fluidic channel and a second signal from a competitive immunoassay associated with the target analyte in the same fluidic sample in the fluidic channel, and (b) determining ratio of the first signal to the second signal to provide a measure of concentration of the target analyte in the fluidic sample, wherein the measure is applicable to a high dynamic range of concentrations of the target analyte.
1. A system for performing an assay on a sample, comprising:
an assay device including:
a capillary channel for containing the sample, and
a microarray immobilized to a surface of the capillary channel, the microarray including (a) a first spot including a first capture molecule for specifically binding to a target analyte in the sample as part of a sandwich immunoassay involving the first capture molecule, the target analyte, and a detect reagent in the sample, and (b) a second spot including a second capture molecule for specifically binding to the detect reagent in a competitive assay; and
an analysis module including:
an interrogation unit for measuring a first signal from the first spot and a second signal from the second spot,
a ratio calculator for calculating a ratio of the first signal to the second signal, as measured or after background subtraction, the ratio being proportional to concentration of the target analyte for a high dynamic range of concentrations of the target analyte, and
an interface for outputting the ratio as a measure of the concentration.
2. The system of claim 1 , the microarray further comprising at least one third spot including a third capture molecule for measuring background.
3. The system of claim 1 , the surface being a surface of a planar waveguide for guiding light to evanescently illuminate the microarray to read out a first fluorescence signal from the first sandwich immunoassay and a second fluorescence signal from the competitive assay.
4. The system of claim 1 ,
the capillary channel further comprising:
an inlet port for receiving the sample upstream of the microarray, and
an outlet port downstream from the microarray;
the capillary channel defining a direction of flow of the sample from an inlet port to an outlet port of the fluidic channel; and
the first spot and the second spot being placed in a row of the microarray parallel to the direction of flow.
5. The system of claim 4 , the surface being a surface of a planar waveguide for guiding light to evanescently illuminate the microarray, with equal evanescent illumination for the first spot and the second spot, to read out (a) a first fluorescence signal from the sandwich immunoassay and (b) a second fluorescence signal from the competitive assay.
6. The system of claim 5 , the row further comprising a third spot including a third molecule for measuring a background contribution to each of the first fluorescence signal and the second fluorescence signal.
7. The system of claim 1 , the target analyte being a first antigen, the detect reagent being an antibody with specific affinity for binding with the target analyte, the first capture molecule being a first antibody with specific affinity for binding with the target analyte, and the second capture molecule being a second antigen with specific affinity for binding with the detect reagent.
8. The system of claim 1 , the high dynamic range exceeding range of proportionality between the first signal and the concentration.
9. The system of claim 1 , the high dynamic range being at least four orders of magnitude.