The dotLab System combines two well-known principles—optical diffraction and affinity reagents—for the quantitative measurement of binding events in real time. Affinity reagents are prepatterned on the surface of a disposable dotLab Sensor to create the diffraction grating.
Assays can employ direct covalent attachment of the target, avidin-biotin coupling, or general protein-recognition reagents. Many assays can be performed label free; however the dotLab System has an extensive sensitivity range and incorporates amplification strategies for low-level analyses.
Existing sensors allow for replicate analysis of single analytes. Sensors under development with user addressable spots allow multiplexing of up to eight analytes in a single sample.
The patterned capture molecules interact with sample in a 10 µL flow channel. The instrument automatically delivers samples and reagents from a standard 96-well plate or bulk containers. Complex matrices including blood, plasma, lysates, and homogenates can normally be used directly without preparation or purification. Sample tubing is integrated with the disposable sensor to minimize risk from plugging or precipitation.
A continuous laser beam interrogates the capture molecules and creates a baseline diffraction intensity. As molecules in the sample bind to the surface, diffraction efficiency is improved in proportion to size and concentration.
A prism molded into the base of the disposable sensor allows operation in total internal reflection mode. This means the measurement beam never passes through the sample, effectively eliminating interference and the need for a separate reference channel.
Binding is determined quantitatively by monitoring the change in intensity of the diffractive signal. The dotLab System displays results graphically in real time. Generation of calibration curves is supported to facilitate the determination of unknowns.
The dotLab technology can be used for a range of applications including the study of biomolecular interactions, protein expression, assay development, and routine immunoassay. The latter two applications as they relate to real-time immunoassays are briefly described in this article.
Quantitative immunoassays are based on interactions between antibodies, the antigen, and other assay components. The process of developing an assay often begins with the qualification of reagents, followed by antibody ranking and selection to identify the best reagents for the application. Antibodies may be produced in animals by inoculation or recombinantly expressed. Additional stages of assay development include identification of the best antibody pairs for sandwich assays, eliminating nonspecific binding and cross-reactivity through the proper choice of reagents, and determining optimal reagent and buffer concentrations.