Traditional ELISA (analog) readout systems require large volumes that ultimately dilute reaction product, requiring millions of enzyme labels to generate signals that are detectable utilizing conventional plate readers. Sensitivity is therefore limited to the picomolar (i.e., pg/mL) range and above. Single-molecule analysis provides a resolution that simply cannot be obtained with bulk ensemble measurements. Single molecule measurements are digital in nature: Each molecule generates a signal that can be counted. It is much easier to measure the presence or absence of signal than to detect the absolute amount of signal—that is, counting is easier than integrating.
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