The Single Molecule Array (Simoa), developed by Quanterix, is an advancement in immunoassay technology designed to measure extremely low concentrations of biomarkers. This platform allows for the detection and quantification of proteins at levels previously considered too low for standard laboratory methods. By transitioning from a traditional bulk measurement to a single-molecule counting process, Simoa provides a tool to observe biological changes at the earliest stages of disease.
How the Single Molecule Array Works
The Simoa process begins similarly to a standard immunoassay, utilizing paramagnetic beads coated with capture antibodies specific to the target protein in a sample. These microscopic beads bind the target molecules, and a second detection antibody, labeled with a reporter enzyme, is added to complete the sandwich-like immunocomplex. This complex then undergoes a physical separation step that enables single-molecule detection.
The prepared beads are loaded onto a disc containing an array of over 200,000 femtoliter-sized microwells. Each well is designed to hold a single bead, effectively isolating the individual immunocomplexes from one another. This isolation transforms the assay from an analog measurement into a digital one.
A fluorogenic substrate is then added, and the microwells are sealed with a layer of oil to trap the substrate and the bead within the femtoliter volume. If a target molecule is present on the bead, the attached enzyme reporter rapidly converts the substrate into a fluorescent product. Because the reaction occurs in such a small, isolated volume, the concentration of the fluorescent signal becomes exceptionally high, making it detectable even if only one enzyme molecule is present. The system then images the array, digitally counting each microwell as either “on” (fluorescent) or “off” (non-fluorescent).
The Leap in Sensitivity
Simoa achieves exceptional sensitivity by changing how the signal is interpreted, moving away from the analog measurement used by traditional methods like standard ELISA. Conventional immunoassays quantify the target protein by measuring the total intensity of light emitted from a bulk solution. This analog approach is limited by background noise from non-specific binding of reagents, which obscures the faint signal produced by very low concentrations of target molecules.
The Simoa platform bypasses this limitation by isolating and counting individual binding events, effectively eliminating the noise contributed by the bulk solution. By confining the enzyme reaction to the minute volume of a femtoliter well, the fluorescent product is highly concentrated. This digital counting method allows for the reliable detection of biomarkers at concentrations in the femtomolar range, representing up to a 1,000-fold increase in sensitivity over older techniques.
The ability to detect vanishingly small quantities of protein means that biomarkers previously undetectable in easily obtained samples like blood can now be accurately quantified. This expands the dynamic range of the assay, allowing researchers to measure extremely low levels of proteins in the digital mode and higher levels in an analog mode. This hybrid approach ensures the assay remains accurate across a broad spectrum of concentrations.
Key Areas of Research and Diagnosis
The ultra-sensitivity of the Simoa technology has had a transformative effect on the study of diseases where biomarkers are present at very low levels in the blood. In the field of neurology, the assay enables the measurement of proteins released from damaged nerve cells, such as Neurofilament Light (NfL) chain, directly in plasma or serum. This capability allows for the monitoring of conditions like multiple sclerosis, traumatic brain injury, and Alzheimer’s disease using a simple blood draw instead of more invasive procedures.
Specific neurological biomarkers like phosphorylated Tau 181 (p-tau181) have been measured in blood for the early detection and monitoring of neurodegeneration, with the assays receiving FDA Breakthrough Device designation. In oncology, Simoa’s sensitivity supports the detection of circulating tumor markers at earlier stages of cancer development. Measuring extremely low levels of these markers may allow for earlier diagnosis, risk assessment, and tracking of treatment effectiveness.
The technology is also useful in monitoring the side effects of cancer treatments, such as chemotherapy-induced peripheral neuropathy, by quantifying NfL in blood. Furthermore, in infectious disease research, the system is used for precise viral load monitoring, including the detection of specific viral proteins like the SARS-CoV-2 N protein.