In 2013, Quanterix Inc. (Lexington, MA) introduced the SIMOA HD-1 for research use only. SIMOA is an acronym for single-molecule array. At introduction, visionaries expected that single-molecule assays would be useful in elucidating the etiology of many diseases such as cancer, including relapse after therapy.
Kevin Hrusovsky, CEO of Quanterix, opened a webinar on December 19, 2017, with a review of recent applications of SIMOA run on the Quanterix HD-1 Analyzer. Hrusovsky provided examples showing that improving ELISA detection limits into the low fg/mL range is useful in oncology, neurology, inflammation, and infectious diseases. I was particularly impressed with applicability in neurology. With prior technology, the blood/brain barrier was recognized as being almost impenetrable for biomarkers getting in or out. But with SIMOA, one can detect single-molecule detection sensitivity in a reaction volume of only 50 × 10-15 L. The volume is so small that a single molecule can produce sufficient product for fluorescence detection.
Starting in 2013, SIMOA technology appeared to be useful in developing rule-in diagnostics for Alzheimer’s disease (AzD). This is based on detection of tau protein and plasma neurofilament light chain (NfL). Since then, SIMOA assays for tau and NfL have been useful in confirming traumatic brain injury (TBI) in collegiate football players, as well as AzD. The blood/brain barrier is less permeable than other organs; hence it requires much more sensitive detection limits to detect the low transport (Figure 1).
Figure 1 – Timeline of publications enabled SIMOA technology in neurology. SIMOA’s improved LOD enables assay of proteins crossing the blood/brain barrier.SIMOA for therapeutics
In therapeutics, one common mantra is “early detection improves outcome.” However, many diseases do not produce detectable quantities of disease-related biomarkers until the disease has progressed to stage 3 or 4, as illustrated in Figure 2. Part of the problem is that today’s technology lacks lower detection limits that might enable earlier detection.
Figure 2 – Preventive healthcare is enabled by improved lower limit of detection (LOD). The early (green) stage is the region of homeostasis. The blue area describes the region of presymptomatic illness. Screening with SIMOA assays with low LOD is often useful here. The red region is typical of performance of symptom-triggered healthcare using legacy ELISA technology.Quanterix has developed a novel technology that extends ELISA detection limits downward by a factor of 10-3 , sometimes more. In Figure 2, the horizontal color bar illustrates transition from homeostasis (green) to yellow (presymptomatic disease) to red for stages 3 and 4 (reactive sick care). The region represented by conventional ELISA detection technology is in the red box on the left and bottom right. The blue box illustrates how improved detection limits (ranging from 0.01 pg/mL to 9 pg/mL) will increase the number of detectable potential biomarkers. The FDA has approved over 200 diagnostic immunoassay tests from about 1300 proteins detected with immunoassays (red box, bottom). Quanterix has developed more than 80 assays specifically optimized for SIMOA technology on the HD-1.
The improved lower detection capability developed by Quanterix is illustrated in the bar chart in Figure 2 for several ELISA assays, including PSA, tau, TNF-α, and a series of interleukins (IL,) etc. Note that the Quanterix limits of quantitation are usually much lower than the band of normalcy.
The improved detection and quantitation limits are achieved by starting with conventional ELISA technology, which has a reaction volume of about 100 µL or more. To get a signal, one uses tens of microliters of sample containing millions of molecules of sample and reagent. This gives an LDL of 10 pM.
The key feature of SIMOA technology is reduction of the reaction volume to 50 × 10-15 L. Every well contains a single analyte molecule or is empty. Each of the 24 SIMOA sample chips contains 216,000 sealed sample wells that are imaged and counted by fluorescence imaging. At low concentration, 10-19M, most wells are empty, and the amount is derived from Poisson counting statistics. As the concentration increases, more wells are filled (about 10-15 M). When the amount increases above one sample molecule per well, the intensity of light in each well can be used to extend the measurement range upward by almost one log. Combining the counting and analog modes yields a dynamic range of more than 4 logs for most assays. Data quality is good. Typical assay precision on a single instrument is 1.5–8.9% for 11 runs on four instruments located at three different sites.
And one more thing…
Quanterix’s first instrument, the HD-1 Analyzer, provides a completely automated workflow that combines complex ELISA sample preparation with a multicolor fluorescence imaging and analysis. This instrument has been successful in demonstrating the utility of SIMOA technology over the last four years. Hrusovsky presented a slide that shows that most major pharmaceutical developers have purchased at least one HD-1.
In the December 2017 webinar, Quanterix introduced the SR-X instrument, which provides flexibility in workflow compared to the HD-1. The HD-1 is a completely automated instrument about the size of a double refrigerator. Throughput is governed by the slowest step in the entire, sequential analytical workflow. Most of the size is attributed to the pipettor, shaker, washer, and reagent and waste storage. The SR-X separates these functions utilizing a benchtop plate washer and incubator.
Sample prep prior to the SIMOA plate is performed off-line from the SR-X imager (Figure 3). Sample is pipetted manually. Magnetic beads provide the capture step. ELISA reagents are added according to the assay protocol. Quanterix also introduced washer and resuspension stations for processing magnetic beads in 96-well format. Decoupling the sample prep from the reader improves workflow flexibility. Repetitive wash and resuspend protocols do not tie up the imager. Typical protocols require 1–2 hours with about 30 minutes of hands-on time. Imaging with the SR-X is about two hours with only 5 minutes of hands on time.
Figure 3 – ELISA sample prep workflow for SR-X. Quanterix provides washer and shaker common in ELISA magnetic bead assays.For counting, the SR-X pipettes the sample and solution of magnetic beads to one of 24 sectors of a circular SIMOA multiwell disc (the same disc used on the HD-1 Analyzer). Each sector contains 216,000 wells. The assay mixture, including substrate, is distributed to the well field, and the wells are sealed with transparent oil. After a short incubation, the SIMOA plate is ready for detection with the high-speed SR-X fluorescence imager. Results from the SR-X and the HD-1 are comparable in LOD and %CV. Response curves overlay when the same assays are run. Both systems have a place in today’s fragmented market.
Summary
In summary, the Quanterix webinar was one of the most interesting of 2017. The speakers reported intriguing applications in cancer therapy, neurology, and more. This demonstrates that SIMOA technology is accepted by scientists and regulators. The emphasis on preventive medicine seems to be well-placed; the detection limits of many assays seem to be sufficient for useful early-stage detection.
Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/Labcompare; e-mail: [email protected]