Conventional ELISA (analog) readout systems necessitate huge volumes that eventually dilute the reaction product. This leads to the need for millions of enzyme labels to produce signals that can be detected using standard plate readers. Hence, sensitivity is restricted to the picomolar range (that is, pg/mL) and beyond.
The resolution offered by single-molecule analysis cannot be achieved with bulk ensemble measurements. By nature, single-molecule measurements are digital: each molecule produces a countable signal. Measuring the presence or absence of signal is considerably easy than detecting the absolute amount of signal. In other words, it is easier to count than to integrate.
Single-Molecule Arrays from Quanterix
Quanterix has devised a technique to simultaneously detect thousands of single-protein molecules. The technique involves using the same reagents as a traditional ELISA and has been employed to measure proteins in a range of different matrices (for example, plasma, serum, cell extracts, urine, cerebrospinal fluid, etc.) at femtomolar (i.e., fg/mL) concentrations.
The technique improves sensitivity by about 1000 times. Arrays of femtoliter-sized reaction chambers, called single-molecule arrays (Simoa™), with the ability to isolate and detect single enzyme molecules are used for this approach.
As the volumes of the arrays are roughly two billion times smaller when compared to a traditional ELISA, the fluorescent product gets accumulated quickly in the presence of a labeled protein. Since diffusion is eliminated, it is easy to observe the higher local concentration of the product. The detection limit can be reached by using only a single molecule (Figure 1).
Figure 1. Top—Analog measurements give increasing intensity as the concentration increases. Bottom—In contrast, digital measurements are independent of intensity and simply rely on a signal/no signal readout.
The Process and Results
In this single-molecule immunoassay, the first step involves attaching antibody capture agents to the surface of paramagnetic beads (with a diameter of 2.7 μm), which will be used to concentrate a dilute solution of molecules. Conventionally, the beads consist of roughly 250,000 attachment sites, so each bead can be imagined to have a “lawn” of capture molecules.
Addition of the beads to the sample solution is done in such a way that there are more numbers of beads when compared to target molecules. In general, 500,000 beads will be added to a 100-μL sample.
The addition of a large number of beads offers two benefits. First, at a bead-to-molecule ratio of approximately 10:1, the percentage of beads containing a labeled immunocomplex obeys Poisson distribution. When the protein concentrations are low, the Poisson distribution signifies that each bead will capture a single immunocomplex or none.
For instance, upon capturing 1 fM of a protein in 0.1 mL (60,000 molecules) and labeling it on 500,000 beads, one protein molecule is carried by 12% of the beads while the remaining 88% do not carry any protein molecules.
Second, the bead-to-bead distance becomes small since there are a large number of beads in solution, thereby enabling every molecule to encounter a bead within a minute. Diffusion of the target analyte molecules, even large proteins, takes place rapidly at this time scale. In theory, all the molecules should involve in multiple collisions with multiple beads.
In this way, the slow attachment to a fixed capture surface is prevented and the binding efficiency increases drastically. Then, the beads are washed to eliminate proteins that are nonspecifically bound, which are then incubated with biotinylated detection antibody and subsequently with β-galactosidase–labeled streptavidin. Thus, each bead that captures a single protein molecule is labeled with an enzyme. Beads that do not capture a protein molecule stay label-free.
Instead of being loaded into an ensemble readout, the beads are loaded into arrays of 216,000 femtoliter-sized wells sized to hold only one bead per well (with a width of 4.25 μm and a depth of 3.25 μm) (Figure 2).
Figure 2. Simoa™ disk containing 24 array assemblies arranged radially. Each array contains 216,000 femtoliter-sized wells, which can contain individual beads with or without an associated immunocomplex.
Beads are added when the substrate is present, and then, wells are sealed with oil and imaged. Simoa™ allows enzyme labels of very low concentrations to be detected by restricting the fluorophores produced by individual enzymes to considerably small volumes of about 40 fL. This ensures that the local concentration of fluorescent product molecules is high. Once a target analyte is captured (i.e., the formation of an immunocomplex), the captured enzyme label transforms the substrate into a fluorescent product (Figure 3).
Figure 3. Loading, sealing, and imaging of single paramagnetic beads in arrays of femtoliter-sized wells. (A) Beads, a fraction of which are associated with captured and enzyme-labeled protein molecules, are introduced into the array. (B) Beads settle by gravity onto the surface of the array, and a fraction of them fall into microwells. The remainder lie on the surface. (C) Oil is introduced into the channel to displace the aqueous medium and excess beads and seal the wells. (D) Sealed wells are imaged. Fluorescent signals are generated in sealed wells that contain beads associated with captured and enzyme-labeled protein molecules.
The analyte concentration in the sample is proportional to the ratio of the number of wells that contain an enzyme-labeled bead to the total number of wells that contain a bead. An increase in the signal can be demonstrated by obtaining two fluorescence images of the array, thus validating that a true immunocomplex is present, and enabling beads related to a single enzyme molecule (an “on” well) to be differentiated from those unrelated to an enzyme (an “off” well).
By counting the number of wells that contain a bead as well as fluorescent product in relation to the total number of wells that contain beads, the concentration of the protein in the test sample can be determined. Since Simoa™ allows protein concentration to be determined digitally instead of measuring the total analog signal, this technique of detecting single immunocomplexes has been called digital ELISA.
The potential of digital ELISA to quantify very low protein concentrations when compared to traditional ELISA is due to two effects: (1) Simoa™’s high sensitivity to enzyme label and (2) the low level of background signal that can be realized by the digitization of protein detection.
In the case of antibodies with known affinity, the immunoassay’s sensitivity will be determined using the assay background. The decreased label concentration and high label sensitivity enable the nonspecific binding to the capture surface to be reduced, thus leading to a significantly lower background signal.
The Simoa™ technology that forms the core of the platform created by Quanterix will allow the detection and measurement of biomarkers that were earlier challenging or impossible to quantify, thus paving the way for novel applications to fulfill major unmet requirements in life science research, in-vitro diagnostics, and biopharma. For instance, fewer than 150 proteins with FDA approval are being used, but the human proteome consists of more than 2,500 secreted proteins.
A majority of the “missing” proteins fall just below the detection limit of the finest ELISAs. As a result, highly sensitive measurements will possibly lead to earlier detection of cancer and infectious diseases, as well as the determination of a range of new biomarkers that are beneficial for in vitro diagnostics and companion diagnostics.
Quanterix is on a mission to change the way in which healthcare is provided by giving researchers the ability to closely examine the continuum from health to disease. In order to make this vision a reality, we brought together the most experienced management team, renowned scientists, industry leading investors and expert advisors, to form a collaborative ecosystem, united through the common goal of advancing the science of precision health.
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