Detecting thousands of single protein molecules

Traditional analog ELISA readout systems need great volumes, which inevitably dilute reaction products, adding the requirement of millions of enzyme labels to develop signals that can be detected with conventional plate readers. This limits sensitivity to the picomolar (i.e., pg/mL) range and higher. Single-molecule analysis offers a solution that cannot be obtained using bulk ensemble measurements. Single-molecule measurements are digital in that every molecule creates a countable signal. Measuring the presence or absence of a signal is more straightforward than detecting the absolute amount of signal. Simply put, counting is simpler than integrating.

Quanterix has created a method for the detection of thousands of single protein molecules at the same time. This method uses the same reagents as the traditional ELISA and has been utilized to measure the proteins in various matrices, including plasma, serum, cerebrospinal fluid, cell extracts, urine, and others at femtomolar (fg/mL) concentrations, and provided roughly 1000x improved sensitivity.

This method uses arrays of femtoliter-sized reaction chambers, known as single-molecule arrays (Simoa®), capable of isolating and detecting single enzyme molecules. The array volumes are around two billion times smaller than a conventional ELISA, resulting in a quick buildup of fluorescent product if a labeled protein is present. This elevated local product concentration can be easily observed when diffusion is defeated. One molecule is required to obtain the detection limit (Fig. 1).

The first step to the single-molecule immunoassay is to attach antibody capture agents to the surface of paramagnetic beads (2.7 mm diameter) that concentrate a dilute molecule solution. The beads commonly comprise around 250,000 attachment sites and can be thought of as having a “lawn” of capture molecules. The beads are introduced to the sample solution to ensure the number of beads is many times greater than the target molecules.

It is typical to add 500,000 beads to a 100- μL sample. This offers two advantages. Firstly, using a rough 10:1 bead-to-molecule ratio, the percentage of beads with a labeled immunocomplex maintains a Poisson distribution.

At low protein concentrations, the Poisson distribution dictates that each bead will capture either a single immunocomplex or none. For example, if 1 fM of protein in 0.1 mL (60,000 molecules) is captured and labeled on 500,000 beads, then 12 % of the beads will carry one protein molecule, and 88 % will not carry any protein molecules. Secondly, with many beads in solution, the bead-to-bead distance is limited, ensuring each molecule interacts with a bead in under a minute. Diffusion of the target analyte molecules, including larger proteins, will happen quickly, and theoretically, all molecules should have several encounters with many beads.

In this way, the process avoids slow binding to a fixed capture surface and substantially increases binding efficiency. Washing of the beads removes non-specifically bound proteins. The beads are then incubated with a biotinylated detection antibody followed by β-galactosidase–labeled streptavidin. Every bead that has captured a single protein molecule is labeled with an enzyme, and beads without a molecule are label-free.

Instead of an ensemble readout, beads are arranged into arrays of 216,000 femtoliter-sized wells, which are sized to hold just one bead per well (4.25 mm width, 3.25 mm depth) (Fig. 2). Beads are added with a substrate, and then the wells are sealed with oil and imaged.

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. Image Credit: Quanterix

Simoa® allows for detecting very low concentrations of enzyme labels by confining the fluorophores created by individual enzymes to very small volumes (~40 fL), guaranteeing an elevated local concentration of fluorescent product molecules.

If a target analyte is captured (or an immunocomplex is formed), then the substrate will convert to a fluorescent product by the captured enzyme label (Fig. 3). There is a correspondence between the ratio of the wells with an enzyme-labeled bead and the total wells with analyte concentration in the sample. Through the acquisition of two fluorescence images of the array, an increase in the signal can be demonstrated, which confirms the existence of a true immunocomplex. Beads that are associated with a single enzyme molecule (an “on” well) can be differentiated from any not associated with an enzyme (an “off” well).

The concentration of protein in the test sample is decided by counting the number of wells that contain both a bead and fluorescent product in relation to the total number of wells that contain beads.

Simoa® allows concentration to be decided digitally instead of by measuring the total analog signal, making this method of detecting single immunocomplexes termed digital ELISA.

Digital ELISA can measure much lower protein concentrations versus conventional ELISA because of two effects: (1) the heightened sensitivity of Simoa® to enzyme label and (2) the low amount of background signal achieved through digitization of protein detection.

In the case of antibodies of a given affinity, the assay background decides the immunoassay sensitivity. The high label sensitivity and decreased label concentration reduce non-specific binding to the capture surface, which results in a significantly reduced background signal.

Figure 2. Simoa^® disc 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. Image Credit: Quanterix

The Simoa® technology at the core of the Quanterix platform allows for detecting and quantifying biomarkers that have proved difficult or impossible to measure. This opens the door to new applications that address greatly unmet desires in life science research, biopharma, and in vitro diagnostics. For instance, less than 150 proteins with FDA approval are used today, but the human proteome consists of over 2,500 secreted proteins.

Most of these “missing” proteins lie under the detection limit of the best ELISAs. Increased sensitivity in measurements is likely to result in earlier detection of cancer and infectious disease and in identifying new biomarkers useful for in vitro and companion diagnostics.

Figure 3. Loading, sealing, and imaging of single paramagnetic beads in arrays of femtoliter-sized wells. (A) Beads, a fraction 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 lies 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. Image Credit: Quanterix

About Quanterix

From discovery to diagnostics, Quanterix’s ultrasensitive biomarker detection is fueling breakthroughs only made possible through its unparalleled sensitivity and flexibility. Quanterix’s Simoa^® technology has delivered the gold standard for earlier biomarker detection in blood, serum or plasma, with the ability to quantify proteins that are far lower than the Limit of Quantification (LoQ) of conventional analog methods.

Its industry-leading precision instruments, digital immunoassay technology and CLIA-certified Accelerator laboratory have supported research that advances disease understanding and management in neurology, oncology, immunology, cardiology and infectious disease. Quanterix has been a trusted partner of the scientific community for nearly two decades, powering research published in more than 2,500 peer-reviewed journals.

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