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1.

Figure 1. Digital ELISA based on arrays of femtoliter wells. From: Single-Molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations.

(A) Capturing and labeling single protein molecules on beads using standard ELISA reagents. (B) Loading of beads into femtoliter well arrays for isolation and detection of single molecules. (C) SEM image of a small section of a femtoliter well array after bead loading. 2.7-μm-diam. beads were loaded into an array of wells with diameters of 4.5 μm and depths of 3.25 μm. (D) Fluorescence image of a small section of the femtoliter well array after signals from single enzymes are generated. While the majority of femtoliter chambers contain a bead from the assay, only a fraction of those beads possess catalytic enzyme activity, indicative of a single, bound protein. The concentration of protein in bulk solution is correlated to the percentage of beads that have bound a protein molecule.

David M. Rissin, et al. Nat Biotechnol. ;28(6):595-599.
2.

Figure 3. Sub-femtomolar detection of proteins in serum using digital ELISA. From: Single-Molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations.

Plots of % active beads against analyte concentration for: (A) Human PSA spiked into 25% serum; and (B) Human TNF-α spiked into 25% serum. The concentrations plotted on the x-axes refer to the final concentration of spiked protein in the diluted sample. The plots on the left hand side show the assay response over the concentration range tested in log-log space. The plots on the right hand side show the assay response in the femtomolar range in linear-linear space to illustrate the limit of detection and linearity of response. Error bars are plotted as the standard deviation over three replicates. LODs were determined by extrapolating the concentration from the signal equal to background signal plus three standard deviations of the background signal; the dashed lines show the signal at the LOD.

David M. Rissin, et al. Nat Biotechnol. ;28(6):595-599.
3.

Figure 4. Digital detection of PSA in serum samples of patients who had undergone radical prostatectomy. From: Single-Molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations.

Concentrations of PSA in serum samples from RP patients (), healthy control samples (), and Bio-Rad PSA control samples (▲) determined using digital ELISA. RP patient samples (SeraCare Life Sciences, Milford, MA) all had undetectable PSA levels as measured by a leading clinical diagnostic assay (ADVIA Centaur); the green line represents the detection limit of the ADVIA Centaur PSA assay (100 pg/mL or 3 pM). All 30 patient samples were above the detection limit of the PSA digital ELISA, shown by the red line (0.006 pg/mL or ~200 aM), with the lowest patient PSA concentrations measured at 0.014 pg/mL (~400 aM) using digital ELISA. Patient samples with the lowest PSA levels were detectable, but approached the LOD of the assay resulting in a large imprecision in the concentration determined (high dose %CV). The digital ELISA was validated for specificity to PSA using control standards (Bio-Rad) and serum from healthy individuals (ProMedDx) that had been assayed using the ADVIA Centaur PSA assay (See Supplementary Table 3).

David M. Rissin, et al. Nat Biotechnol. ;28(6):595-599.
4.

Figure 2. Digitization of enzyme-linked complexes greatly increases sensitivity compared to bulk, ensemble measurements. From: Single-Molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations.

(A) Log-log plot of signal output (% active beads for SiMoA, or r.f.u. for plate reader) as a function of the concentration of SβG captured on biotinylated beads. SβG concentrations for the ensemble readout ranged from 3 fM to 300 fM, with a detection limit of 15 × 10−15 M (15 fM; green line). For the SiMoA assay, SβG concentrations ranged from 350 zM to 7 fM, demonstrating a linear response of greater than 4 logs, with a calculated detection limit of 220 × 10−21 M (220 zM; red line). Error bars are based on the standard deviation over three replicates for both technologies. LODs were determined by extrapolating the concentration from the signal equal to background signal plus three standard deviations of the background signal. (B) The imprecision of SiMoA is determined by the Poisson noise of counting single events. The intrinsic variation (Poisson noise) of counting single active beads is given by √n. Comparing the Poisson noise associated coefficient of variation (%CV = √n/n) with the SiMoA %CV over three measurements confirmed that the imprecision of the assay is determined by counting error.

David M. Rissin, et al. Nat Biotechnol. ;28(6):595-599.

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