We report a method for isolating individual paramagnetic beads in arrays of femtoliter-sized wells and detecting single enzyme-labeled proteins on these beads using sequential fluid flows in microfabricated polymer array assemblies. Arrays of femtoliter-sized wells were fabricated in cyclic olefin polymer (COP) using injection moudling based on DVD manufacturing. These arrays were bonded to a complementary fluidic structure that was also moulded in COP to create an enclosed device to allow delivery of liquids to the arrays. Enzyme-associated, paramagnetic beads suspended in aqueous solutions of enzyme substrate were delivered fluidically to the array such that one bead per well was loaded by gravity. A fluorocarbon oil was then flowed into the device to remove excess beads from the surface of the array, and to seal and isolate the femtoliter-sized wells containing beads and enzyme substrate. The device was then imaged using standard fluorescence imaging to determine which wells contained single enzyme molecules. The analytical performance of this approach compared favourably to the standard method, i.e., glass arrays mechanically sealed against a silicone gasket, such that prostate specific antigen (PSA) could be detected from 0.02 pg/mL up to 100 pg/mL. The use of an enclosed fluidic device to isolate beads in single-molecule arrays offers a multitude of advantages for low-cost manufacturing, ease of automation, and instrument development to enable applications in biomarker validation and medical diagnosis.

Amyloid β (Aβ) peptides are proteolytic products from amyloid precursor protein (APP) and are thought to play a role in Alzheimer disease (AD) pathogenesis. While much is known about molecular mechanisms underlying cerebral Aβ accumulation in familial AD, less is known about the cause(s) of brain amyloidosis in sporadic disease. Animal and postmortem studies suggest that Aβ secretion can be up-regulated in response to hypoxia. We employed a new technology (Single Molecule Arrays, SiMoA) capable of ultrasensitive protein measurements and developed a novel assay to look for changes in serum Aβ42 concentration in 25 resuscitated patients with severe hypoxia due to cardiac arrest. After a lag period of 10 or more hours, very clear serum Aβ42 elevations were observed in all patients. Elevations ranged from approximately 80% to over 70-fold, with most elevations in the range of 3-10-fold (average approximately 7-fold). The magnitude of the increase correlated with clinical outcome. These data provide the first direct evidence in living humans that ischemia acutely increases Aβ levels in blood. The results point to the possibility that hypoxia may play a role in the amyloidogenic process of AD.

Measurement of prostate specific antigen (PSA) in prostate cancer patients following radical prostatectomy (RP) has been hindered by the limit of quantification of available assays. Because radical prostatectomy removes the tissue responsible for PSA production, postsurgical PSA is typically undetectable with current assay methods. Evidence suggests, however, that more sensitive determination of PSA status following RP could improve assessment of patient prognosis and response to treatment and better target secondary therapy for those who may benefit most. We developed an investigational digital immunoassay with a 2–logs–lower limit of quantification than current ultrasensitive third–generation PSA assays. We developed reagents for a bead–based ELISA for use with high–density arrays of femtolitervolume wells. Anti–PSA capture beads with immunocomplexes and associated enzyme labels were singulated within the wells of the arrays and interrogated for the presence of enzymatic product. We characterized analytical performance, compared its accuracy with a commercially available test, and analyzed longitudinal serum samples from a pilot study of 33 RP patients. The assay exhibited a functional sensitivity (20% interassay CV) <0.05 pg/mL, total imprecision <10% from 1 to 50 pg/mL, and excellent agreement with the comparator method. All RP samples were well within the assay measurement capability. PSA concentrations following surgery were found to be predictive of prostate cancer recurrence risk over 5 years. The robust 2–log improvement in limit of quantification relative to current ultrasensitive assays and the validated analytical performance of the assay allow for accurate assessment of PSA status after RP.

The quantitative measurement of inflammatory cytokines in blood has been limited by insufficient sensitivity of conventional immunoassays. This limitation has prevented the widespread clinical monitoring of cytokine concentrations in chronic inflammatory diseases. We applied a sensitive, single molecule detection technology to measure TNF–α and IL–6 in the plasma of patients with Crohn’s disease (CD), before and after treatment with anti–TNF–α therapy. Plasma from 17 patients with CD was collected prior to initiation of anti–TNF–α therapy, and the Crohn’s disease activity index (CDAI) was determined for each patient. A sub–set of these patients returned for followup 12 weeks after treatment started. Plasma from age– and gender–matched controls was also collected. Digital ELISAs were developed for TNF–α and IL–6, and the plasma concentrations of these cytokines were determined using digital ELISA. The limits of detection of the TNF–α and IL–6 digital ELISAs were 0.008 pg/mL and 0.006 pg/mL, respectively. Both cytokines were detected in all samples using digital ELISA and the concentrations of TNF–α and IL–6 in the plasma of patients with CD were (3.6±0.9) pg/mL and (10.9±11.2) pg/mL, respectively. TNF–α levels in patients and healthy controls were not significantly different, but the IL–6 levels in plasma were significantly elevated in patients compared to controls. After therapy, the mean reduction of the concentrations of free TNF–α and IL–6 were 46% and 58%, respectively. Digital ELISA provided the first quantitative measurements of TNF–α and IL–6 concentrations in the plasma of all patients in a population with CD. The changes in cytokine concentrations after therapy–which could be quantified because of the high sensitivity of digital ELISA–could be used for monitoring therapeutic efficacy.

We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10^-19 M to 10^-13 M, for use in high sensitivity enzyme–linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multistep immunosandwich approach, isolating the beads in an array of 50–femtoliter wells (Single Molecule Array, SiMoA), and detecting bead–associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than 1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of “on” or “off” beads (digital regime). At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement.

The ability to detect single protein molecules^1,2 in blood could accelerate the discovery and use of more sensitive diagnostic biomarkers. To detect low–abundance proteins in blood, we captured them on microscopic beads decorated with specific antibodies and then labeled the immunocomplexes (one or zero labeled target protein molecules per bead) with an enzymatic reporter capable of generating a fluorescent product. After isolating the beads in 50–fL reaction chambers designed to hold only a single bead, we used fluorescence imaging to detect single protein molecules. Our single–molecule enzyme–linked immunosorbent assay (digital ELISA) approach detected as few as ~10–20 enzyme–labeled complexes in 100 μL of sample (~10^-19 M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10^-15 M) much lower than conventional ELISA^3–5. Digital ELISA detected prostate–specific antigen (PSA) in sera from patients who had undergone radical prostatectomy at concentrations as low as 14 fg/mL (0.4 fM).