Protein aggregation is a central feature of many neurodegenerative diseases, yet methods to characterize aggregate size in complex biological samples remain limited. Here, we show that fluorescence intensity from individual single-molecule array (Simoa) microwells encodes size-dependent information beyond conventional digital quantification. Using defined synthetic tau assemblies, we establish that increasing aggregate size produces higher microwell brightness. Applying this technique to human brain homogenate reveals a shift toward larger tau aggregates in Alzheimer's disease compared to age-matched controls, in agreement with orthogonal measurements by single-molecule super-resolution microscopy. Brightness profiling further captures time-dependent aggregate size increase in a neuronal cell model, demonstrating sensitivity to dynamic changes in aggregation. Although resolution is limited between similarly sized small species, Simoa brightness robustly reports population-level shifts in aggregate size distributions. These findings repurpose a widely used ultrasensitive detection platform to provide high-throughput structural as well as quantitative insight into protein aggregation in biological systems.

A novel improvement to Quanterix Corporation Single molecule array (Simoa) SR-X instrument has been shown to offer up to 100-fold increased sensitivity compared with standard Simoa HD-X and SR-X instruments. We independently validated this increased sensitivity using the Simoa interleukin 17A (IL17A) assay. IL17A was measured in 32 paired Alzheimer’s disease (AD; n = 13) and control (CTRL; n = 19) serum and cerebrospinal fluid (CSF) samples. Mean IL17A concentrations were compared in both biofluids measured using four Simoa instruments - one HD-X instrument and three SR-X instruments modified to improve the efficiency of reading beads. HD-X quantified IL17A in 0% of CSF samples and 37.5% of serum samples, with mean serum IL17A concentrations of 0.126pg/mL in AD and 0.228pg/mL in CTRLs. In contrast, the modified SR-X instruments A, B and C quantified CSF IL17A in 84.4%, 96.9% and 96.9% of CSF samples, respectively, with mean AD concentrations of 2.92fg/mL, 5.95fg/mL and 7.25fg/mL, respectively, and mean CTRL concentrations of 3.60fg/mL, 4.75fg/mL and 9.02fg/mL, respectively. Furthermore, the modified SR-X instruments A, B and C all quantified IL17A in 100% of serum samples, with mean serum AD concentrations of 0.145pg/mL, 0.285pg/mL, and 0.249pg/mL, respectively, and mean CTRL concentrations of 0.300pg/mL, 0.406pg/mL and 0.362pg/mL, respectively. There were no statistically significant intra-instrument differences between AD compared with CTRLs. All three modified SR-X serum concentrations showed a strong positive statistically significant correlation with HD-X serum concentrations. All three modified SR-X serum concentrations also showed a strong positive statistically significant correlation with one another. Finally, all three modified SR-X instruments showed no correlation between serum and CSF concentrations, in line with previous literature. Increasing the bead reading efficiency of the SR-X exhibits increased sensitivity compared with the standard Simoa HD-X instrument, allowing the quantification of IL17A in CSF and a greater yield of serum samples.

We report methods that improve the manipulation of magnetic beads using digital microfluidics (DMF) that can enhance the performance of single molecule array (Simoa) digital protein assays in miniaturized analytical systems. Despite significant clinical and biomedical applications for digital protein detection, the development of miniaturized Simoa systems has been limited by the requirements for use of large sample volumes (∼100 μL) and low numbers of beads (∼5000) for high sensitivity tests. To address these challenges, we improved the integration of DMF with Simoa-based assays by developing strategies for loading mixtures of sample and beads into DMF networks using methods relying on either virtual channels or small liquid segments that were applied either in parallel or in a stepwise manner. We have also demonstrated a dedicated densifying electrode technique that captures low numbers of beads within a droplet, allowing high bead retention with minimal residual volumes of liquid. Based on these improvements, we optimized the front-end assay processing of beads using DMF and demonstrated a method to detect tumor necrosis factor α (TNF-α) by Simoa that showed equivalent performance to a microtitre plate assay. The new strategies described here form a step toward integrating DMF and Simoa for a wide range of applications.

Digitalizing the signals generated from single protein molecules has significantly improved the sensitivity of immunoassays compared to traditional analog "bulk" measurements. The single molecule array (Simoa) technology, for instance, leverages counting of single molecules on magnetic beads to detect low-abundance proteins in biofluids. While existing digital detection platforms are ultra-sensitive, they typically require compartmentalization and complex and bulky analysis equipment, limiting their applicability in resource-limited settings. Here, we introduce a compartmentalization-free digital detection technique, that allows for much more straightforward detection analysis. We applied this method to a model assay for detecting the SARS-CoV-2 spike protein and compared its performance to alternative techniques. We optimized the new method for digital microfluidics and present preliminary results using an automated system to analyze undiluted human saliva samples, with imaging performed on a portable optical system. We propose that future iterations of the scheme introduced here have the potential to enable a wide range of applications beyond the laboratory.

We describe progress towards developing a low-cost, simple, and compact imaging system for Digital Bead Assays (DBA) for use in Point-of-Care (POC) diagnostic systems. DBA — such as digital ELISA using single molecule arrays (Simoa) — have emerged as a key advance for the sensitive detection of proteins down to attomolar concentrations, i.e., single-digit numbers of proteins in a droplet of blood or another clinical sample. These assays have enabled unique clinical research and diagnostic measurements, e.g., the measurement in blood of protein biomarkers of neurological conditions, enabling “blood tests for the brain” that can detect Alzheimer’s disease 16 years before dementia symptoms arise. For DBA to have its maximum impact on society, it must be available in low-cost, compact equipment that can be used by anyone around the world. For this goal to become a reality, low-cost and simple imaging systems are needed. In this paper, we will describe a concept for a low-cost, simple, and compact DBA imager. We will describe evaluation of low-cost optics — such as cell-phone optics — and cameras, and how image analysis methods can be used to generate useful data from the lower resolution images provided by these systems.

This paper reviews methods for detecting proteins based on molecular digitization, i.e., the isolation and detection of single protein molecules or singulated ensembles of protein molecules. The single molecule resolution of these methods has resulted in significant improvements in the sensitivity of immunoassays beyond what was possible using traditional "analog" methods: the sensitivity of some digital immunoassays approach those of methods for measuring nucleic acids, such as the polymerase chain reaction (PCR). The greater sensitivity of digital protein detection has resulted in immuno-diagnostics with high potential societal impact, e.g., the early diagnosis and therapeutic intervention of Alzheimer's Disease. In this review, we will first provide the motivation for developing digital protein detection methods given the limitations in the sensitivity of analog methods. We will describe the paradigm shift catalyzed by single molecule detection, and will describe in detail one digital approach - which we call digital bead assays (DBA) - based on the capture and labeling of proteins on beads, identifying "on" and "off" beads, and quantification using Poisson statistics. DBA based on the single molecule array (Simoa) technology have sensitivities down to attomolar concentrations, equating to ∼10 proteins in a 200 μL sample. We will describe the concept behind DBA, the different single molecule labels used, the ways of analyzing beads (imaging of arrays and flow), the binding reagents and substrates used, and integration of these technologies into fully automated and miniaturized systems. We provide an overview of emerging approaches to digital protein detection, including those based on digital detection of nucleic acids labels, single nanoparticle detection, measurements using nanopores, and methods that exploit the kinetics of single molecule binding. We outline the initial impact of digital protein detection on clinical measurements, highlighting the importance of customized assay development and translational clinical research. We highlight the use of DBA in the measurement of neurological protein biomarkers in blood, and how these higher sensitivity methods are changing the diagnosis and treatment of neurological diseases. We conclude by summarizing the status of digital protein detection and suggest how the lab-on-a-chip community might drive future innovations in this field.

We report methods that improve the quantification of digital bead assays (DBA)─such as the digital enzyme-linked immunosorbent assay (ELISA)─that have found widespread use for high sensitivity measurement of proteins in clinical research and diagnostics. In digital ELISA, proteins are captured on beads, labeled with enzymes, individual beads are interrogated for activity from one or more enzymes, and the average number of enzymes per bead (AEB) is determined based on Poisson statistics. The widespread use of digital ELISA has revealed limitations to the original approaches to quantification that can lead to inaccurate AEB. Here, we have addressed the inaccuracy in AEB due to deviations from Poisson distribution in a digital ELISA for Aβ-40 by changing the AEB calculation from a fixed threshold between digital counting and average normalized intensity to a smooth, continuous combination of digital counting and intensity. We addressed issues with determining the average product fluorescence intensity from single enzymes on beads by allowing outlier, high intensity arrays to be removed from average intensities, and by permitting the use of a wider range of arrays. These approaches improved the accuracy of a digital ELISA for tau protein that was affected by aggregated detection antibodies. We increased the dynamic range of a digital ELISA for IL-17A from AEB ∼25 to ∼130 by combining long and short exposure images at the product emission wavelength to create virtual images. The methods reported will significantly improve the accuracy and robustness of DBA based on imaging─such as single molecule arrays (Simoa)─and flow detection.

We have developed an ultrasensitive multiplexed immunoassay using 384-well microtiter plates capable of detecting proteins at subfemtomolar concentrations that requires as little as 2.5 μL of sample. Arrays of up to 4 capture antibodies were patterned on the bottom of the wells of a 384-well plate either by directly printing the capture antibodies or by printing anti-peptide tag anchor antibodies and incubating these arrays with capture antibodies conjugated to the corresponding peptide tags ("customized" assays). Samples were incubated with the antibody arrays and shaken orbitally at 2000 rpm to achieve the greatest sensitivity. Chemiluminescence (CL) from immunocomplexes labeled with horseradish peroxidase was imaged across the entire plate to quantify the amount of protein bound to each antibody spot of the arrays. The 384-well assay had a throughput 5-fold greater than 96-well plates that was achieved from simultaneous imaging of CL in all 384-wells and the use of automated pipettors to allow parallel processing of 384 assays. We developed 4 assays based on the 384-well CL ELISA: a direct print assay for IL-10 (limit of detection (LOD) = 0.075 fM); a customized assay for IL-6 (0.22 fM); a customized pharmacokinetic (PK) assay for measuring adalimumab (7.3 pg/mL); and a customized 4-plex assay for IL-5 (0.1 fM), IL-6 (0.52 fM), IL-10 (0.2 fM), and TNF-α (3.2 fM). The sensitivity and precision of the cytokine assays were comparable to current ultrasensitive protein detection methods in 96-well formats. The PK assay for adalimumab was 650 times more sensitive than a commercially available 96-well plate ELISA. We used the 384-well CL ELISAs to measure endogenous levels of the cytokines in the serum and plasma of healthy humans: the mean concentrations and precision were comparable to those from 96-well immunoassays. This 384-well format with subfemtomolar sensitivity will enable ultrasensitive multiplexed immunoassays to be performed with higher throughput and lower sample volumes than currently possible, a particularly important capability for clinical studies in drug development.

Free-roaming domestic cats (i.e., cats that are owned or unowned and are considered 'at large') are globally distributed non-native species that have marked impacts on biodiversity and human health. Despite clear scientific evidence of these impacts, free-roaming cats are either unmanaged or managed using scientifically unsupported and ineffective approaches (e.g., trap-neuter-release [TNR]) in many jurisdictions around the world. A critical first initiative for effective, science-driven management of cats must be broader political and legislative recognition of free-roaming cats as a non-native, invasive species. Designating cats as invasive is important for developing and implementing science-based management plans, which should include efforts to prevent cats from becoming free-roaming, policies focused on responsible pet ownership and banning outdoor cat feeding, and better enforcement of existing laws. Using a science-based approach is necessary for responding effectively to the politically charged and increasingly urgent issue of managing free-roaming cat populations.

For Rapa Nui (Easter Island) and its largest islet, Motu Nui, the change of the species assemblage over time was analysed, and a trait-based approach to evaluate the potential losses in seabird function across the past centuries was applied. At a finer scale, the seasonal changes in seabird species composition in the current seabird assemblage was assessed to better understand the dynamics of the long-term inferred patterns. For Rapa Nui, the composition of the seabird assemblage between the prehistorical, historical, and current time has changed significantly. The most critical change, probably associated with human colonization, was observed between prehistoric and current times. The current diminished number of nesting seabird species was probably the result of local extirpation without evidence of colonization by new species. For Motu Nui, changes in species composition were also followed by changes in trait structure, which were smaller than observed in Rapa Nui. This is probably due to the presence of a relatively high number of related species (i.e. Procellariids) with high similarities in their foraging behaviour. The nesting seabird assemblages in Rapa Nui and Motu Nui differ in exposure to risk; thus, conservation strategies applied to the islands should be planned on a fine spatial scale. For Rapa Nui, which is an urban wildlife area with several invasive species and a low number of remaining native seabird species, management should focus on fencing and pets control. For Motu Nui, management should instead focus on the establishment of quarantine and other biosecurity tools to avoid both the entry and proliferation of new invasive species.

•Measured effect of orchid biodiversity decline on traditional knowledge & culture.•Knowledge survey of 120 individuals from two ethnic groups & three age groups.•Four domains of culturally-important orchid knowledge were negatively impacted.•Price speculation encouraged knowledge acquisition contrary to traditional culture.•Population decline negatively impacted cultural valuation of traditional knowledge.

Circulating microRNAs are biomarkers reported to be stable and translational across species. MicroRNA-122 (miR-122) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). We developed a single molecule, dynamic chemical labeling (DCL) assay to directly detect miR-122 in blood. The DCL assay specifically measured miR-122 directly from 10 μL of serum or plasma without any extraction steps, with a limit of detection of 1.32 pM that enabled the identification of DILI. Testing of 192 human serum samples showed that DCL accurately identified patients at risk of DILI after acetaminophen overdose (area under ROC curve 0.98 (95% CI; 0.96-1), P < 0.0001). The DCL assay also identified liver injury in rats and dogs. The use of specific captured beads had the additional benefit of stabilizing miR-122 after sample collection, with no signal loss after 14 days at room temperature, in contrast to PCR that showed significant loss of signal. RNA sequencing demonstrated the presence of multiple miR-122 isomiRs in the serum of patients with DILI that were at low concentration or not present in healthy individuals. Sample degradation over time produced more isomiRs, particularly rapidly with DILI. PCR was inaccurate when analyzing miR-122 isomiRs, whereas the DCL assay demonstrated accurate quantification. We conclude that the DCL assay can accurately measure miR-122 to diagnose liver injury in humans and other species and can overcome microRNA stability and isomiR challenges.

We have developed a customizable contact printed multiplex immunoassay capable of simultaneously measuring up to five analytes with attomolar sensitivities. This enzyme-linked immunosorbent assay (ELISA) was based on spotting different antibodies in a circular pattern at the bottom of a microtiter plate well. Unlike traditional antibody printing for ELISA that prints a capture antibody specific to a target of interest, in this ELISA we printed unique "anchor" antibodies at the well surface, each having a high affinity for a specific peptide target. By coupling each peptide to a unique assay capture antibody, this array of anchor antibodies enabled a customizable contact printed multiplex immunoassay workflow. As a proof of concept, we developed a 5-plex assay measuring interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 22 (IL-22), and tumor necrosis factor alpha (TNF-α). Measurements of these five analytes in serum and plasma correlated well between the method utilizing the anchor antibodies and peptides and the traditional capture antibody printing approach, with r2 values of 0.99, 0.93, 0.99, 0.96, and 0.75 for IL-5, IL-6, IL-10, IL-22, and TNFα, respectively. This approach makes customizable multiplex ultrasensitive ELISA available to laboratories without access to the precision printing instrumentation and will be useful for antibody screening, custom assay development, biomarker detection, and protein profiling for diagnostic applications.

We report the development of digital enzyme-linked immunosorbent assays (ELISAs) based on single molecule arrays (Simoa) with improved sensitivities over conventional digital ELISA, enabling detection of proteins at sub-attomolar concentrations. The improvements in sensitivity were based on using fewer beads to capture the target proteins (≤5000 vs.∼500 000 beads) that increased the ratio of molecules to beads, and increasing the fraction of beads that were analyzed (bead read efficiency) from ∼5% to ∼50%. Bead read efficiency was increased by: a) improving the loading of beads into arrays of microwells by combining capillary and magnetic forces in a method called magnetic-meniscus sweeping (MMS); b) using a centrifugal washer to minimize bead loss during the assay; and, c) improved optics and image analysis to enable the analysis of more microwells. Using this approach, we developed an assay for IL-17A with a limit of detection (LOD) of 0.7 aM, 437-fold more sensitive than standard digital ELISA. A digital ELISA with improved sensitivity was used to measure IL-17A in 100 serum and plasma samples with 100% detectability, compared to 51% for standard digital ELISA. Low numbers of capture beads yielded improved LODs for IL-12p70 (0.092 aM), p24 (9.1 aM), and interferon alpha (45.9 aM). IL-4 and PSA showed no improvements in sensitivity using fewer beads, primarily due to low antibody loading on beads and increased non-specific binding, respectively. The results were consistent with a kinetic model of binding that showed that combining capture antibodies with high on-rates with high antibodies per bead yields the greatest improvement in sensitivity.

This paper describes the need for technologies that improve analytical sensitivity to proteins to better define and monitor the progression from heath to disease over the course of an individual's life. These technologies have the potential to allow the early diagnosis of disease, and trigger treatments at the time when they have the greatest opportunity to be effective. We will describe a technology that we have developed for high sensitivity protein detection, namely, single molecule arrays (Simoa). Simoa is based on the capture of protein molecules on magnetic beads, labeling each protein with an enzyme, and counting of single enzyme labels on beads that are isolated in arrays of femtoliter wells. Simoa has enabled the detection of proteins at subfemtomolar concentrations in a variety of biological fluids. We describe the impact of higher sensitivity of proteins using Simoa on: less invasive testing; earlier detection of disease; providing biomarker baseline profiles for healthy individuals; testing of small sample volumes; monitoring of therapeutic efficacy; faster tests; and detection of proteins in complex samples. We also provide a perspective of how new technologies that allow the low-cost manufacture and miniaturization of Simoa could drive the next wave of analytical devices, including wearables.

The concentration of cytokines in blood are often at pictogram per mL and sub-picogram per mL. Recent advances in the sensitivity of immunoassays, such as single molecule arrays (Simoa), have allowed the detection of many of these cytokines that were previously undetectable in blood. While the limits of detection (LOD) of these new technologies are in the femtogram per mL range, the concentrations of cytokines in many healthy individuals can still not be detected. In fact, the quantitative measurement of several important cytokines are <50% in healthy individuals. We will report on advances to improve the sensitivity of the Simoa technology into the attogram per mL range that improve the quantitative measurement of a broad range of cytokines.

Background. Most Clostridioides difficile toxinogenic strains produce both toxins A and B (A(+)B(+)), but toxin A-negative, toxin B-positive (A(-)B(+)) variants also cause disease. We report the identification of a series of pathogenic clinical C. difficile isolates that produce high amounts of toxin A with low or nondetectable toxin B. Methods. An ultrasensitive, quantitative immunoassay was used to measure toxins A and B in stool samples from 187 C. difficile infection (CDI) patients and 44 carriers. Isolates were cultured and assessed for in vitro toxin production and in vivo phenotypes (mouse CDI model). Results. There were 7 CDI patients and 6 carriers who had stools with detectable toxin A (TcdA, range 23-17 422 pg/mL; 5.6% of samples overall) but toxin B (TcdB) below the clinical detection limit (<20 pg/mL; median TcdA:B ratio 17.93). Concentrations of toxin A far exceeded B in in vitro cultures of all 12 recovered isolates (median TcdA:B ratio 26). Of 8 toxin A>>B isolates tested in mice, 4 caused diarrhea, and 3 of those 4 caused lethal disease. Ribotyping demonstrated strain diversity. TcdA-predominant samples were also identified at 2 other centers, with similar frequencies (7.5% and 6.8%). Conclusions. We report the discovery of clinical pathogenic C. difficile strains that produce high levels of toxin A but minimal or no toxin B. This pattern of toxin production is not rare (>5% of isolates) and is consistently observed in vitro and in vivo in humans and mice. Our study highlights the significance of toxin A in human CDI pathogenesis and has important implications for CDI diagnosis, treatment, and vaccine development.

Featured Article: Rissin DM, Kan CW, Campbell TG, Stuart SC, Fournier DR, Song L, et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol 2010;28:595–9.3 Proteins are the functional units of life. Berson et al. opened the field of protein measurements with the development of RIAs in the 1950s (1). In 1971, Engvall and Perlmann reported the ELISA (2) that has since been the staple for protein measurements. In 2010, when our article featured here was published, there were 325 proteins detectable in blood, of which 171 were Food and Drug Administration–approved tests of clinical value (3). Most of these proteins are measured using ELISA. When the human genome was sequenced, it was predicted that there should be nearly 4000 proteins in the human bloodstream (4). Where are the missing proteins? ELISA enabled detection down to picomolar concentrations, but this limit of quantification was insufficient to fully access the human blood proteome. The single-molecule array (Simoa) …

Circulating microRNAs are biomarkers reported to be stable and translational across species. miR-122 (miR-122-5p) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). Our objective was to develop an extraction-free and amplification-free detection method for measuring miR-122 that has translational utility in context of DILI. We developed a single molecule dynamic chemical labelling (DCL) assay based on miR-122 hybridization to an abasic peptide nucleic acid probe that contained a reactive amine instead of a nucleotide at a specific position in the sequence. The single molecule DCL assay specifically measured miR-122 directly from 10 µL of serum or plasma without any extraction steps, with a fit-for-purpose limit of detection of 1.32 pM. In 192 human serum samples, DCL accurately identified patients at risk of DILI (area under ROC curve 0.98 (95%CI 0.96-1), P<0.0001). The miR-122 assay also quantified liver injury in rats and dogs. When DCL beads were added to serum, the miR-122 signal was stabilised (no loss of signal after 14 days at room temperature). By contrast, there was substantial degradation of miR-122 in the absence of beads (≈60% lost in 1 day). RNA sequencing demonstrated the presence of multiple miR-122 isomiRs with DILI that were at low concentration or not present in healthy patient serum. Sample degradation over time produced more isomiRs, particularly rapidly with DILI. PCR was inaccurate when analysing miR-122 isomiRs, whereas the DCL assay demonstrated accurate quantification. In summary, the DCL assay can accurately measure miR-122 directly from serum and plasma to diagnose liver injury in humans and other species, and can overcome important microRNA biomarker analytical and biological challenges.