ABSTRACT
A novel supramolecular photoactuator in the form of a thin film of centimetric size has been developed as an alternative to traditional liquid crystal elastomers (LCE) involving azobenzene (AZO) units or photochromic microcrystals. This thin film is produced through spin coating without the need for alignment or crosslinking. The photoactuator combines a photochromic dithienylethene (DTE) functionalized with ureidopyrimidinone (UPy) units, and a telechelic thermoplastic elastomer, also functionalized with UPy, allowing quadruple hydrogen bonding between the two components. Upon alternating ultraviolet (UV) and visible light exposure, the film exhibits reversible bending and color changes, studied using displacement and absorption tracking setups. For the first time, the photomechanical effect (PME) is quantitatively correlated with photochromism, showing that DTE units drive the movement under both UV (photocyclization) and visible (photoreversion) light. In situ illumination techniques reveal that the PME arises from photoinduced strain within 160 nm UPy-bonded DTE domains, which expand and contract by approximately 50% under UV and visible light, respectively. The semicrystalline nature of the elastomer and a robust supramolecular network connecting both components are critical in converting microscopic photostrain into macroscopic actuation.
ABSTRACT
Amorphous solid dispersions (ASD) are known to enhance the absorption of poorly water-soluble drugs. In this work we synthesise well-defined Polyvinylpyrrolidone (PVP) to establish the impact of dispersity and chain-end functionality on the physical properties of Curcumin (CUR)/PVP ASD. Thermodynamic characterisation of synthesised PVP emphasises a strong effect of the dispersity on the glass transition temperature (Tg), 50 °C higher for synthesised PVP than for commercial PVP K12 of same molar mass. This increase of Tg affects the thermodynamic properties of CUR/PVP ASD successfully formulated up to 70 wt% of CUR by milling or solvent evaporation. The evolution of both the Tg and CUR solubility values versus CUR content points out the development of fairly strong CUR-PVP interactions that strengthen the antiplasticising effect of PVP on the Tg of ASD. However, for ASD formulated with commercial PVP this effect is counterbalanced at low CUR content by a plasticising effect due to the shortest PVP chains. Moreover, the overlay of the phase and state diagrams highlights the strong impact of the polymer dispersity on the stability of CUR/PVP ASD. ASD formulated with low dispersity PVP are stable on larger temperature and concentration ranges than those formulated with PVP K12.
Subject(s)
Curcumin , Polymers , Povidone , Solubility , Transition TemperatureABSTRACT
Mitotic bookmarking transcription factors (TFs) are thought to mediate rapid and accurate reactivation after mitotic gene silencing. However, the loss of individual bookmarking TFs often leads to the deregulation of only a small proportion of their mitotic targets, raising doubts on the biological significance and importance of their bookmarking function. Here we used targeted proteomics of the mitotic bookmarking TF ESRRB, an orphan nuclear receptor, to discover a large redundancy in mitotic binding among members of the protein super-family of nuclear receptors. Focusing on the nuclear receptor NR5A2, which together with ESRRB is essential in maintaining pluripotency in mouse embryonic stem cells, we demonstrate conjoint bookmarking activity of both factors on promoters and enhancers of a large fraction of active genes, particularly those most efficiently reactivated in G1. Upon fast and simultaneous degradation of both factors during mitotic exit, hundreds of mitotic targets of ESRRB/NR5A2, including key players of the pluripotency network, display attenuated transcriptional reactivation. We propose that redundancy in mitotic bookmarking TFs, especially nuclear receptors, confers robustness to the reestablishment of gene regulatory networks after mitosis.
Subject(s)
Chromatin , Transcription Factors , Animals , Mice , Transcription Factors/genetics , Transcription Factors/metabolism , Mitosis/genetics , Regulatory Sequences, Nucleic Acid , Mouse Embryonic Stem Cells/metabolismABSTRACT
Compartmentalization and binding-triggered conformational change regulate many metabolic processes in living matter. Here, we have synergistically combined these two biorelevant processes to tune the Diels-Alder (DA) reactivity of a synthetic self-complexing host-guest molecular switch CBPQT4+ -Fu, consisting of an electron-rich furan unit covalently attached to the electron-deficient cyclobis(paraquat-p-phenylene) tetrachloride (CBPQT4+ , 4Cl- ) host. This design allows CBPQT4+ -Fu to efficiently compartmentalize the furan ring inside its host cavity in water, thereby protecting it from the DA reaction with maleimide. Remarkably, the self-complexed CBPQT4+ -Fu can undergo a conformational change through intramolecular decomplexation upon the addition of a stronger binding molecular naphthalene derivative as a competitive guest, triggering the DA reaction upon addition of a chemical regulator. Remarkably, connecting the guest to a thermoresponsive lower critical solution temperature (LCST) copolymer regulator controls the DA reaction on command upon heating and cooling the reaction media beyond and below the cloud point temperature of the copolymer, representing a rare example of decreased reactivity upon increasing temperature. Altogether, this work opens up new avenues towards combined topological and supramolecular control over reactivity in synthetic constructs, enabling control over reactivity through molecular regulators or even mild temperature variations.
ABSTRACT
CONTEXT.: Repeated surgery is necessary for 20% to 40% of breast conservation surgeries owing to the unavailability of any adjunctive, accurate, and objective tool in the surgeon's hand for real-time margin assessment to achieve the desired balance of oncologic and cosmetic outcomes. OBJECTIVE.: To assess the feasibility of using a multispectral autofluorescence imaging device for discriminating malignant neoplasm from normal breast tissue in pathology as a critical step in the development of a device for intraoperative use, and to demonstrate the device's utility for use in processing and prioritizing specimens during frozen section and in the pathology grossing room. DESIGN.: We performed a preliminary assessment of our device, called the TumorMAP system, on 172 fresh tissue blocks from 115 patients obtained from lumpectomy specimens at the time of initial gross examination and compared the device results with gold standard pathology evaluation. RESULTS.: The preliminary results demonstrate the potential of our device in detecting breast cancer in fresh tissue samples with a sensitivity of 82%, a specificity of 91%, a positive predictive value of 84%, and a negative predictive value of 89%. CONCLUSIONS.: Our results suggest that the TumorMAP system is suitable for the detection of malignant neoplasm in freshly excised breast specimens and has the potential to evaluate resection margins in real time.
ABSTRACT
The straightforward coupling between a triazolinedione (TAD) unit and citronellyl derivatives via an Alder-ene reaction has been exploited to tailor the physicochemical surface properties of glassy carbon (GC) surfaces in an ultrafast and additive-free manner. For this purpose, we first covalently grafted a TAD precursor onto GC via electrochemical reduction of an in situ generated diazonium salt, which was then electrochemically oxidized into the desired GC-bonded TAD unit. A kinetic study of the modification of this reactive layer with an electroactive ferrocene probe proved that a complete functionalization was obtained in merely 1 minute. Further modification experiments with a fluorinated probe demonstrated that the surface properties can be swiftly tailored on demand. The different modification steps, as well as the efficiency of this strategy, were investigated by electrochemistry, contact angle goniometry, and X-ray photoelectron spectroscopy analysis.
ABSTRACT
Histones are punctuated with small chemical modifications that alter their interaction with DNA. One attractive hypothesis stipulates that certain combinations of these histone modifications may function, alone or together, as a part of a predictive histone code to provide ground rules for chromatin folding. We consider four features that relate histone modifications to chromatin folding: charge neutralisation, molecular specificity, robustness and evolvability. Next, we present evidence for the association among different histone modifications at various levels of chromatin organisation and show how these relationships relate to function such as transcription, replication and cell division. Finally, we propose a model where the histone code can set critical checkpoints for chromatin to fold reversibly between different orders of the organisation in response to a biological stimulus.
Subject(s)
Genome, Human/physiology , Histone Code/physiology , Histones/genetics , Animals , Chromatin/genetics , Chromatin/metabolism , Histones/metabolism , HumansABSTRACT
Cellular response to reactive oxygen species (ROS) includes both reversible redox signaling and irreversible nonenzymatic reactions which depend on the nature and concentration of the ROS involved. Changes in thiol/disulfide pairs affect protein conformation, enzymatic activity, ligand binding, and protein-protein interactions. During spermatogenesis and epididymal maturation, there are ROS-dependent modifications of the sperm chromatin and flagellar proteins.The spermatozoon is regulated by redox mechanisms to acquire fertilizing ability. For this purpose, controlled amounts of ROS are necessary to assure sperm activation (motility and capacitation). Modifications of the thiol groups redox status of sperm proteins are needed for spermatozoon to achieve fertilizing ability. However, when ROS are produced at high concentrations, the established oxidative stress promotes pathological changes affecting sperm function and leading to infertility. Sperm proteins are sensitive to high levels of ROS and suffer modifications that impact on motility, capacitation, and the ability of the spermatozoon to recognize and bind to the zona pellucida and damage of sperm DNA. Thiol oxidation, tyrosine nitration, and S-glutathionylation are highlighted in this review as significant redox-dependent protein modifications associated with impairment of sperm function and alteration of paternal genome leading to infertility. Peroxiredoxins, the primary antioxidant protection in spermatozoa, are affected by most of the protein modifications described in this review. They play a significant role in both physiological and pathological processes in mammalian spermatozoa.
Subject(s)
Reactive Oxygen Species/metabolism , Spermatozoa/metabolism , Humans , Infertility, Male/etiology , Male , Peroxiredoxins , Protein Processing, Post-TranslationalABSTRACT
Multi-stimuli responsive nanogels based on biocompatible hydrophilic polymers have emerged as promising drug delivery systems to improve anticancer therapy with hydrophobic drugs, through increase of circulating-time in the bloodstream, tumor-targeting and reduction of systemic toxicity. This paper reports on the synthesis, characterization and biological perspectives of light- and thermoresponsive hyaluronic acid (HA)-based nanogels containing coumarin as the photocleavable group. Newly synthesized nanogels exhibited interesting features: formation by a temperature-triggered self-assembly process, successful incorporation of poorly water-soluble molecules, light-responsiveness as demonstrated by a significant shift in the critical aggregation temperature after light irradiation, efficient internalization by cancer cells overexpressing the CD44 receptor of HA, ability to circulate for a prolonged period of time in the bloodstream after intravenous injection in mice and considerable detection in tumor tissues. Our findings indicate that coumarin-containing HA-based nanogels may be promising delivery systems for anticancer chemotherapy.
Subject(s)
Antineoplastic Agents/administration & dosage , Coumarins/chemistry , Drug Delivery Systems , Hyaluronic Acid/chemistry , Nanoparticles , Animals , Chlorocebus aethiops , Female , HeLa Cells , Humans , Hydrogels , Mice , Mice, Nude , Vero Cells , Xenograft Model Antitumor AssaysABSTRACT
In this article, we report on the reversible tethering of end-functionalized polymers onto catechol-based titanium platforms by exploiting the reversible Diels-Alder (DA) cycloaddition reaction. For this purpose, furan and maleimide end-functionalized polymers, prepared via reversible addition-fragmentation chain transfer polymerization, were covalently grafted through a DA reaction onto reactive titanium platforms elaborated from catechol-based anchors incorporating either the furan or the maleimide moiety. As a proof of concept, a hydrophilic poly(oligo(ethylene glycol)acrylate) (POEGA) and a hydrophobic poly(2,2,2-trifluoroethyl acrylate) (PTFEA) were grafted onto titanium surfaces and subsequently detached by exploiting the thermoreversible nature of the DA reaction [i.e., retro Diels-Alder (rDA)]. These polymers were interchanged by performing a second DA reaction, thereby allowing the titanium surface wettability to be switched from hydrophobic to hydrophilic on demand. The grafting of furan/maleimide end-functionalized polymers onto functionalized (maleimide/furan, respectively) catechol-based titanium platforms and the subsequent rDA/DA sequence used for switching the titanium surface were evidenced by attenuated total reflectance-Fourier transform-infrared spectroscopy, X-ray photoelectron spectroscopy, and contact angle measurements.
ABSTRACT
Histone modifications alone or in combination are thought to modulate chromatin structure and function; a concept termed histone code. By combining evidence from several studies, we investigated if the histone code can play a role in higher-order folding of chromatin. Firstly using genomic data, we analyzed associations between histone modifications at the nucleosome level. We could dissect the composition of individual nucleosomes into five predicted clusters of histone modifications. Secondly, by assembling the raw reads of histone modifications at various length scales, we noticed that the histone mark relationships that exist at nucleosome level tend to be maintained at the higher orders of chromatin folding. Recently, a high-resolution imaging study showed that histone marks belonging to three of the five predicted clusters show structurally distinct and anti-correlated chromatin domains at the level of chromosomes. This made us think that the histone code can have a significant impact in the overall compaction of DNA: at the level of nucleosomes, at the level of genes, and finally at the level of chromosomes. As a result, in this article, we put forward a theory where the histone code drives not only the functionality but also the higher-order folding and compaction of chromatin.
ABSTRACT
Disease detection at the molecular level is driving the emerging revolution of early diagnosis and treatment. A challenge facing the field is that protein biomarkers for early diagnosis can be present in very low abundance. The lower limit of detection with conventional immunoassay technology is the upper femtomolar range (10(-13) M). Digital immunoassay technology has improved detection sensitivity three logs, to the attomolar range (10(-16) M). This capability has the potential to open new advances in diagnostics and therapeutics, but such technologies have been relegated to manual procedures that are not well suited for efficient routine use. We describe a new laboratory instrument that provides full automation of single-molecule array (Simoa) technology for digital immunoassays. The instrument is capable of single-molecule sensitivity and multiplexing with short turnaround times and a throughput of 66 samples/h. Singleplex and multiplexed digital immunoassays were developed for 16 proteins of interest in cardiovascular, cancer, infectious disease, neurology, and inflammation research. The average sensitivity improvement of the Simoa immunoassays versus conventional ELISA was >1200-fold, with coefficients of variation of <10%. The potential of digital immunoassays to advance human diagnostics was illustrated in two clinical areas: traumatic brain injury and early detection of infectious disease.
Subject(s)
Automation, Laboratory/instrumentation , Automation, Laboratory/methods , Clinical Laboratory Techniques/instrumentation , Clinical Laboratory Techniques/methods , Immunoassay/instrumentation , Immunoassay/methods , Humans , Reproducibility of Results , Sensitivity and Specificity , Signal Processing, Computer-Assisted/instrumentation , Time FactorsABSTRACT
Organisms adapt their physiology and behavior to the 24-h day-night cycle to which they are exposed. On a cellular level, this is regulated by intrinsic transcriptional-translational feedback loops that are important for maintaining the circadian rhythm. These loops are organized by members of the core clock network, which further regulate transcription of downstream genes, resulting in their circadian expression. Despite progress in understanding circadian gene expression, only a few players involved in circadian transcriptional regulation, including transcription factors, epigenetic regulators, and long noncoding RNAs, are known. Aiming to discover such genes, we performed a high-coverage transcriptome analysis of a circadian time course in murine fibroblast cells. In combination with a newly developed algorithm, we identified many transcription factors, epigenetic regulators, and long intergenic noncoding RNAs that are cyclically expressed. In addition, a number of these genes also showed circadian expression in mouse tissues. Furthermore, the knockdown of one such factor, Zfp28, influenced the core clock network. Mathematical modeling was able to predict putative regulator-effector interactions between the identified circadian genes and may help for investigations into the gene regulatory networks underlying circadian rhythms.
Subject(s)
Circadian Rhythm , Gene Expression Profiling , Gene Regulatory Networks , RNA, Long Noncoding/genetics , Transcription Factors/genetics , Transcriptional Activation , Animals , Computer Simulation , Epigenesis, Genetic , Fibroblasts/metabolism , Mice , Models, Genetic , NIH 3T3 CellsABSTRACT
During meiosis, homologous chromosomes associate to form the synaptonemal complex (SC), a structure essential for fertility. Information about the epigenetic features of chromatin within this structure at the level of superresolution microscopy is largely lacking. We combined single-molecule localization microscopy (SMLM) with quantitative analytical methods to describe the epigenetic landscape of meiotic chromosomes at the pachytene stage in mouse oocytes. DNA is found to be nonrandomly distributed along the length of the SC in condensed clusters. Periodic clusters of repressive chromatin [trimethylation of histone H3 at lysine (Lys) 27 (H3K27me3)] are found at 500-nm intervals along the SC, whereas one of the ends of the SC displays a large and dense cluster of centromeric histone mark [trimethylation of histone H3 at Lys 9 (H3K9me3)]. Chromatin associated with active transcription [trimethylation of histone H3 at Lys 4 (H3K4me3)] is arranged in a radial hair-like loop pattern emerging laterally from the SC. These loops seem to be punctuated with small clusters of H3K4me3 with an average spread larger than their periodicity. Our findings indicate that the nanoscale structure of the pachytene chromosomes is constrained by periodic patterns of chromatin marks, whose function in recombination and higher order genome organization is yet to be elucidated.
Subject(s)
Chromatin/chemistry , Chromatin/metabolism , Chromosomes, Mammalian/metabolism , Microscopy/methods , Pachytene Stage , Animals , Centromere/metabolism , Histones/metabolism , Lysine/metabolism , Methylation , Mice , Models, Biological , Synaptonemal Complex/metabolism , Transcription, GeneticABSTRACT
Epigenetic mechanisms determine the access of regulatory factors to DNA during events such as transcription and the DNA damage response. However, the global response of histone modifications and chromatin accessibility to UV exposure remains poorly understood. Here, we report that UV exposure results in a genome-wide reduction in chromatin accessibility, while the distribution of the active regulatory mark H3K27ac undergoes massive reorganization. Genomic loci subjected to epigenetic reprogramming upon UV exposure represent target sites for sequence-specific transcription factors. Most of these are distal regulatory regions, highlighting their importance in the cellular response to UV exposure. Furthermore, UV exposure results in an extensive reorganization of super-enhancers, accompanied by expression changes of associated genes, which may in part contribute to the stress response. Taken together, our study provides the first comprehensive resource for genome-wide chromatin changes upon UV irradiation in relation to gene expression and elucidates new aspects of this relationship.
Subject(s)
Chromatin Assembly and Disassembly/radiation effects , Chromatin/metabolism , DNA Damage , Epigenesis, Genetic/radiation effects , Ultraviolet Rays/adverse effects , Animals , Chromatin/genetics , Chromatin/pathology , Mice , NIH 3T3 CellsABSTRACT
We report a system and assay for performing fully-automated measurement of 6 proteins simultaneously with single molecule sensitivity. The system combines handling of samples, reagents, and consumables, with a module for imaging single molecule arrays (Simoa) to enable immunoassays that have high sensitivity (~fg/mL), are multiplexed, and are fully-automated. A 6-plex cytokine Simoa assay for IL-6, TNF-α, GM-CSF, IL-10, IL-1ß, and IL-1α was developed on the system. The assays had limits of detection in the range 0.01-0.03pg/mL, and the average imprecision (CV) of the Simoa signal was 4.2%. This assay was used to measure the concentrations of these cytokines in the plasma of patients with Crohn's Disease (CD), before and after treatment with anti-TNF-α antibody drugs, and in the serum of Type 1 diabetics. Concentrations of TNF-α and IL-6 in the CD samples determined using the fully-automated, multiplex Simoa assay had good correlation with the manual, single-plex assays previously reported. Drug treatment caused reductions in the mean concentration of all 6 cytokines in the plasma of CD patients. The concentrations of 4 cytokines were significantly higher in diabetics compared to healthy controls. The system could enable the widespread, multiplexed measurement of protein biomarkers with low abundance.
Subject(s)
Automation , Cytokines/blood , Immunoassay/methods , Crohn Disease/blood , Diabetes Mellitus/blood , Enzyme-Linked Immunosorbent Assay/instrumentation , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunoassay/instrumentation , Reproducibility of Results , WorkflowABSTRACT
Assemblies of huntingtin (HTT) fragments with expanded polyglutamine (polyQ) tracts are a pathological hallmark of Huntington's disease (HD). The molecular mechanisms by which these structures are formed and cause neuronal dysfunction and toxicity are poorly understood. Here, we utilized available gene expression data sets of selected brain regions of HD patients and controls for systematic interaction network filtering in order to predict disease-relevant, brain region-specific HTT interaction partners. Starting from a large protein-protein interaction (PPI) data set, a step-by-step computational filtering strategy facilitated the generation of a focused PPI network that directly or indirectly connects 13 proteins potentially dysregulated in HD with the disease protein HTT. This network enabled the discovery of the neuron-specific protein CRMP1 that targets aggregation-prone, N-terminal HTT fragments and suppresses their spontaneous self-assembly into proteotoxic structures in various models of HD. Experimental validation indicates that our network filtering procedure provides a simple but powerful strategy to identify disease-relevant proteins that influence misfolding and aggregation of polyQ disease proteins.
Subject(s)
Algorithms , Nerve Tissue Proteins/metabolism , Protein Aggregation, Pathological/metabolism , Protein Folding , Amino Acid Sequence , Animals , Brain/metabolism , Brain/pathology , Cell Line, Tumor , Drosophila/genetics , Drosophila/metabolism , Huntingtin Protein , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , PC12 Cells , Protein Binding , RatsABSTRACT
Alpha-solenoids are flexible protein structural domains formed by ensembles of alpha-helical repeats (Armadillo and HEAT repeats among others). While homology can be used to detect many of these repeats, some alpha-solenoids have very little sequence homology to proteins of known structure and we expect that many remain undetected. We previously developed a method for detection of alpha-helical repeats based on a neural network trained on a dataset of protein structures. Here we improved the detection algorithm and updated the training dataset using recently solved structures of alpha-solenoids. Unexpectedly, we identified occurrences of alpha-solenoids in solved protein structures that escaped attention, for example within the core of the catalytic subunit of PI3KC. Our results expand the current set of known alpha-solenoids. Application of our tool to the protein universe allowed us to detect their significant enrichment in proteins interacting with many proteins, confirming that alpha-solenoids are generally involved in protein-protein interactions. We then studied the taxonomic distribution of alpha-solenoids to discuss an evolutionary scenario for the emergence of this type of domain, speculating that alpha-solenoids have emerged in multiple taxa in independent events by convergent evolution. We observe a higher rate of alpha-solenoids in eukaryotic genomes and in some prokaryotic families, such as Cyanobacteria and Planctomycetes, which could be associated to increased cellular complexity. The method is available at http://cbdm.mdc-berlin.de/~ard2/.
Subject(s)
Genomics , Proteins/physiology , Protein Conformation , Proteins/chemistry , Proteins/geneticsABSTRACT
Ribosomal loci represent a major tool for investigating environmental diversity and community structure via high-throughput marker gene studies of eukaryotes (e.g. 18S rRNA). Since the estimation of species' abundance is a major goal of environmental studies (by counting numbers of sequences), understanding the patterns of rRNA copy number across species will be critical for informing such high-throughput approaches. Such knowledge is critical, given that ribosomal RNA genes exist within multi-copy repeated arrays in a genome. Here we measured the repeat copy number for six nematode species by mapping the sequences from whole genome shotgun libraries against reference sequences for their rRNA repeat. This revealed a 6-fold variation in repeat copy number amongst taxa investigated, with levels of intragenomic variation ranging from 56 to 323 copies of the rRNA array. By applying the same approach to four C. elegans mutation accumulation lines propagated by repeated bottlenecking for an average of ~400 generations, we find on average a 2-fold increase in repeat copy number (rate of increase in rRNA estimated at 0.0285-0.3414 copies per generation), suggesting that rRNA repeat copy number is subject to selection. Within each Caenorhabditis species, the majority of intragenomic variation found across the rRNA repeat was observed within gene regions (18S, 28S, 5.8S), suggesting that such intragenomic variation is not a product of selection for rRNA coding function. We find that the dramatic variation in repeat copy number among these six nematode genomes would limit the use of rRNA in estimates of organismal abundance. In addition, the unique pattern of variation within a single genome was uncorrelated with patterns of divergence between species, reflecting a strong signature of natural selection for rRNA function. A better understanding of the factors that control or affect copy number in these arrays, as well as their rates and patterns of evolution, will be critical for informing estimates of global biodiversity.
Subject(s)
Nematoda/genetics , RNA, Ribosomal/genetics , Animals , Caenorhabditis elegans/classification , Caenorhabditis elegans/genetics , Nematoda/classification , PhylogenyABSTRACT
We have developed a method that enables the multiplexed detection of proteins based on counting single molecules. Paramagnetic beads were labeled with fluorescent dyes to create optically distinct subpopulations of beads, and antibodies to specific proteins were then immobilized to individual subpopulations. Mixtures of subpopulations of beads were then incubated with a sample, and specific proteins were captured on their specific beads; these proteins were then labeled with enzymes via immunocomplex formation. The beads were suspended in enzyme substrate, loaded into arrays of femtoliter wells--or Single Molecule Arrays (Simoa)--that were integrated into a microfluidic device (the Simoa disc). The wells were then sealed with oil, and imaged fluorescently to determine: a) the location and subpopulation identity of individual beads in the femtoliter wells, and b) the presence or absence of a single enzyme associated with each bead. The images were analyzed to determine the average enzyme per bead (AEB) for each bead subpopulation that provide a quantitative parameter for determining the concentration of each protein. We used this approach to simultaneously detect TNF-α, IL-6, IL-1α, and IL-1ß in human plasma with single molecule resolution at subfemtomolar concentrations, i.e., 200- to 1000-fold more sensitive than current multiplexed immunoassays. The simultaneous, specific, and sensitive measurement of several proteins using multiplexed digital ELISA could enable more reliable diagnoses of disease.