David M. Rissin

Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations

The ability to detect single protein molecules 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. 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).

Stochastic inhibitor release and binding from single-enzyme molecules

Inhibition kinetics of single-β-galactosidase molecules with the slow-binding inhibitor d-galactal have been characterized by segregating individual enzyme molecules in an array of 50,000 ultra small reaction containers and observing substrate turnover changes with fluorescence microscopy. Inhibited and active states of β-galactosidase could be clearly distinguished, and the large array size provided very good statistics. With a pre-steady-state experiment, we demonstrated the stochastic character of inhibitor release, which obeys first-order kinetics. Under steady-state conditions, the quantitative detection of substrate turnover changes over long time periods revealed repeated inhibitor binding and release events, which are accompanied by conformational changes of the enzymes catalytic site. We proved that the rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk-reaction kinetics.

Distinct and long-lived activity states of single enzyme molecules.

Individual enzyme molecules have been observed to possess discrete and different turnover rates due to the presence of long-lived activity states. These stable activity states are thought to result from different molecular conformations or post-translational modifications. The distributions in kinetic activity observed in previous studies were obtained from small numbers of single enzyme molecules. Due to this limitation, it has not been possible to fully characterize the different kinetic and equilibrium binding parameters of single enzyme molecules. In this paper, we analyze hundreds of single beta-galactosidase molecules simultaneously; using a high-density array of 50,000 fL-reaction chambers, we confirm the presence of long-lived kinetic states within a population of enzyme molecules. Our analysis has isolated the source of kinetic variability to kcat. The results explain the kinetic variability within enzyme molecule populations and offer a deeper understanding of the unique properties of single enzyme molecules. Gaining a more fundamental understanding of how individual enzyme molecules work within a population should provide insight into how they affect downstream biochemical processes. If the results reported here can be generalized to other enzymes, then the stochastic nature of individual enzyme molecule kinetics should have a substantial impact on the overall metabolic activity within a cell.

Optical imaging fiber-based live bacterial cell array biosensor

A live cell array biosensor was fabricated by immobilizing bacterial cells on the face of an optical imaging fiber containing a high-density array of microwells. Each microwell accommodates a single bacterium that was genetically engineered to respond to a specific analyte. A genetically modified Escherichia coli strain, containing the lacZ reporter gene fused to the heavy metal-responsive gene promoter zntA, was used to fabricate a mercury biosensor. A plasmid carrying the gene coding for the enhanced cyan fluorescent protein (ECFP) was also introduced into this sensing strain to identify the cell locations in the array. Single cell lacZ expression was measured when the array was exposed to mercury and a response to 100nM Hg(2+) could be detected after a 1-h incubation time. The optical imaging fiber-based single bacterial cell array is a flexible and sensitive biosensor platform that can be used to monitor the expression of different reporter genes and accommodate a variety of sensing strains.

Hypoxia due to cardiac arrest induces a time-dependent increase in serum amyloid β levels in humans.

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.

Use of Colorimetric Test Strips for Monitoring the Effect of Hemodialysis on Salivary Nitrite and Uric Acid in Patients with End-Stage Renal Disease: A Proof of Principle

BACKGROUND Initial screening of potential biomarkers for monitoring dialysis was performed with saliva samples collected from patients with end-stage renal disease (ESRD). A more thorough analysis of the most promising markers identified in the initial screening was conducted with saliva samples acquired at hourly intervals throughout dialysis to monitor analyte concentrations as dialysis progressed. We observed that salivary nitrite (NO(2)(-)) and uric acid (UA) concentrations consistently decreased as dialysis proceeded. METHODS Solution-based colorimetric-detection chemistries for NO(2)(-) and UA were converted to a test strip format to produce a simple method for semiquantitatively measuring NO(2)(-) and UA concentrations in the clinic or at the patients home. We assessed the test strips with saliva samples collected from both ESRD patients undergoing dialysis and healthy control volunteers to qualitatively monitor the effect of dialysis on salivary NO(2)(-) and UA. We used computer software to analyze digital images of the resulting test strip color intensities. RESULTS Test strip measurements showed that mean salivary concentrations of NO(2)(-) and UA were decreased in ESRD patients by 86% and 39%, respectively, compared with 15% and 9% for time-matched controls. Comparison of test strip results with calibrated solution-based assays suggests that the test strips can semiquantitatively measure salivary concentrations of NO(2)(-) and UA. CONCLUSIONS The colorimetric test strips monitored changes in salivary NO(2)(-) and UA concentrations that occurred in ESRD patients during dialysis. The test strips may prove useful for noninvasively evaluating dialysis progress and may also be useful for monitoring renal disease status.

Multiplexed single molecule immunoassays

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-1β 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.

The Simoa HD-1 Analyzer: A Novel Fully Automated Digital Immunoassay Analyzer with Single-Molecule Sensitivity and Multiplexing

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.

Microsensor arrays for saliva diagnostics.

Abstract:  Optical fiber microarrays have been used to screen saliva from patients with end‐stage renal disease (ESRD) to ascertain the efficacy of dialysis. We have successfully identified markers in saliva that correlate with kidney disease. Standard assay chemistries for these markers have been converted to disposable test strips such that patients may one day be able to monitor their clinical status at home. Details of these developments are described. In addition, saliva from asthma and chronic obstructive pulmonary disease (COPD) patients is being screened for useful diagnostic markers. Our goal is to develop a multiplexed assay for these protein and nucleic acid biomarkers for diagnosing the cause and severity of pulmonary exacerbations, enabling more effective treatment to be administered. These results are reported in the second part of this article.

A fully-automated, six-plex single molecule immunoassay for measuring cytokines in blood.

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-1β, 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 Crohns 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.