Ricin is an extremely potent ribosomal inactivating protein listed as a Category B select agent. Although ricin intoxication is not transmittable from person to person, even a single ricin molecule can lead to cell necrosis because it inactivates 1500 ribosomes/min. Since there is currently no vaccine or therapeutic treatment for ricin intoxication, ultrasensitive analytical assays capable of detecting ricin in a variety of matrixes are urgently needed to limit exposure to individuals as well as communities. In this paper, we present the development and application of a single-molecule array (Simoa) for the detection of ricin toxin in human urine and serum. Single-domain antibodies (sdAbs), among the smallest engineered binding fragments, were chemically coupled to the surface of paramagnetic beads for the sensitive detection of ricin toxin. The Simoa was able to detect ricin at levels of 10 fg/mL, 100 fg/mL, and 1 pg/mL in buffer, urine and serum, respectively, in a fraction of the assay time need using immuno-polymerase chain reaction (IPCR). Using a fully automated state-of-the-art platform, the Simoa HD-1 analyzer, the assay time was reduced to 64 min.

The delivery of exogenous agents can enable noninvasive disease monitoring, but existing low-dose approaches require complex infrastructure. In this paper, we describe a microdose-scale injectable formulation of nanoparticles that interrogate the activity of thrombin, a key regulator of clotting, and produce urinary reporters of disease state. We establish a customized single molecule detection assay that enables urinary discrimination of thromboembolic disease in mice using doses of the nanoparticulate diagnostic agents that fall under regulatory guidelines for “microdosing.”

Dengue virus serotype 2-positive plasma was applied to two indirect single-molecule arrays (Simoas) for the detection of anti-dengue virus IgG and IgM. The Simoas were 1,000 and 10,000 times more sensitive than enzyme-linked immunosorbent assays (ELISAs) for the detection of IgG and IgM, respectively. Using Simoas, serology may be used for the detection of dengue virus infection in the acute phase.T he potential for early diagnosis of infectious disease in the acute phase of illness relies on the detection of ultralow concentrations of circulating pathogens and related biomarkers, such as host immunoglobulin, cytokines, DNA, and RNA. Subfemtomolar detection of proteins was recently demonstrated by capturing single molecules of disease-related biomarkers onto magnetic beads and then amplifying the signal by enzymatic amplification. The assay, coined single-molecule array (Simoa), has facilitated the ultrasensitive detection of prostate-specific antigen (PSA) (1), HIV p24 protein (2), bacterial genomic DNA (3), tumor necrosis factor ␣ (TNF-␣), interleukin 6 (IL-6), IL-1␣, and IL-1␤ (4), and synthetic urinary biomarkers (5) using submilliliter clinical sample volumes (1).Dengue virus is a 50-nm arthropod-borne positive-sense RNA virus and is reemerging as one of the most globally important infectious diseases. The incidence of dengue virus infections has increased rapidly since the 1950s, currently affecting more than 100 countries worldwide (6), with an estimated 3 billion persons at risk of infection (7). When based solely on clinical observations, dengue is difficult to diagnose, as the disease state frequently mimics other febrile illnesses, such as influenza, measles, meningococcal infections, and enteric infections (8). Current dengue diagnostic methods include virus isolation, nucleic acid and antigen detection, and serological tests; however, the performance of each method is predominantly dependent on assay sensitivity and the day of sample collection due to the complicated kinetics of viremia, NS1, and host antibody response. The reemergence of dengue and the complex clinical manifestations of infection make the need for rapid and accurate diagnostics of critical importance for clinical precision, control and prevention, and outbreak surveillance. In response to such a need, we developed two proof-ofconcept indirect Simoas for the ultrasensitive detection of type 2 anti-dengue virus IgG and IgM and compared the Simoa sensitivities to two commercially available EuroImmun dengue ELISA kits.The indirect Simoa for the detection of either anti-dengue virus IgG or IgM follows precisely the same assay format as an indirect ELISA, utilizing inactivated dengue virus type 2 antigencoated magnetic beads for the capture of dengue virus-specific antibodies. Each captured antibody is detected by the addition of a biotinylated detector antibody specific to human IgG or IgM and labeled using streptavidin-␤-galactosidase (S␤G). Individual beads are then isolated in 46-fl reaction wells in the presence of a fluorogenic sub...