H. single detection event of an analyte molecule does not contain enough analytical information. Therefore, it is necessary to make either many parallel measurements on a larger area or many sequential measurements in the same detection volume. 2.?Enzyme Labels ELISA has been successfully transformed into single\molecule immunoassays. The generation of thousands of fluorescent molecules per enzyme label molecule multiplied by up to a million photons per fluorophore generates a very strong signal that can be detected by simple wide\field fluorescence microscopy. In particular, \galactosidase from is an excellent enzyme label for single\molecule applications because it is robust and can turn over up to 1000 substrate molecules per second. Based on this strategy, Rotman28 reported the very first single\molecule experiment already in 1961. The enzymatic substrate turnover, however, is a kinetic process and requires time, which leads to product diffusion. Consequently, the signal is not detectable at the same location as the analyte. There are two options to spatially allocate the signal to the analyte. 2.1. Assays Based on Localized Product Deposition In the easiest case, the Epifriedelanol enzymatic reaction generates a product that precipitates around the analyte. The group of Suzuki29 designed a digital sandwich immunoassay on beads by using a conjugate of detection antibody and horseradish peroxidase. The enzyme label converted a fluorescence\labeled tyramide substrate to a short\lived radical which immediately coupled the fluorescent labels only to the analyte\bearing beads. This led to a high local fluorescence signal at the site Epifriedelanol of analyte binding. It was noted, however, that the detection of the tyramide signal on the beads by flow cytometry was less efficient than that of digital ELISAs in confined environments. 2.2. Assays in Confined Environments Alternatively, the enzyme label converts a substrate to a soluble fluorescent product. In this case, the reaction must be confined in a very small compartment in order to prevent product Mouse monoclonal antibody to Keratin 7. The protein encoded by this gene is a member of the keratin gene family. The type IIcytokeratins consist of basic or neutral proteins which are arranged in pairs of heterotypic keratinchains coexpressed during differentiation of simple and stratified epithelial tissues. This type IIcytokeratin is specifically expressed in the simple epithelia lining the cavities of the internalorgans and in the gland ducts and blood vessels. The genes encoding the type II cytokeratinsare clustered in a region of chromosome 12q12-q13. Alternative splicing may result in severaltranscript variants; however, not all variants have been fully described diffusion.30 The concentration of the reaction product exceeds the detection threshold in small confined volumes. For example, a single molecule of \galactosidase enclosed in a volume of 50?fL (50?m3) can produce a fluorophore concentration of 2?m in 1?min, which can be easily detected via conventional epifluorescence microscopy.31 Enzymatic reactions were confined in water\in\oil emulsion28 or microfluidic droplets, fused silica capillaries,32 virus capsids,33 lipid vesicles,34 or so\called femtoliter arrays. In particular, femtoliter arrays and water\in\oil microfluidic droplets have found analytical applications. 2.2.1. Femtoliter Arrays Femtoliter arrays consist of a large number of homogeneous wells fabricated in the surface of optical fiber bundles,35 fused silica slides,36 or polydimethylsiloxane (PDMS).37 The Walt group38 pioneered single\molecule immunoassays based on a fluorogenic enzymatic reaction in femtoliter arrays, which was commercialized by Quanterix. In the so\called Simoa platform (single\molecule arrays, Figure?2), magnetic beads with a capture antibody were dispersed in a sample. The bead concentration was typically much higher than the analyte concentration. The beads were magnetically separated and incubated with a biotinylated antibody, followed by the addition of a streptavidinC\galactosidase conjugate. A high bead\to\analyte ratio resulted in a small fraction of beads labeled with a single enzyme molecule and a large excess of unlabeled beads. The beads were loaded with a fluorogenic substrate onto a femtoliter array and sealed with a gasket or oil film. A highly fluorescent product accumulated only in wells that contained a bead with a bound analyte molecule. The analyte concentration was determined digitally by counting the number of fluorescent wells. Prostate\specific antigen (PSA) and tumor necrosis factor\ (TNF\) were detected with a limit of detection (LOD) of 1 1.5?fg?mL?1 (50?am) and 2.5?fg?mL?1 (150?am), respectively. Other clinically relevant analytes included cancer biomarkers,39 urinary biomarkers,40 p24 protein of HIV,41 and the neurofilament light chain protein (NFL), a neuronal injury marker of various neurodegenerative conditions and Epifriedelanol brain injuries. Traditionally, cerebrospinal fluid must be obtained for analysis by lumbar puncture because the concentration of NFL in the blood is too low for a conventional ELISA. Shahim et?al.42 developed an ultrasensitive immunoassay with an LOD of 0.29?pg?mL?1, which enabled NFL measurements in the serum of healthy subjects. Olivera et?al.43 determined tau protein in blood Epifriedelanol plasma (LOD of 0.012?pg?mL?1) to examine the relationship between increased tau protein levels and chronical neurological and psychological symptoms in military personnel after a traumatic brain injury. The ultrasensitive detection of biothreats such as ricin has also been demonstrated.44 Open in a separate window Figure.