Synthesis Of Novel Substrates Research Articles

Lysosomal diseases: diagnostic update

Technological developments in newborn and population screening, biomarker discovery for monitoring treatment and rapid high throughput DNA sequencing are having a great impact on the diagnostic procedure for symptomatic patients with lysosomal storage diseases. The use of dried blood spots, initially for newborn screening, has stimulated the introduction of automated, rapid and more sensitive methods for the assay of lysosomal enzymes, including the synthesis of novel substrates. Storage products and secondary metabolites in urine and cells can be identified and measured very accurately and sensitively by high performance liquid chromatography and tandem mass spectrometry. This has enhanced the preliminary metabolite screen for LSDs and facilitated the diagnosis of transport defects. Fast, reliable and affordable high throughput DNA sequencing, such as whole or selected exome sequencing, is helping to make diagnoses in difficult cases, to reveal novel gene defects, to widen the clinical spectrum of diseases and possibly to identify modifying genetic factors. Bioinformatics will be necessary to handle the data generated by these new technologies. Notwithstanding, these technical innovations, accurate and reliable diagnosis will still depend on the knowledge and experience of skilled laboratory staff.

T7 RNA polymerase as a self-replicating label for antigen quantification

Enzymes are used widely as labels in binding assays for protein analytes, because they provide signal amplification. Efforts at improving the assay sensitivity have been focused mainly on the synthesis of novel substrates, e.g. fluorogenic and chemiluminogenic ones. We report the investigation of T7 RNA polymerase (T7RP) as a label with unique characteristics for antigen quantification. In an in vitro, coupled (one-step) transcription/translation reaction, T7RP catalyzes the expression of an enzyme-coding DNA template to produce free enzyme (luciferase) in solution. We demonstrate that the generated luciferase is linearly related to the input T7RP in a range covering over four orders of magnitude. It is also shown that T7RP exhibits a significant level of self-replication (100-fold) in vitro by acting on a DNA template comprising the T7RP cDNA downstream of a T7 promoter. By combining the self-replication reaction with the expression of luciferase DNA, as low as 1400 T7RP molecules are detectable. Furthermore, the T7RP is biotinylated, complexed with streptavidin and used for antigen quantification in a microtiter well-based assay with high sensitivity and reproducibility.

Naphthyl dioxetane phosphates: synthesis of novel substrates for enzymic chemiluminescent assays

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNaphthyl dioxetane phosphates: synthesis of novel substrates for enzymic chemiluminescent assaysB. Edwards, A. Sparks, J. C. Voyta, R. Strong, O. Murphy, and Irena BronsteinCite this: J. Org. Chem. 1990, 55, 25, 6225–6229Publication Date (Print):December 1, 1990Publication History Published online1 May 2002Published inissue 1 December 1990https://doi.org/10.1021/jo00312a036RIGHTS & PERMISSIONSArticle Views510Altmetric-Citations48LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (752 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts