A smartphone readout system for gold nanoparticle-based lateral flow assays: application to monitoring of digoxigenin.

For modern approaches in precision medicine, fast and easy-to-use point-of-care diagnostics (POCs) are essential. Digoxin was chosen as an example of a drug requiring close monitoring. Digoxin is a cardiac glycoside used for the treatment of tachycardia with a narrow therapeutic window of 0.5-2.0 ng·mL-1, and toxic effects are common for concentrations above 2.5 ng·mL-1. For monitoring of blood concentration levels and treatment of intoxication, highly selective antibodies for digoxin and its hapten, digoxigenin, are available. A smartphone readout system is described for measuring digoxigenin in human serum using a common gold nanoparticle lateral flow assay (LFA). The R-package GNSplex, which also includes a Shiny app for quantitative test interpretation based on linear models, is used for image analysis. Images of lateral flow strips were taken with an iPhone camera and a simple darkbox made from black cardboard. Sensitivity and accuracy of the quantitative smartphone system as well as analytical parameters such as limit of detection (LOD) were determined and compared to data obtained with a high resolution BioImager. The data show that the smartphone based digoxin assay yields reliable quantitative results within the clinically relevant concentration range. Graphical abstract For therapeutic drug monitoring and point of care diagnostics we introduce the open source R-package GNSplex for smartphone readout and interpretation of lateral flow assays. The cardiac glycoside dixogin was used as target for this quantitative smartphone reader.

Structural and docking studies of Leucaena leucocephala Cinnamoyl CoA reductase.

Lignin, a major constituent of plant call wall, is a phenolic heteropolymer. It plays a major role in the development of plants and their defense mechanism against pathogens. Therefore Lignin biosynthesis is one of the critical metabolic pathways. In lignin biosynthesis, the Cinnamoyl CoA reductase is a key enzyme which catalyzes the first step in the pathway. Cinnamoyl CoA reductase provides the substrates which represent the main transitional molecules of lignin biosynthesis pathway, exhibits a high in vitro kinetic preference for feruloyl CoA. In present study, the three-dimensional model of cinnamoyl CoA reductase was constructed based on the crystal structure of Grape Dihydroflavonol 4-Reductase. Furthermore, the docking studies were performed to understand the substrate interactions to the active site of CCR. It showed that residues ARG51, ASN52, ASP54 and ASN58 were involved in substrate binding. We also suggest that residue ARG51 in CCR is the determinant residue in competitive inhibition of other substrates. This structural and docking information have prospective implications to understand the mechanism of CCR enzymatic reaction with feruloyl CoA, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well.

Sequence analysis, in silico modeling and docking studies of caffeoyl CoA-O-methyltransferase of Populus trichopora.

Caffeoyl coenzyme A-O-methyltransferases (CCoAOMTs) which are characterized under class I plant OMTs, methylates CoA thioesters, with an in vitro kinetic preference for caffeoyl CoA. CCoAOMTs exhibit association with lignin biosynthesis by showing a prime role in the synthesis of guaiacyl lignin and providing the substrates for synthesis of syringyl lignin. The sequence analysis of CCoAOMT from Populus trichopora exhibits 58 nucleotide substitutions, where transitions overcome transversions. Validation of homology models of both CCoAOMT1 and 2 isoforms reveals that 92.4% and 96% residues are falling in the most favorable region respectively in the Ramachandran plot, indicating CCoAOMT2 as the more satisfactory model, and the overall quality factor of both isoforms is 98.174. The structural architecture analysis is showing very good packing of residues similar to protein crystal structures data. The active site residues and substrate-product interactions showed that CCoAOMT2 possesses more affinity toward caffeoyl CoA, feruloyl CoA, 5-hydroxy feruloyl CoA and sinapoyl CoA than CCoAOMT1, therefore it exist in a more active conformation. The affinity of CCoAOMT2 with feruloyl CoA is highest among all the affinities of both CCoAOMT isoforms with their substrates and products. This information has potential implications to understand the mechanism of CCoAOMT related enzymatic reactions in Populus trichopora, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well.