Non-Enzymatic Sensing of Creatinine for Early Detection of Chronic Kidney Disease

Chronic kidney disease (CKD) affects a 8-16 % of the global population. Creatinine is a useful biomarker for screening and detecting CKD. Creatinine concentrations in urine outside the normal clinical range (4.4-13.3 mM) gives an indication of kidney conditions including CKD, and requires further evaluation by the nephrologists in secondary care clinical settings. The fact that CKD is asymptomatic, it is critical to detect it at earlier stages and prevent its rogression with medicines. Currently Jaffe reaction method of creatinine detection is the most widely used technique. Although, this significantly suffers from low specificity and high interferences from molecules including ascorbic acid. Enzymatic methods, although are highly specific, but have problems with high cost and low stability due to use of enzymes. Therefore, there is an urgent need to develop low cost and simple non-invasive creatinine detection methods, which is the focus of this PhD project.We have developed a novel simple sensing mechanism based on bare glassy carbon electrode (GCE) which can detect creatinine electrochemically (LOD 60 µM and sensitivity 1.50 µA/mM) and colorimetrically (LOD 6.63 µM). An application for a patent has been made for this work. Additionally, nickel-based systems have also been studied to detect creatinine, although there is high interference to urea and requires further optimisation. SPE based creatinine sensing methods designed (LOD 60 µM) as part of a preliminary study in this project including surface modifications, aims to use creatinine sensing for POCT applications in future. Market testing of the concepts used in this project has informed us to evaluate albumin sensing

An alkaline ferrocyanide non-enzymatic electrochemical sensor for creatinine detection

Creatinine is a useful metabolite for the evaluation of Chronic Kidney Disease (CKD). Creatinine concentrations in urine outside of the healthy range of 4 to 15 mM may indicate CKD and other kidney conditions. Early detection and monitoring can provide lifesaving intervention, and as such there is a need to develop rapid, cheap and selective electrochemical creatinine sensors. A simple non-enzymatic creatinine sensing platform enabled by the electrocatalytic response of the metabolite to ferricyanide in alkaline conditions is presented. Studies were made on unmodified glassy carbon (GCE) and screen printed carbon electrodes (SPCE), both giving sensitivity of 21±5 µA mM-1 cm-2 and limit of detection of ca. 60 µM

Mesalazine based topical hydrogel formulation enhances anti-oxidant and cytokine activity in wounded STZ-induced mice

Background: The present study is targeted to elucidate the wound healing potential of mesalazine in STZ induced diabetic mice by comparing various antioxidant, pro-inflammatory cytokine levels and other wound healing parameters at 3,7 and 14th day. Methods: Full thickness excisional wounds of 6mm size were created on the dorsal side of STZ induced mice and topical treatment of mesalazine based different hydrogels was applied for 14 days. Wound tissues were excised on day 3, 7 and 14 for various wound healing parameters. Results: Delayed in wound healing was observed in diabetic group which eventually got accelerated after application of ethosome based mesalazine loaded hydrogel designated as D5 in study. Significant wound contraction rate was observed in D5 group and tissue hydroxyproline and tensile strength was also elevated after treatment with mesalazine loaded ethosomal hydrogel. The level of ROS was found to be significantly decreased in SOD, CAT and GSH experiment. LPO level were found to be elevated in D5 group. Finding of levels of pro-inflammatory cytokine suggested significant decrease and showed significant elevation in vascular endothelial growth factor (VEGF).  Conclusion: D5 group of ethosome based mesalazine loaded hydrogel showed promising results in controlling all the factors to their normal range and was effective in accelerating wound 51healing in diabetic mice.  Keywords: Diabetic wounds; hydrogels; antioxidants; cytokines; VEGF; Mesalazin

Ancient DNA Reveals Late Pleistocene Existence of Ostriches in Indian Sub-Continent

<div><p>Ancient DNA (aDNA) analysis of extinct ratite species is of considerable interest as it provides important insights into their origin, evolution, paleogeographical distribution and vicariant speciation in congruence with continental drift theory. In this study, DNA hotspots were detected in fossilized eggshell fragments of ratites (dated ≥25000 years B.P. by radiocarbon dating) using confocal laser scanning microscopy (CLSM). DNA was isolated from five eggshell fragments and a 43 base pair (bp) sequence of a 16S rRNA mitochondrial-conserved region was successfully amplified and sequenced from one of the samples. Phylogenetic analysis of the DNA sequence revealed a 92% identity of the fossil eggshells to <i>Struthio camelus</i> and their position basal to other palaeognaths, consistent with the vicariant speciation model. Our study provides the first molecular evidence for the presence of ostriches in India, complementing the continental drift theory of biogeographical movement of ostriches in India, and opening up a new window into the evolutionary history of ratites.</p></div

List of eggshell specimens from India with associated genes and predicted taxon.

<p>List of eggshell specimens from India with associated genes and predicted taxon.</p

Primer sequences used for PCR and sequencing of 16S and Hypervariable regions of mitochondrial DNA.

<p>Primer sequences used for PCR and sequencing of 16S and Hypervariable regions of mitochondrial DNA.</p

Ostrich Eggshell.

<p>Pictorial view and radial cross section showing various layers, shell thickness and pore diameter.</p

Quantification of fossilized DNA.

<p>Bioanalyzer image showing bands of 65 bp and 43 bp in GK/KH/010 and GK/RN/011 respectively. Concentration of DNA in pg/ml of five samples, calculated by bioanalyzer.</p

Alignment of partial 16s rRNA gene sequences of samples and ratites and evolutionary relationships of taxa.

<p>(A) and (B) Alignment of ratites sequences and sample GK/RN/011 and GK/RU/009. Dots signify identity to the <i>Struthio camelus</i> sequence. (C) The evolutionary history was inferred using the Neighbor-Joining method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164823#pone.0164823.ref042" target="_blank">42</a>]. The bootstrap consensus tree inferred from 1000 replicates [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164823#pone.0164823.ref043" target="_blank">43</a>] is taken to represent the evolutionary history of the taxa analyzed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164823#pone.0164823.ref043" target="_blank">43</a>]. The evolutionary distances were computed using the Maximum Composite Likelihood method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164823#pone.0164823.ref044" target="_blank">44</a>] and are in the units of the number of base substitutions per site. The analysis involved 7 nucleotide sequences. The codon positions which were included: 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 87 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164823#pone.0164823.ref045" target="_blank">45</a>].</p

Continental drifting and vicariant speciation of ratites.

<p>Breakup of supercontinent Gondwanaland during Early Cretaceous and biogeographical movement of ratites to different continents.</p