Highly Sensitive Electrochemiluminescence Assay for Cardiac Troponin I and Adenosine Triphosphate by using Supersandwich Amplification and Bifunctional Aptamer

Abstract

A highly sensitive electrochemiluminescence (ECL) assay was developed for sequential detection of cardiac troponin I (cTnI) and adenosine triphosphate (ATP) through supersandwich amplification and bifunctional aptamer. The bifunctional aptamer (S1) contained ATP binding aptamer and cTnI binding aptamer. A gold electrode was modified with the specific peptide of cTnI through a self-assembly technique. A sandwichtype conjugate (peptide < cTnI > S1) was formed when the peptide-modified electrode was successively reacted with cTnI and S1. Then, hybridizations between S1 and two ss-DNA auxiliary probes, in which one is complementary with ATP binding aptamer and the other is ATP binding aptamer, led to the formation of long-range ds-DNA on the electrode surface. In the presence of ECL indicator Ru(phen) 3 2 + , a large amount of Ru (phen) 3 2 + was intercalated into ds-DNA grooves, resulting in amplification of the ECL signals. The ECL assay was successfully developed for the detection of cTnI in the range of 8.0 10 À13 to 1.0 10 À11 g/mL based on the increased ECL intensity. After detecting cTnI, ATP was detected in the range of 30 to 500 nM, based on switching structures of aptamers from ds-DNA to ss-DNA/target complex. A low detection limit of 0.3 pg/mL and 10 nM for cTnI and ATP, respectively, was obtained. The employment of a bifunctional aptamer probe and supersandwich signal amplification is promising for the sensitive detection of multiple targets.

Isolation, expression and biochemical characterization of recombinant hyoscyamine-6β-hydroxylase from Brugmansia sanguinea – tuning the scopolamine production

“…Since scopolamine content in plants is directly correlated to hyoscyamine-6β-hydroxylase content and activity, engineering of this enzyme and the exploration of more potent H6H plant species in order to boost scopolamine yield has attracted considerable interest. [7][8][9][10][11][12][13][14][15][16][17][18] B. sanguinea is a flowering shrub from the Datureae tribe, used in the Andes region as a potent hallucinogenic and sedative drug for various medicinal and ritual practices. 22 It has been identified as a potent scopolamine producer based on plant part drug content analysis although closer genetic and biochemical details are missing.…”

“…5,6 To overcome this low efficiency, several working groups have reported attempts to increase the expression level of the key enzyme H6H in various plant species. [7][8][9][10][11][12][13][14][15][16] Another useful approach involves the recombinant cloning and expression of H6H in Escherichia coli 3,17 or Saccharomyces cerevisiae 18 with subsequent in vitro transformation of 1 by the purified enzyme. In this regard, site-specific mutagenesis is able to enhance enzyme activity, but identifying "hotspots" for targeted mutagenesis is presently random since structural information about H6H is very limited.…”

Point-of-Care Compatibility of Ultra-Sensitive Detection Techniques for the Cardiac Biomarker Troponin I—Challenges and Potential Value

“…This approach had a LOD of 0.79 ng/L and a linear detection range of 1–10 ng/L. A different ECL assay uses peptides as the recognition element and aptamers as the basis to create a super-sandwich for signal amplification [58]. This is a technique which is gradually becoming more popular, tackling sensitivity issues associated with standard sandwich assays [59].…”

“…Figure 3 and Figure 4: [37,38,48,49,50,57,58,61,65,66,68,69,70,71,74,84,85,86,87,88,89,93,94,95,96,97,104,105,111,112,115,116,117,120,121,122,123,125,126,127,128,130,132,133,134,136,138,144,145,148,150,151,159,161,174,175,176,184,185,186,187,188,189,190,…”

Sensitive impedimetric detection of troponin I with metal–organic framework composite electrode

“…Signicant attention has been paid by the researchers in the recent past to develop electrochemical and uorescent biosensors for the analysis of cTnI. [8][9][10][11][12][13] In particular, electrochemical biosensors provide better sensitivity and quantitative estimations while also requiring relatively fewer amounts of nanomaterials and recognition biomolecules during their fabrication. 14 The performance of electrochemical biosensors is largely dependent upon the transducer surface.…”