Amplified detection of genome-containing biological targets using terminal deoxynucleotidyl transferase-assisted rolling circle amplification

2018 ◽  
Vol 54 (6) ◽  
pp. 682-685 ◽  
Author(s):  
Yi-Chen Du ◽  
Yan-Jun Zhu ◽  
Xiao-Yu Li ◽  
De-Ming Kong
Keyword(s):  
Rolling Circle Amplification ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Rolling Circle ◽  
Biological Targets

We proposed a terminal deoxynucleotidyl transferase (TdT)-assisted rolling circle amplification (RCA) strategy for the amplified detection of genome-containing biological targets.

scholarly journals On the Electrochemical Detection of Alpha-Fetoprotein Using Aptamers: DNA Isothermal Amplification Strategies to Improve the Performance of Weak Aptamers

Biosensors ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 46
Author(s):  
Ramón Lorenzo-Gómez ◽  
Daniel González-Robles ◽  
Rebeca Miranda-Castro ◽  
Noemí de-los-Santos-Álvarez ◽  
María Jesús Lobo-Castañón
Keyword(s):  
Dissociation Constant ◽  
Electrochemical Detection ◽  
Magnetic Beads ◽  
Rolling Circle Amplification ◽  
Binding Constants ◽  
Alpha Fetoprotein ◽  
Isothermal Amplification ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tumor Biomarker ◽  
Rolling Circle

Affinity characterization is essential to develop reliable aptamers for tumor biomarker detection. For alpha-fetoprotein (AFP), a biomarker of hepatocellular carcinoma (HCC), two DNA aptamers were described with very different affinity. In this work, we estimate the dissociation constant of both of them by means of a direct assay on magnetic beads modified with AFP and electrochemical detection on carbon screen-printed electrodes (SPCE). Unlike previous works, both aptamers showed similar dissociation constant (Kd) values, in the subµM range. In order to improve the performance of these aptamers, we proposed the isothermal amplification of the aptamers by both terminal deoxynucleotidyl transferase (TdT) and rolling circle amplification (RCA). Both DNA amplifications improved the sensitivity and also the apparent binding constants from 713 nM to 189 nM for the short aptamer and from 526 nM to 32 nM for the long aptamer. This improvement depends on the true affinity of the binding pair, which ultimately limits the analytical usefulness.

scholarly journals Improving the Analytical Performance of Weak Aptamers: DNA Isothermal Amplification Approaches

Proceedings ◽  
2020 ◽  
Vol 60 (1) ◽  
pp. 26
Author(s):  
Ramón Lorenzo-Gómez ◽  
Rebeca Miranda-Castro ◽  
Noemí de-los-Santos-Álvarez ◽  
María Jesús Lobo-Castañón
Keyword(s):  
Magnetic Beads ◽  
Rolling Circle Amplification ◽  
Binding Constants ◽  
Isothermal Amplification ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tumor Biomarker ◽  
Dna Aptamers ◽  
Rolling Circle ◽  
Neutrophil Gelatinase Associated Lipocalin ◽  
Potential Biomarker

Affinity characterization is an essential but time-consuming task to develop reliable aptamers for tumor biomarker detection and is not always thoroughly addressed. For neutrophil gelatinase-associated lipocalin (NGAL), a potential biomarker of pancreatic cancer, two DNA aptamers were described with very different affinity. Likewise, another pair of DNA aptamers was developed with very different affinity for alpha-fetoprotein (AFP), a biomarker of hepatocellular carcinoma. In this work, we estimated the dissociation constant of these aptamers by means of a direct assay on magnetic beads modified with biomarker and electrochemical detection on screen-printed carbon electrodes. In order to improve the performance of these aptamers, we proposed the isothermal amplification of the aptamers for both biomarkers by rolling circle amplification (RCA). In the case of AFP aptamers, we also tried terminal deoxynucleotidyl transferase (TdT), a template-independent amplification. Both DNA amplifications improved the sensitivity and the apparent binding constants of the aptamers for the two cancer biomarkers. Nevertheless, this improvement depends on the true affinity of the binding pair, which ultimately limits their analytical usefulness.

scholarly journals Rolling Circle Amplification as a Universal Method for the Analysis of a Wide Range of Biological Targets

2021 ◽  
Vol 47 (6) ◽  
pp. 1172-1189
Author(s):  
R. R. Garafutdinov ◽  
A. R. Sakhabutdinova ◽  
A. R. Gilvanov ◽  
A. V. Chemeris
Keyword(s):  
Rolling Circle Amplification ◽  
Universal Method ◽  
Rolling Circle ◽  
Biological Targets ◽  
Wide Range

Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog
Keyword(s):  
Magnetic Nanoparticles ◽  
Ferromagnetic Resonance ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Resonance Field ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Serum Samples ◽  
Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>

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