Influence of citrate buffer and flash heating in enhancing the sensitivity of ratiometric genosensing of Hepatitis C virus using plasmonic gold nanoparticles

Hrishikesh Shashi Prakash,Jayati Ray Dutta,Pranay Amruth Maroju,Naga Sai Sriteja Boppudi,Aniket Balapure,Ramakrishnan Ganesan

doi:10.1186/s40486-021-00134-3

Hrishikesh Shashi Prakash, Jayati Ray Dutta + Show 4 more

Open Access

https://doi.org/10.1186/s40486-021-00134-3

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Abstract

Gold nanoparticles (Au NPs) based technology has been shown to possess enormous potential in the viral nucleic acid diagnosis. Despite significant advancement in this domain, the existing literature reveals the diversity in the conditions employed for hybridization and tagging of thiolated nucleic acid probes over the Au NPs. Here we employ the probe sequence derived from the Hepatitis C virus to identify the optimal hybridization and thiol-Au NP tagging conditions. In a typical polymerase chain reaction, the probes are initially subjected to flash heating at elevated temperatures to obtain efficient annealing. Motivated by this, in the current study, the hybridization between the target and the antisense oligonucleotide (ASO) has been studied at 65 °C with and without employing flash heating at temperatures from 75 to 95 °C. Besides, the efficiency of the thiolated ASO’s tagging over the Au NPs with and without citrate buffer has been explored. The study has revealed the beneficial role of flash heating at 95 °C for efficient hybridization and the presence of citrate buffer for rapid and effective thiol tagging over the Au NPs. The combinatorial effect of these conditions has been found to be advantageous in enhancing the sensitivity of ratiometric genosensing using Au NPs.

Highlights

Owing to the innate plasmonic and electronic properties, gold nanoparticles (Au Gold nanoparticles (NPs)) gained significant attention in several biological applications [1–7]
Further quantification of the bands corresponding to the hybridized Deoxyribonucleic acid (DNA) using ImageJ software (Version 1.8.0_172) revealed a 7.5% higher hybridization efficacy with the sample subjected to a flash heating of 95 °C for 120 s, as opposed to the sample that was not subjected to any flash heating (Fig. 1b)
It was found that the discrimination between the target and control at the ratiometric values was more pronounced when citrate buffer was used. This reveals that the use of citrate buffer is useful in enhancing the conjugation efficiency even when the DNA to Au NPs ratio is as low as 3.5:1, while the available literature reports used a ratio of 20:1 and higher [29]

Summary

Introduction

Owing to the innate plasmonic and electronic properties, gold nanoparticles (Au NPs) gained significant attention in several biological applications [1–7]. When the thiol-tagged DNAs were attempted to conjugate with the surface of citrate-capped Au NPs, due to the chargecharge repulsion, the efficacy of the binding was found to be poor [25, 26]. Salt-aging technique was developed, in which the externally added electrolyte such as NaCl minimizes the charge-charge repulsion between the citrate units as well as with the phosphate moieties of the incoming DNAs and thereby creating adequate void space for the thiolated DNA to approach the surface of Au NPs [27, 28]. The salt-aging process requires 24–48 h for efficient conjugation [19] This has been overcome by strategies like tuning the pH of the Au NPs using citrate buffers, instant dehydration in butanol (INDEBT), etc. The necessity of citrate buffer for the cases with low DNA to Au NPs ratio has so far not been explored

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