Author: Wang, Shiyao
Title: Development of a universal fluorescent nanoprobe for monitoring isothermal nucleic acid amplification reaction
Advisors: Lee, Thomas (BME)
Yip, Shea Ping (HTI)
Degree: Ph.D.
Year: 2021
Subject: Nucleic acids
Gene amplification
Hong Kong Polytechnic University -- Dissertations
Department: Department of Biomedical Engineering
Pages: xxiv, 108 pages : color illustrations
Language: English
Abstract: Isothermal nucleic acid amplification techniques have received increasing attention in central and clinical laboratories as well as decentralized settings for accurate and ultrasensitive detection of pathogens such as viruses and bacteria. These isothermal techniques have the advantages of simple temperature control and short reaction time compared with the current gold standard of polymerase chain reaction (thermocycling-based amplification of specific target sequence). The ability to monitor the progress of the amplification reaction in real-time, typically by incorporating organic fluorophores into the reaction mixture, eliminates additional detection time and enables quantification over a wide dynamic range. Fluorescent semiconductor nanocrystals (quantum dots) have proved to be promising substitutes for organic fluorophores by virtue of their superior brightness and photostability. Efforts have been made to couple quantum dot fluorescent detection probe with isothermal amplification, however, almost all platforms involved modifying quantum dots with target-specific oligonucleotide sequences (complementary to target/amplified sequences) via special attachment chemistries. Apart from laborious, time-consuming, and costly preparation, different oligonucleotide-modified quantum dot probes are needed for different targets. To date, the development of universal quantum dot probes (i.e., without attachment of oligonucleotide) for monitoring isothermal amplification reactions remains unexplored. In this thesis, 3 types of universal quantum dot probes, including cysteamine-, 2-mercaptoethanesulfonate-, and mercaptoacetic acid-modified quantum dots, were developed for loop-mediated isothermal amplification (LAMP) assays.
We discovered that cysteamine-modified quantum dots (amine modification) were dispersed in a negative LAMP sample (without target DNA sequence) but were precipitated in a positive LAMP sample (with target DNA sequence). We investigated the effects of the LAMP reaction components (both reactants and products) on the dispersion/precipitation of cysteamine-modified quantum dots. The dispersion was attributed to the electrostatic repulsion between cysteamine-modified quantum dots (positively charged; amine modification), while the precipitation was attributed to the entrapment of cysteamine-modified quantum dots within magnesium pyrophosphate crystals (positive LAMP reaction product; electrostatic attraction between positively charged amine and negatively charged pyrophosphate). This assay was highly sensitive (limit of detection of 500 copies using lambda DNA as template; 42 aM with a reaction volume of 20 μL). Besides, the applicability of this assay platform for real sample analysis was demonstrated with avian influenza virus (H9 subtype; reverse transcription LAMP, RT-LAMP, for RNA template). The assay results were consistent with the gold standard test (real-time reverse transcription polymerase chain reaction, RT-qPCR).
We also discovered that sodium 2-mercaptoethanesulfonate-modified quantum dots (sulfonate modification) and mercaptoacetic acid-modified quantum dots (carboxyl modification) were dispersed in a negative LAMP sample (without target DNA sequence) but were precipitated in a positive LAMP sample (with target DNA sequence). Unlike cysteamine-modified quantum dots, these 2 types of quantum dots (sulfonate and carboxyl modifications) were negatively charged. It should be noted that magnesium ion present in the LAMP reaction mixture could complex with sulfonate/carboxyl, causing interparticle aggregation and undesired precipitation.
Nevertheless, the presence of tris(hydroxymethyl)aminomethane and Tween 20 (isothermal reaction buffer components) rendered sulfonate-/carboxyl-modified quantum dots dispersed by means of steric hindrance. On the other hand, the precipitation in the positive LAMP sample was attributed to the entrapment of sulfonate-/carboxyl-modified within magnesium pyrophosphate crystals (positive LAMP reaction product; complexation of sulfonate/carboxyl and pyrophosphate with magnesium ion). Both assay platforms (sulfonate-/carboxyl-modified quantum dots) had a limit of detection of 250 copies (lambda DNA as template; 21 aM with a reaction volume of 20 μL). We further demonstrated the applicability of the 2 assay platforms for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; the causative agent of coronavirus disease 2019, COVID-19; RT-LAMP for RNA template). The assay results were in good agreement with RT-qPCR.
Having the advantages of simple preparation, excellent universality (without oligonucleotide modification of quantum dot; same quantum dot probe for different target sequences) and photostability of these 3 newly developed quantum dot probes, their widespread adoption in LAMP-based nucleic acid testing can make significant contribution to a wide variety of applications such as medical diagnostics, food safety monitoring, and environment surveillance.
Rights: All rights reserved
Access: open access

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