Facile surface functionalization of gold nanoparticles and quantum dots for precipitation-based real-time monitoring of isothermal DNA amplification

Abstract

Deoxyribonucleic acid (DNA) testing is currently one of the most powerful methods for a wide range of applications, including medical diagnostics, food safety testing, and environmental surveillance. The gold standard technique is real-time polymerase chain reaction, which features simultaneous amplification and detection. These assays are carried out almost exclusively in resource-rich central laboratories due to the use of sophisticated, bulky, and expensive instrument. Besides, sample transportation to and queuing in central laboratories delay treatment/response decision. Hence, there is a huge demand for achieving decentralized (point-of-care/field) testing with new assay platform that is simple, portable, and low cost. For amplification, isothermal technique is a promising alternative to thermocycling-based polymerase chain reaction. For detection, nanomaterials have proved to be superior to traditional reporter molecules (organic fluorophores). Nevertheless, simultaneous isothermal amplification and nanomaterial-based detection has not been demonstrated to date. The main challenges are that previously reported nanomaterial probes (functionalized with oligonucleotides) were not compatible with amplification reactions, resulting in a loss of detection property of the nanomaterial probes and/or enzyme inhibition. In this thesis, two real-time loop-mediated isothermal amplification (real-time LAMP) assay platforms were developed based on gold nanoparticles (AuNPs) and quantum dots (QDs) with facile surface functionalization (not with oligonucleotides). The first assay platform with AuNPs enabled visual readout of the LAMP reaction. LAMP operates at a constant temperature of 60-65 °C, generating a billion copies of a specific DNA sequence within 1 hour. Another favorable feature of LAMP is the release of pyrophosphate ion (P₂O₇ ⁴⁻) as a reaction byproduct, which complexes with magnesium ion (Mg²⁺, enzyme cofactor) to form a precipitate (but visible to the naked eye only under special lighting conditions). It was found that AuNPs co-modified with thiolated poly(ethylene glycol) and 11-mercaptoundecanoic acid (PEG/MUA-AuNPs) were dispersed as a red solution in Mg²⁺ but appeared as a red precipitate in Mg₂P₂O₇. Steric hindrance by PEG effectively stabilized the particles against Mg²⁺-induced aggregation (complexation of MUA's carboxyl group with free Mg²⁺). On the other hand, PEG did not hinder the binding between PEG/MUA-AuNPs and Mg₂P₂O₇ crystals. When PEG/MUA-AuNPs were incorporated into a LAMP reaction mixture, as expected, a negative sample (without target DNA) appeared as a red dispersion whereas a positive sample (with target DNA) appeared as a red precipitate. This assay scheme could detect down to 500 copies of target DNA (~40 aM; reaction volume of 20 μL; the most sensitive among all the reported AuNP-based colorimetric DNA detection platforms). It should be emphasized that PEG/MUA-AuNPs did not have any inhibition effect on LAMP. Real-time LAMP was performed with a homemade simple, palm-sized, low-cost prototype device (temperature control and 520 nm transmittance measurement of the supernatant). The second assay platform with QDs enabled fluorescence readout of the LAMP reaction. It was hypothesized that the dispersion/precipitation behavior of PEG/MUA-AuNPs in LAMP could be observed with other types of nanomaterials. Remarkably, CdSeS/ZnS QDs modified with cysteamine (Cys-QDs) exhibited similar dispersion/precipitation behavior, i.e., dispersion in Mg²⁺ and precipitation in Mg₂P₂O₇. The binding mechanism underlying the precipitation phenomenon was found to be electrostatic in nature. Specifically, cysteamine's positively charged amine group was bound to negatively charged P₂O₇ ⁴⁻, thereby entrapping Cys-QDs inside Mg₂P₂O₇ crystals. When Cys-QDs were incorporated into a LAMP reaction mixture, a negative sample (without target DNA) exhibited uniform fluorescence throughout the solution whereas a positive sample (with target DNA) exhibited fluorescence only at the bottom of the solution (supernatant was clear). This assay scheme could detect down to 200 copies of target DNA. To conclude, the two assay platforms developed in this thesis possessed the advantages of high sensitivity and specificity, simplicity, short turnaround time, worry-free carryover contamination control, and low cost. Therefore, they are readily applicable to numerous applications such as infectious disease detection. Effort is being made to validate the new platforms for avian influenza detection in field samples.