A precipitation-based universal detection platform for DNA amplification reaction using bovine serum albumin-modified gold nanoparticles and reduced graphene oxide

xx, 90 pages : color illustrationsPolyU Library Call No.: [THS] LG51 .H577M BME 2017 WangThe detection of nucleic acid plays an increasingly significant role in various fields, including medical diagnosis, food safety testing, and biowarfare agent detection. The gold standard technique is real-time polymerase chain reaction, which involves fluorescent signal readout. Due to the use of the complicated and expensive instrument, these assays are performed in central laboratories. Toward decentralized testing (point-of-care/field testing), which can provide timely treatment/response decision, a simple signal readout is essential. Gold nanoparticles (AuNPs) have been intensively investigated for DNA detection by virtue of their result readout by the naked eye. Typically, these assays involve the use of two sets of oligonucleotide-modified gold nanoparticles (oligo-AuNPs) (each being complementary to half of a target sequence), by which target recognition and signal readout are coupled. In the absence of the target, oligo-AuNPs are dispersed and the solution appears red in color, while in the presence of the target, oligo-AuNPs are cross-linked/aggregated and the solution turns purple. However, it should be noted that oligo-AuNPs are prepared using thiol-modified oligonucleotides, which is expensive. Besides, the red-to-purple color change can sometimes be difficult to differentiate by the naked eye. In this thesis, a new type of AuNPs (bovine serum albumin-modified AuNPs, BSA-AuNPs; responsible for signal readout) together with reduced graphene oxide (rGO; responsible for target recognition) are utilized for the detection of a target DNA sequence based on the precipitation-based readout. In the absence of the target, single-stranded oligonucleotide probe binds with rGO via π-stacking interaction and stabilizes rGO from salt-induced aggregation. Upon the addition of BSA-AuNPs, a red dispersion is obtained. While in the presence of the target, the double-stranded probe-target hybrid does not bind with rGO. Upon the addition of salt followed by BSA-AuNPs, a red precipitate is obtained (co-precipitation of BSA-AuNPs with the aggregated rGO). The limit of detection for oligonucleotide target was 5 nM. Moreover, this assay scheme is readily applicable to polymerase chain reaction (PCR) and helicase-dependent amplification (HDA) product detection, thereby achieving the limits of detection of 83 fM and 21 aM, respectively. Taken together, this new assay platform has the advantages of facile and low-cost preparation (BSA-AuNPs versus oligo-AuNPs), simple result readout, and ultrasensitive detection. It is well suited for decentralized testing and, to the best of our knowledge, is the first attempt of utilizing the co-precipitation of BSA-AuNPs with rGO for nucleic acid detection.Department of Biomedical EngineeringM.Phil., Department of Biomedical Engineering, The Hong Kong Polytechnic University, 2017MPhi