Application of multiple response optimization design to quantum dot-encoded microsphere bioconjugates hybridization assay

Abstract

The optimization of DNA hybridization for genotyping assays is a complex experimental problem that depends on multiple factors such as assay formats, fluorescent probes, target sequence, experimental conditions, and data analysis. Quantum dot-doped particle bioconjugates have been previously described as fluorescent probes to identify single nucleotide polymorphisms even though this advanced fluorescent material has shown structural instability in aqueous environments. To achieve the optimization of DNA hybridization to quantum dot-doped particle bioconjugates in suspension while maximizing the stability of the probe materials, a nonsequential optimization approach was evaluated. The design of experiment with response surface methodology and multiple optimization response was used to maximize the recovery of fluorescent probe at the end of the assay simultaneously with the optimization of target-probe binding. Hybridization efficiency was evaluated by the attachment of fluorescent oligonucleotides to the fluorescent probe through continuous flow cytometry detection. Optimal conditions were predicted with the model and tested for the identification of single nucleotide polymorphisms. The design of experiment has been shown to significantly improve biochemistry and biotechnology optimization processes. Here we demonstrate the potential of this statistical approach to facilitate the optimization of experimental protocol that involves material science and molecular biology.

Investigation and Development of Quantum Dot-Encoded Microsphere Bioconjugates for DNA Detection by Flow Cytometry

“…Following the development of stable QDEMbioconjugates presented here, an allelic suspension oligonucleotide hybridization assay adapted to commercialized QDEMs was developed. A novel optimization approach was used that simultaneously maximize hybridization efficiency and the stability of the materials [42].…”

Integrated Quantum Dot Barcode Smartphone Optical Device for Wireless Multiplexed Diagnosis of Infected Patients

“…In the previous chapter we demonstrated quantum dot barcodes' advantages over that of traditional organic fluorophore particles' for point-of-care multiplex detection. Opticallyencoded microbeadsinfused with quantum dots or otherwisein literature have so far been used in conjunction with flow cytometers 29,50,51,115 , microfluidic chips 76,[116][117][118] , or optical fiber systems 23,24 . While many have become established laboratory practices, these techniques are not suitable for diagnostic applications in resource-limited settings.…”

Cytotoxicity and immune response of CdSe/ZnS Quantum dots towards a murine macrophage cell line

“…To date, QDs have been widely used in applications ranging from medicine to material science due to their unique tunable optical properties. [15][16][17][18] However, with the continuous growth of QD research and applications, it is of paramount importance for us to understand their potential toxicity in vitro and in vivo. 7 To understand some aspects of this issue, the present study investigated the effects of the commercially available and frequently used carboxyl CdSe/ZnS QDs on the viability and function of the macrophages.…”