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

Abstract

The development of screening assays continues to be an active area of research in molecular diagnostics. Fluorescent microspheres conjugated to biomarkers (nucleic acids, proteins, lipids, carbohydrates) and analyzed on flow cytometer instruments offered a new approach for multiplexed detection platform in a suspension format. Quantum dots encoded into synthetic microspheres have the potentials to improve current screening bioassays and specifically suspension array technology. In this paper, commercialized quantum dot-encoded microsphere were evaluated and optimized as fluorescent probes to address some of the limitations of suspension array technologies. A comprehensive study was undertaken to adapt the bioconjugation procedure to the quantum dot-encoded microsphere structural and optical properties. Both the leaching-out of quantum dots and microspheres degradation under bioconjugation experimental conditions were minimized. A rapid, efficient and reproducible conjugation method was developed for the detection of single-stranded DNA with the commercialized quantum dot-encoded microsphere. Approximately ten thousand microspheres were conjugated to short amino-modified DNA sequences in one hour with high efficiency. The bioconjugated microspheres acting as fluorescent probes successfully detected a DNA target in suspension with high specificity. Quantum dot-encoded microsphere commercial products are limited which strongly prevents reproducible and comparative studies between laboratories. The method developed here contributes to the understanding of quantum dot-encoded microsphere reactivity, and to the optimization of adapted experimental procedure. This step is essential in the development of this new fluorescent probe technology for multiplex genotyping assay and molecular diagnostic applications.

Polystyrene Microparticles with Convergently Grown Mesoporous Silica Shells as a Promising Tool for Multiplexed Bioanalytical Assays

“…Regarding sequence specificity, control experiments with single, double, and quadruple base mismatches revealed that DNA-S4-PS40 showed a comparable performance as has been reported before in the literature, only determined by the nucleic acid hybridization behavior based on pairing vs mismatching (Section 4.2, including Table S6 and Figure S11, Supporting Information). ,, …”

“…Such an effect thus has a more pronounced impact at lower concentrations. Overall, the multiplexed assays were successful in proving that this type of doped polymer core/functionalized mesoporous shell particles can be used as a powerful tool in simple single-particle assay formats, competing well with reported approaches, ,, which usually rely on larger beads (3–6 μm) and utilize molecular beacons or longer strands, while offering higher simplicity, versatility, and modularity. The use of smaller beads such as those reported here entails faster reaction kinetics and allows for small reaction volumes, increasing throughput, and being more economical.…”

Polystyrene Core–Silica Shell Particles with Defined Nanoarchitectures as a Versatile Platform for Suspension Array Technology

“…57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 28 encoded microspheres and determined similar detection limits but had to perform several washing steps after hybridization. 58 In both cases, polymer beads with diameters larger than 3 µm were used as spherical substrates. In our direct experiment avoiding time-consuming washing steps by a simple and fast Mix-and-Read assay, we were able to detect <1 fmol, i.e., ca.…”

RNA and DNA Diagnostics

“…For example, the magnetic barcode encoding of microspheres provides a platform for rapid Mycobacterium tuberculosis nucleic acid detection out of clinical samples (Liong et al 2013). Multiplex improvements are addressed by combining quantum-dot encoding and microspheres as novel optical encoding strategy Thiollet et al 2012). QD is also emerging in the area of "encoding hybrids", for example, the QD-and gold nanoparticle-encoded polystyrene microspheres (Quach et al 2011).…”