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Functional core/shell particles are highly sought after in analytical chemistry, especially in methods suitable for single-particle analysis such as flow cytometry because they allow for facile multiplexed detection of several analytes in a single run. Aiming to develop a powerful bead platform of which the core particle can be doped in a straightforward manner while the shell offers the highest possible sensitivity when functionalized with (bio)chemical binders, polystyrene particles were coated with different kinds of mesoporous silica shells in a convergent growth approach. Mesoporous shells allow us to obtain distinctly higher surface areas in comparison with conventional nonporous shells. While assessing the potential of narrow- as well as wide-pore silicas such as Mobil composition of matter no. 41 (MCM-41) and Santa Barbara amorphous material no. 15 (SBA-15), especially the synthesis of the latter shells that are much more suitable for biomolecule anchoring was optimized by altering the pH and both, the amount and type of the mediator salt. Our studies showed that the best performing material resulted from a synthesis using neutral conditions and MgSO4 as an ionic mediator. The analytical potential of the particles was investigated in flow cytometric DNA assays after their respective functionalization for individual and multiplexed detection of short oligonucleotide strands. These experiments revealed that a two-step modification of the silica surface with amino silane and succinic anhydride prior to coupling of an amino-terminated capture DNA (c-DNA) strand is superior to coupling carboxylic acid-terminated c-DNA to aminated core/shell particles, yielding limits of detection (LOD) down to 5 pM for a hybridization assay, using labeled complementary single-stranded target DNA (t-DNA) 15mers. The potential of the use of the particles in multiplexed analysis was shown with the aid of dye-doped core particles carrying a respective SBA-15 shell. Characteristic genomic sequences of human papillomaviruses (HPV) were chosen as the t-DNA analytes here, since their high relevance as carcinogens and the high number of different pathogens is a relevant model case. The title particles showed a promising performance and allowed us to unequivocally detect the different high- and low-risk HPV types in a single experimental run.
Bioanalytical, diagnostic, and security applications require the fast and sensitive determination of a steadily increasing number of analytes or events in parallel in a broad variety of detection formats.[1,2] Ideal candidates for spectral encoding and multiplexing schemes are luminescent nanocrystals like semiconductor quantum dots (QDs), particularly Cd-containing II/VI QDs with their narrow and symmetric emission bands. With the availability of relatively simple and inexpensive instrumentation for time-resolved fluorescence measurements, similar strategies utilizing the compound-specific parameter fluorescence lifetime or fluorescence decay kinetics become increasingly attractive.[3-5] The potential of different types of QDs like II/VI, III/V and Cd-free ternary QDs such as AgInS (AIS) QDs for lifetime-based encoding and multiplexing has been, however, barely utilized, although the lifetimes of these nanocrystals cover a time windows which is barely accessible with other fluorophores. Here we present a brief insight into the photophysics of AIS QDs and show the potential of dye- and QD-encoded beads for lifetime-based encoding and detection schemes in conjunction with flow cytometry and fluorescence lifetime imaging microscopy