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Suspension arrays play a major role in recent developments of multiplexed proteomic and genomic assays. Using for instance cytometry, this microparticle-based analytical method features several advantages compared with flat array technologies such as facile fabrication of the analytical platform, high analytical surface area, fast reaction kinetics and a high analytical throughput.
Since the first immunoassay for soluble antigens employing flow cytometry in 1982 by Lisi et al., enormous improvements were made concerning the assay chemistry and multiplexing capabilities of this technique. More recently, bead-based applications in microfluidic devices were studied which show promising abilities in point-of-need testing technologies.
However, for optimum performance highly mono-disperse and low-density particles are required to guarantee proper correlation and to prevent sedimentation. Usually polymeric particles, such as polystyrene and PMMA spheres, fulfill these requirements and offer good accessibility in conventionally equipped laboratories. Major limitations of these beads however reside with the possibilities for post-modification of the surface and the sensitivity of the polymeric network to the presence of (traces of) organic solvents. A way to circumvent these issues while retaining the key requirements on applications in suspension arrays is the coating of the polymer particles with a silica shell. This strategy not only increases the thermal and chemical stability but facilitates further tailoring of the surface properties with a large variety of commercially available silanes.
This contribution presents a facile synthetic approach to highly mono-disperse polystyrene@silica core-shell particles, bearing simple means to control the size of the particles and the silica shell thickness. Main parameters such as surface area and shell structure are discussed in correlation to analytical benefits of the particles. For the latter, standard flow cytometry applications with (bio)analytical suspension assays are performed.
Multiplexed bead-based array formats play an increasingly important role in analytical laboratories. Due to the high surface-to-volume ratio, fast reaction kinetics and modular assay design, these sensor formats are applied in clinical diagnostic, drug development and classical biosensors with great success.
As the spherical platform, researchers utilize micron sized particles made from polymeric or silica material. Such beads are commercially available from vendors such as BD or Luminex. However, we have encountered several problematic issues which accompany these platforms: first, bigger sized beads, which are required for particle handling reasons, are difficult to prepare with high monodispersity - a key requirement for cytometric application. Second, plain beads, made from either polymer or silica, have each several disadvantages such as inferior scattering properties in case of silica or limited flexibility for coupling strategies in the case of latex beads.
In order to overcome this problem, we have developed a versatile core-shell (CS) platform which consists of a polymeric core with a structurally controlled silica shell. In our approach, the core building block can be easily prepared with high yields and high monodispersity in a dispersion polymerization from approximately 500 nm to 1.3 µm. Then, silica is coated in a classical sol-gel process to protect the core with a stable yet modifiable surface (see SEM image in Figure 1, platform). Here, we combine ideal scattering properties and easy preparation of the polymeric core with the chemical flexibility of a silica surface. Moreover, the additional shell domain adds density to the composite, which makes particle handling feasible also for nanometer sized beads.
In this contribution we present proof-of-principle results of competitive immunoassays with fluorescence detection using our CS beads – each performed in mix-and-read fashion without washing steps. All sizes are applicable in cytometric read-out which and can be used for size encoding (see Set 1 to 3 in Figure 1, size encoding). However, further multiplexing for a set of at least 20 parameters can be achieved by swelling hydrophobic dyes into the core. To the same time, precise tuning of the surface with mixed silane layers allowed us to improve the selectivity towards small molecules in competitive immunoassays significantly (see example in Figure 1, Application). We believe that our platform allows researchers to gain access to superior assay performance in combination with a low-threshold approach for the synthesis of the spherical platform.
Suspension arrays play an important role in recent developments of multiplexed proteomic and genomic assays. Using cytometry, this microparticle-based analytical method features several advantages compared with flat array technologies such as facile fabrication of the analytical platform, high analytical surface area, fast reaction kinetics and a high analytical throughput.
Since the first immunoassay for soluble antigens employing flow cytometry in 1982 by Lisi et al., enormous improvements were made concerning the assay chemistry and multiplexing capabilities of this technique. Recently, bead-based applications in microfluidic devices were studied which show promising abilities in point-of-need technologies.
However, for adequate performance highly monodisperse and low density particles are required to guarantee proper correlation and to prevent sedimentation. Usually polymeric particles, such as polystyrene and PMMA spheres, fulfill these requirements and offer good accessibility in standard equipped laboratories. Major limitations here are rather restricted possibilities for post-modifications of the surface and sensitivity to organic solvents. A way to circumvent these issues, while retaining the key requirements for suspension array applications, is silica coating of polymeric particles. Not only thermal and chemical stability is increased, but further modifications of a well-defined silica surface with a huge variety of commercial available silanes are accessible. To the same time, the polymeric core can be modified with additional functions, such as magnetic properties or optical identification tags.
This contribution presents a synthetic approach to prepare SiO2@PS core-shell particles with architectures ranging from smooth to raspberry and core-satellite assemblies. We use flow cytometry to characterize the shell thickness, monodispersity and available surface area of the particles. Approaches toward multiplexing strategies and single particle analytics using ratiometric signaling via FRET show the great potential of these hybrid composites to address future challenges in SATs.