The BLINK approach to multiplexing is unique. Rather than inventing more fluorescent channels, we imbue our BLINK Beads, which serve as both carrier of reagents and the reaction volume, with the multiplexing functionality. This design means our approach is not limited to the development of further sets of fluorescent channels, fluorophores and quenchers and also addresses the disadvantages of channel multiplexing, namely cross-interference and elaborate assay development.
Our Beads carry a color-code, which consists of various fluorescent particles integrated into the bead (core code) and a background staining of the hydrogel (shell code) assigning beads to their respective subgroup.
Using coded beads increases the level of multiplexing. Additionally, upon thermocycling each individual bead results in an independent reaction volume eliminating cross-interference.
There are several approaches to multiplexing – 1) Sample multiplexing and 2) Target multiplexing with panels.
Sample Multiplexing involves selective encoding of individual samples with encoded beads, allowing for parallel processing of multiple samples in one test assay. For this, the sample’s nucleic acids are brought into contact with a subgroup of beads having a certain color-code. This is done separately for each sample with a respective code, followed by washing and loading with PCR reagents. As soon as the beads are compartmentalised within the PCR oil, each bead represents an independent reaction and the beads of different samples can be pooled for simultaneous amplification reaction and eventual detection. A big advantage of this way of multiplexing (besides the higher throughput per run) is that in case of positive reaction there is no need to resolve the pool and test all samples individually again as by the bead code positive reactions can be assigned to the affected sample(s).
Ultraplexing is quantitative, ultra-sensitive target multiplexing. It is the ability to interrogate a sample for several analytes, a process whereby Beads are encoded with different fluorescent codes being pre-coupled with specific primer pairs (and if applicable, probes) and used in parallel. This enables you to build panels; for example you could couple a specific primer/probe set to blue for influenza, green for RSV and red for coronavirus etc., building a useful respiratory panel. As each bead subgroup is only having one primer pair immobilised to it and hence detects only for one target in its independent reaction volume (bead in oil), this results in a cross interference-free reaction. Given a similar thermoprofile, reactions for different targets can be easily combined in a plug and play manner in one assay. Unlike assays on other platforms, replacement of certain targets does not need cumbersome re-evaluation of the remaining primer pairs as they are not interfering with each other by design. Additionally, this method also allows multiplexing with intercalating dyes as primers can be immobilised and assigned to certain color-coded bead types.
All types of multiplexing can be used for classical digital PCR, real-time PCR or Melting Curve Analysis.
Taken together BLINK Beads allow completely new approaches for multiplexing with a drastic increase of multiplexing grade while minimising the effort in building panels.