They are comprised of a porous matrix that provides a surface for target binding and the space for the actual amplification and detection reaction. Thus a single Bead becomes the tool for a complete analysis workflow, including nucleic acid extraction and purification, target amplification and detection.
Beads equipped with different primers and probes can be combined to form multiplex panels. Each Bead remains an isolated nanoreactor throughout the amplification and detection process, becoming a digital compartment.
Each BLINK Bead forms an isolated reaction compartment (similar to droplets or nano-wells) for performing thousands of independent enzymatic reactions separated from each other.
The PCR amplification on the Beads is a variant of digital PCR, which is based on partitioning the sample into thousands of independent PCR reactions. The BLINK approach utilizes the Beads to provide the partitioning mechanism. This process randomly distributes the target molecules amongst the partitions provided by the Beads.
BLINK Bead nanoreactors integrate nucleic acid extraction, purification, amplification and detection into a single workflow in each Bead compartment.
For nucleic acid capture Beads are equipped with a binding polymer, enabling continuous workflows from sample to result on one and the same carrier. Since the complete captured material is used in the analysis, more precise and sensitive results are achieved. Additionally, workflow complexity is reduced.
All steps from sample lysis to result readout are carried out on one and the same carrier, the BLINK Bead. All material is amplified in a minimised volume distributed across thousands of individual compartments which provide for optimal thermal control, enabling fast cycling on BLINK’s hardware. BLINK’s design achieves accelerated workflows from sample to result.
The accuracy of the quantification is defined using Poisson’s statistics, which models the random distribution of the target into the partitions. However, it is also possible to monitor amplification on Beads in real time. This provides for quantification at high target concentrations when digital amplification becomes unfeasible.
Highly efficient amplification in digital nano-compartments provides for single molecule sensitivity and exquisite quantification. By analysing each partition for the presence (positive reaction) or absence (negative reaction) of a fluorescent signal the absolute number of molecules present in the sample is calculated.
By combining digital and real time analysis it is possible to build assays with an ultra-wide measurement range without sample dilution.
Primers and Probes are reversibly bound to the encoded Beads so that different Beads can be used for sample prep. Once accommodated in oil, the primers and probes are released for amplification and detection. Since each reaction is carried out in an individual compartment, Beads, and as such assays, with different target specificities can be safely combined in a single workflow without cross interference.
Melting curve analysis using intercalating dyes or molecular beacons on BLINK nanoreactor Beads allows for easy SNP detection and quantitation of SNPs or other genetic variants in a digital format.
BLINK Beads can be customized for specific applications with different binding specificities, primers and probes.
BLINK Beads comprise a hydrogel matrix that defines the actual space for amplification and detection. Each Bead is encoded with a fluorescent code and carries magnetic particles for ease of handling. Moreover, the matrix is derivatized with streptavidin providing a simple means for reversibly attaching primer and probe oligonucleotides to the matrix. The hydrogel matrix is coated with a crosslinked polymer for efficient binding of nucleic acids.
In a suitable binding buffer nucleic acids contained in a sample bind to the Bead surface.
A wash step removes un-specifically bound materials leaving purified nucleic acid bound to the Bead.
Beads are loaded with generic amplification & detection reagents (enzymes, buffers, dNTPs).
Beads are suspended in oil
Hydrogel core liquifies at elevated temperature and forms a droplet. Primers and probes are released. Enzymes are activated. PCR begins. All code and magnetic particles are confined within a space formed by the contracting binding layer, thus clearing up the nanoreactor space for amplification and fluorescence detection.
Target specific amplification is performed within the nanoreactor space formed by the Bead.
Due to spatial separation fluorescence signal formed by the amplification reaction can be read simultaneous with the fluorescent Bead code by fluorescence imaging.
Sensitivity and exquisite target quantification.
Beads encoded with a dye for bead segmentation can be used with any primers/probes to design and perform a digital assay with exquisite quantification. When used on the BLINK X platform, digital PCR can be performed in under 15 minutes.
Quantitative, ultra-sensitive multiplexing.
Ultraplexing is highly flexible, extensive detection of multiple targets from a sample with single molecule sensitivity. This process allows each panel member to be quantitated without cross-interference. Beads with different fluorescence codes are equipped with analyte specific reagents and used in parallel.
Each Bead is an individual reaction compartment that carries out the detection reaction without cross-interference by other reagents. This means that different pre-made assays can be combined into test panels without further optimisation and re-validation. Moreover, new markers can be added seamlessly to existing test panels.
Multiple sample parallel processing
Sample Multiplexing involves selective encoding of individual samples with encoded beads, allowing for parallel processing of multiple samples in one test assay. Once the targets are bound to the Beads the detection reaction is carried out simultaneously on all samples in the respective reaction compartments provided by the Beads.
Currently BLINK’s technologies and products are accessible through strategic collaborations with BLINK as a development partner in selected application areas. Test developers and researchers will be able to develop their assays and applications independently on the BLINK X platform utilising validated components while maintaining design flexibility.