BLINK Beads are powering next generation molecular assay design, providing a simplified, robust, rapid and undisrupted sample-to-answer workflow with dramatically improved sample utilization and assay performance.
The Beads are magnetic fluorescence encoded nanoreactors equipped with target specific reagents that facilitate digital multiplex assays. They are comprised of a porous matrix that provides a surface for random distribution and binding of targets and the compartment for an amplification and detection reaction. Beads enable complex analysis that traditionally require multiple separate workflows and assays to be performed with a single test. PCR reactions can be performed within 10 minutes on a simple laboratory instrument, the BLINK X, while the multiplexing capabilities enable more than 50 assays to be performed in parallel on one sample. Importantly, BLINK Beads are still compatible with existent assays and device platforms.
Compartmentalisation is an integral component of any Bead workflow, so each assay can be performed in a digital format. With BLINK Beads, digital PCR becomes a tool that is as accessible and easy-to-use as qPCR.Bead-based dPCR assays are microfluidics-free. Sample compartmentalization is provided by pre-made hydrogel beads and does not require microfluidic droplet generators or expensive nano-well plates with precise amplification volumes.
The performance charactersitics of the Beads, namely single molecule sensitivity and exquisite quantification, are ideally suited to applications such as:
Copy number variation
Rare mutation detection
Gene expression and miRNA analysis
GMO detection
NGS validation and preparation
Water monitoring
Pathogen detection
Each Bead features a functionalised surface for target binding, embedded magnetic nanoparticles for simplified handling, a distinct fluorescent code to serve as an individual address for the assay specific reagents (primers & probes) that are also accommodated on the Bead.
Fluorescence-encoded Beads equipped with different primers and probes can be combined to form complex digital multiplex panels. Compartmentalisation facilitated by the Bead design results in multiplex assays that are free from any cross-interference.
BLINK Beads can be easily loaded with analyte/target specific reagents. These different pre-made Bead assays can be combined freely into complex digital test panels. A variety of digital PCR formats are facilitated with this approach:
Simple dPCR
Ultraplexing (digital Target Multiplexing)
Digital Sample Multiplexing
In addition, Beads allow melt curve analysis post-PCR and real-time PCR analysis for samples with high target load.
Sensitivity and exquisite target quantification.
BLINK Beads encoded with a dye for automatic Bead recognition 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 target multiplexing.
Ultraplexing is highly flexible, extensive detection and quantification 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 amplification and detection reaction without cross-interference by reagents accommodated on different Beads. 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. The digital nature of the assay enables quantification for each sample in the pool. Repeat testing is not required if a sample in a pool is positive.
We have combined sample nucleic acid extraction, multiplex analysis and quantification by digital PCR in a single compact workflow. Complex analysis that traditionally required multiple separate workflows and assays can now be done with a single test.
We improve sample utilization by including all targets captured from a sample into the amplification and detection process, massively improving assay sensitivity and precision.
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. Rare targets are not missed and precisely counted. 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. Our product features self-arrangement of nanoreactors in a monolayer. This process is fast, robust and provides for quick thermocycling, allowing PCR reactions within 10 minutes. BLINK’s design achieves accelerated workflows from sample to result.
For detection and optimal temperature control, our product features self-arrangement of nanoreactors in a monolayer. This process is fast, robust and provides for quick thermocycling, allowing PCR reactions within 10 minutes. The monolayer arrangement simplifies imaging and even enables advanced features such as real-time imaging (qPCR) and melt curve analysis.
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 which is based on binding rather than volume distribution as in conventional digital partitioning. This process randomly distributes the target molecules amongst the partitions provided by the Beads, removing the need for special, disposable microfluidics components.
A unique feature of our approach is the spatial separation of individual PCR reactions in the respective nanoreactors by using pre-coupled primer/probe combinations customised for subsets of Beads. This prevents non-specific amplification by cross-interaction of primer sets and allows massive multiplexing.
The accuracy of the dPCR 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.
Beads are extremely flexible and can be utilised for a variety of analyses. (expand on this introduction)
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Analytes in a known volume of sample are randomly distributed across a known number of Bead nanoreactors, which are used as individual amplification and detection compartments for dPCR.
Beads are incubated with the sample under binding conditions (right binding buffer mixed with sample). Following a wash step to remove any non-specifically bound materials, the Beads are loaded with generic PCR reagents and encapsulated in an oil, separating the Beads and preventing any liquid exchange between Beads. Thereafter the Beads are arranged in a plate designed for exquisite temperature control and fluorescence detection. Simple Bead handling is facilitated by the magnetic nature of the Beads. Following thermocycling and fluorescence imaging a simple algorithm is applied to quantitate the targets present in the sample.
Compartmentalisation is achieved without the need for sophisticated disposable plastics or microfluidics. In other digital formats, the achievable precision of the assay is determined by the mean volume of the compartments; for Bead assays only the number of Beads interrogated with a known volume of sample is relevant. Since targets are bound to the Beads, different sample volumes can be used for digital analysis.
Using Beads for dPCR lowers both the cost and the complexity of the process. Additional sample preparation kits are not needed for “sample-to-answer” assays.
Beads can be equipped with assay specific reagents so that only generic reagents need to be added.
The entire sample volume can be used for analysis, increasing both the sensitivity & precision of the assay.
During PCR, amplification can be monitored by real time fluorescence detection for each Bead, thus each Bead becomes an individual qPCR assay. This provides for quantification by Ct analysis when the digital measurement range is exceeded due to high target concentration in the sample. By combining digital and real-time analysis, an unprecedented measurement of >8 logs range is facilitated, while sample can be quantitated without any diluting steps. Ct analysis also provides for trouble shooting during assay development as the Ct value is a great indicator for sample purity.
Real-time monitoring of Bead-specific fluorescence also provides for melt curve analysis. A Tm value can be assigned to every individual amplification product formed on the Beads and used for specificity analysis. This provides a straightforward technical approach for massively parallel, rapid mutation analysis and assuring specificity for rare or even single amplification events.
Since the assays are built on magnetic nanoreactors, the disposables used for the assays are simple and cost-effective; integration on automated laboratory tools or transfer to point-of-care platforms is straightforward.
BLINK Beads can be customized for specific applications with different primers and probes. Individual Beads quipped with a unique fluorescent code can be loaded with assay specific primers and probes. Following a simple loading procedure the Beads can be equipped with a defined concentration of reagents and individually tested for performance. Then the validated assays can be mixed and processed as panel assay. Individual assays can be provided as freeze-dried lyophilized pellets with a pre-defined number of Beads carrying either one or several different assays.
Beads optimized for nucleic acid binding and purification and subsequent target amplification and detection.
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.
