Pronostics - Researching the next generation of medical diagnostics

UltraPlex™ - Multiplexing Just Went Digital!

UltraPlex™ is a completely automated multiplexing solution composed of barcoded microparticles, UltraCodes, an automated reader system, the UltraPlex™ SmartReader, and an integrated fluid handling robotic system, the UltraPlex™ SmartStation. UltraPlex™ is ideal for a wide range of applications, from ELISAs to multiplex PCR profiling to high throughput screens. The possibilities are endless because almost any kind of assay can be readily ported to the barcoded microparticles at the heart of the UltraPlex™ system.

UltraPlex™, which utilises barcoded microparticles to tag and track from tens to thousands of analytes in a single tube, enables large numbers of biological assays to be undertaken simultaneously, rather than being performed one at a time. Key benefits are, smaller test sample required, more information from each test sample, quicker diagnosis and decreased labour and reagent requirements. Because UltraPlex™ generates a solution based, particle array, it provides the best environment for protein assays used in IVD, drug discovery and proteomics.

UltraCodes

At the heart of UltraPlex™ solutions are UltraCodes, microscopic aluminium particles, each etched with a unique barcode. Each microparticle can act as the substrate for a different assay, reaction or test. Collections of different Ultracodes can be mixed together, the assays performed in parallel in a single vessel, and then be rapidly decoded by the UltraPlex™ SmartReader. UltraCodes are manufactured using well proven, conventional, semiconductor fabrication techniques ensuring extremely high and consistent quality, ease of availability and economic cost, all thanks to the many years of experience and substantial economies of scale associated with the micro-electronics industry.

Unlike dye-bead multiplex systems (where the ‘codes’ set by the ratio of fluorescent dyes within the particle are effectively continuous), barcoding brings all the benefits of digital signal quality: in the rare event of a ‘mis-read’ an ‘unrecognised’ code is noted. The result is that mis-identification of a test particle is effectively reduced to zero, eliminating one of the major limitations of dyebead multiplexing. Similarly, because the code is physically etched through the microparticle, it is permanent and cannot bleach like dye-coded beads. Multiplexing just went digital! Each standard microparticle (inset) is only 0.1mm in length.

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Fig. 1. Reading the binary code - "UltraCode Secure" microparticle

The UltraCodes come in a number of designs, each optimised for different purposes. For many applications, the 5-digit UltraCode Secure microparticle is ideal, offering essentially error-free decoding. Alternatively, where high particle densities are required (such as in high-multiplex applications), the smaller, 2-digit UltraCode Lite, microparticles accelerate reading times, while keeping mis-identification to an acceptable minimum.

One of the major advantages of UltraPlex™ is its facility to enable to high-multiplex (or ‘megaplex’) applications. UltraCode Secure microparticles are available in 100,000 different codes. Other microparticle designs allow even more codes, and Pronostics can readily create new custom microparticle designs optimised for particular applications.

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Fig. 2. shows five different "UltraCode Secure" microparticles each with a unique code

Pronostics have developed a portfolio of attachment chemistries which coat the microparticles to create substrates optimised for different protein binding. Particles treated with our UltraCode Standard Coat have a similar protein binding capacity to cell culture grade plastic, such as that commonly used in ELISA wells (~1µg/cm2). However, custom surfaces, optimised for attachment of other molecules, have been developed and are available for use if appropriate.

A major advantage of the aluminium substrate, used for Ultracodes, is its robust physical properties. UltraCodes™ survive extremes of pH, ionic strength, organic solvents and high temperatures ensuring their compatibility with the widest possible range of analytical methodology. UltraCodes can be stored for at least 2 years at -20ºC, and survive multiple freeze-thaw cycles.

UltraPlex™ Assays

The UltraPlex™ system has already been used to multiplex a wide range of assays which demonstrate its broad applicability. The most advanced applications are in direct immunoassay, where Pronostics has already developed and is currently launching a series of products directed at clinical immunology laboratories. These products clearly demonstrate that UltraPlex™ assays yield sensitivities and specificities at least as good as conventional “one analyte at a time” ELISAs.

To screen a test sample for multiple specific antibodies, the known antigens are separately tagged with different UltraCodes, then mixed together and reacted with the test sample (Figure 3 below, shows an example of 3 multiplexed antigens). If the specific antibodies are present in the sample, they will bind to their respective barcoded antigens. Fluorescent reporter antibodies are used to detect a positive reaction between the sample and the barcoded screening antigens, and result in a fluorescent microparticle (Figure 3 blue and pink are positive results). Barcoded antigens for which no corresponding antibody is present in the sample will not fluoresce (red shows a negative result). This form of assay can be extended across a whole range of tests and sectors, from disease screening, allergy testing, and biomarker analysis.

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Fig. 3. Multiplexed experiment using 3 antigens
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Pronostics continue to develop new products based on direct immunoassays, ranging from allergen-specific IgE assays, to a unique class of profiling diagnostics which use a 64-plex profile of the immune system to diagnose coronary heart disease.

Sandwich ELISAs can be ported to UltraPlex™ as readily as direct immunoassays: for example, a panel of cytokine assays yielded sensitivities around 10pg/ml comparable to conventional competing products.

Applications are not limited to immunoassays. We have demonstrated that biotinylated oligonucleotides can be readily coupled to streptavidin-coated UltraCodes™, and then be specifically hybridized with labelled probes, opening up a wide range of potential applications in multiplex genetic testing.

Similarly, active enzymes (such as proteases), receptors or even live cells can be immobilised on UltraCodes facilitating multiplex high throughput screening approaches where a library can simultaneously be tested for activity against a wide range of targets.

Automated Reading - the UltraPlex™ SmartReader

UltraCodes can be used as the substrate for almost any assay format that yields a fluorescent output. In principle, it is possible to decode the microparticles and simultaneously measure the fluorescent output using conventional laboratory equipment (such as a fluorescence microscope or flow cytometer). Without dedicated decoding software, however, such manual reading is likely to be impractically slow. As a result, Pronostics have developed a dedicated reader, the UltraPlex™ SmartReader, running Pronostics' SmartDecode software, which is fully compatible with virtually all LIM systems.

The UltraPlex™ SmartReader is a self-contained unit, occupying approximately 0.3m2 of bench space, combined with a dedicated compact PC workstation. Although based around a fluorescent microscope, the unit accepts standard 96-well plates and yields up to 27,000 data points per hour at the press of a single button. Each well is scanned in turn and the microparticles are decoded from a phase-contrast image. Simultaneously, the output of the assay is quantified from a fluorescent image of the same field of view. At least 10 particles of each code are measured in each well, and the mean fluorescence reported.

SmartReader is configured with high quality optics and alternative filter sets to read commonly used fluorescent reporters. Our next generation reader, now under development, will allow simultaneous measurement of multiple dyes further expanding UltraPlex™ multiplexing capabilites.

The reader's software, SmartDecode, already outputs the data matrix (which can contain more than 1,000 test results for a 16-plex assay) in a format compatible with all major LIM systems, or in Excel format for manual analysis, and custom output formats can readily be added.

The UltraPlex™ SmartReader is the ideal solution for most medium throughput applications. However, Pronostics has carried out proof-of-principle experiments decoding UltraPlex™ assays on collaborative partners high-end microscope based reader systems, and have shown that data accrual rates 10-fold higher than with UltraPlex™ SmartReader can readily be achieved. Similarly, real time decoding in flow-based reader systems is also practicable and could be developed further for applications demanding very high throughput.

Liquid Handling Robotics - the UltraPlex™ SmartStation

In collaboration with Xiril AG, Pronostics have developed a customised liquid handling robot, the UltraPlex™ SmartStation, for medium to high throughput environments, which fully automates assay performance and exceeds the performance of many autoanalysers.

The UltraPlex™ SmartStation robot has a four-tip head, capable of dispensing 10-1000 µl of liquid (1% CVar and maximum 3% inaccuracy), and incorporates liquidlevel detection technology to ensure reagent transfer. The system uses disposable plastic tips, but can be reconfigured to re-use tips if required for particular applications. Typically, three washes on a 96-well plate take about 10 minutes to perform. The Robot has four (or more, if required) separate liquid reservoirs suitable for complex protocols with multiple detection reagents, with a dead volume of less than 10 ml conserving costly assay components. Importantly, the system has a shaking vacuum manifold optimised for handling UltraCodes™ in conventional filter plates.

Samples can be taken from a wide range of tubes (1.5 to 10 ml volume) or from 96-well plates. The UltraPlex™ SmartStation also incorporates a standard barcode reader (compatible with most standard barcode formats) for sample tube identification. Ideal for high throughput applications, this ensures that the sample ID and data subsequently obtained from it are permanently linked, effectively eliminating sample handling errors. The SmartStation is driven by an integrated PC workstation. In addition to delivering ‘one-button’ convenience to operate standard protocols, it also offers the user immense flexibility in designing new protocols for a wide range of applications.

Next Generation UltraPlex™

Looking to the future, Pronostics will leverage its experience in micro and nanofabrication (which underpinned the development of UltraPlex™ coded microparticles by semi-conductor fabrication techniques) to develop nanosensors capable of generating multiplexed assays and biological fingerprints at even lower cost and higher throughput than is currently possible. Such research is in its very early stages, but through our collaborations such as with Prof. Mark Welland (Head of the Nanoscale Laboratory at the Cavendish Laboratory, Cambridge University) we aim to build on our position as the leaders in low cost generation of multiplexed assays and biological fingerprints.

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