Technology Overview

Firefly BioWorks' hydrogel particles are the ideal assay substrate -- Hydrogel particles made by Firefly BioWorks' patented Optical Liquid Stamping are hydrophilic, bio-inert, and flexible (literally!).  This remarkable material is unlike any other substrate on the biomarker detection market.  The hydrogel particles are porous, which allows target molecules to diffuse and bind in a unique nanoscale three-dimensional scaffold.  This both maximizes signal and widens the assay's dynamic range.  In addition to more signal, you can expect decreased background with hydrogel particles because the porosity is precisely tuned to exclude certain molecular species based on size.  In short, the Firefly™ Discovery Platform enables you to gather a lot more of the signal you seek.

Our microRNA detection method shows single-nucleotide specificity and no sequence bias
-- The Firefly™ microRNA Assay uses a unique post-hybridization ligation-based scheme to fluorescently label bound microRNA targets.  This process not only simplifies the protocol, but eliminates the sequence bias seen in PCR-based approaches.  The entire microRNA detection process is just three steps: (1) Hybridize, (2) Label, and (3) Report.  The Hybridize step binds your microRNA targets to the target-specific probes attached to the hydrogel Firefly™ Particles.  The Label step ligates a universal biotinylated adapter to the captured targets.  A fluorescent reporter binds to the universal adapter during the Report step.  This reporter molecule is ultimately used for microRNA detection in a flow cytometer.

. .

Firefly™ Particles can be detected by standard flow cytometers -- the Firefly™ Discovery Platform readily expands the capabilities of almost any cytometer.  Currently, we are able to support select instruments from BD, Millipore, and Life Technologies.  You can read a cross-cytometer comparison and learn more about our expanding capabilities here.

         

The Firefly™ Discovery advantage -- Firefly BioWorks’ technology can rapidly profile up to 68 microRNAs over large numbers of samples in a variety of biological samples.  Most users prefer purified RNA, but the Firefly™ microRNA Assay has also been demonstrated in cell lysate and tissue digest.  Read more about performance of the Firefly™ microRNA Assay here, and discover how the Firefly™ Discovery Platform compares to competing technologies.

    

 

Optical Liquid Stamping enables manufacturing of complex hydrogel particles with unique barcodes
Firefly BioWorks' core technology is Optical Liquid Stamping (OLS), a microfabrication process that combines photolithography with microfluidics. This revolutionary process, described schematically in the sequence below, uses bursts of light shaped through a mask to rapidly print complex hydrogel microparticles from streams of fluids doped with photosensitive monomers and biomolecules of interest, such as antibodies, DNA oligonucleotides, etc.

       
A microchannel is filled with photopolymerizable fluid. A photomask with the desired particle shape is placed above the microchannel. Light shines through the photomask to initiate hydrogel formation. Firefly particles are collected for final packaging.

This patented method and several bio-assays developed on this platform have been published in leading academic journals, including Science, Nature Materials, Nature Protocols and Analytical Chemistry. The flexibility of this technology is such that particles can be designed from a very large set of parameters, such as:

• Size range of 5-500 μm and broad range of shapes
• Wide range of materials: hydrophilic, hydrophobic, amine or carboxyl terminated, etc.
• Tunable porosity
• Wide array of functional probes for: mRNAs, microRNAs, proteins, etc.
• Multiple functional regions on the same particle
• Ability to encapsulate cells, viruses, enzymes, quantum dots, etc.

                  

Protein Detection

Firefly's porous hydrogel particles are ideally suited for detection of proteins. We have demonstrated mutliplexed detection of protein targets using traditional sandwich assays. Similar to our microRNA assay, all protein detection assays are carried out in 96-well filter plates, taking ~3 hours to complete. We have demonstrated multiplexed protein detection using commercially-available antibody pairs with typical detection limits in the low pg/ml range. Furthermore, we have demonstrated assay specificity via multiplexed detection of phosphorylated versus non-phosphorylated species with varying amount of activation time (shown on the right).

Messenger RNA Assays

The Firefly platform has been used for mRNA detection using standard sample preparation methods including IVT (in vitro transcription) and LMA (ligation-mediated amplification). These assays were validated against the current state-of-the-art bead based system, showing better specificity and higher sensitivity with synthetic targets.

PCR-Coupled Assays

Firefly hydrogel particles are very robust, withstanding the high temperatures used in PCR amplification. The technology has been incorporated into “one-pot” assays where multiplex PCR is performed and amplicons are captured by hydrogel particles present in the PCR mix. The particles can then be scanned for quantification of each amplicon. This process eliminates the need to design amplicons of varying size and perform subsequent gel electrophoresis for multiplex PCR analysis.

Single-Color Scanning

Because the probe and code regions can be spatially separated, it is possible to scan Firefly particles using a single excitation source and detector. This unique attribute makes the platform ideal for applications demanding simple, inexpensive, or portable detection systems. Particles may have complex 2D barcodes (for imaging systems) or 1D barcodes well-suited for rapid flow-through scanning. Firefly has also designed custom software to decode particles and quantify bound target.

Single-color hydrogel particles have been analyzed using static imaging systems such as fluorescence microscopes and commercial microarray scanners. Flow-through platforms used for analysis include custom microfluidic systems, as well as commercial flow-cytometers. This class of single-color particles offers:

  • Static or flow-through analysis with custom or commercially-available equipment;
  • Expandable barcoding from 1 to 1000;
  • Single or multiple probe regions per particle; and
  • Custom decoding software to identify particles and quantify bound target.
  • Technical Note

    Pregibon, D.C. microRNA Profiling with FirePlex® miRSelect: A Technical Overview and Cross-Cytometer Comparison (2012)

    Publications

    The articles listed below describe the application of our technology to biomarker detection. Optical Liquid Stamping can also be employed in areas outside biotechnology. The full publications list is available from the Doyle Lab.