Multiplexing Done Differently: Flexible, Sensitive, And Accurate Detection Of Mirna And Protein Analytes Using Firefly Particle Technology

Detection of molecular biomarkers in biological fluids is used as an indicator of normal biological processes, pathogenic processes, and response to therapeutic intervention 1,2 . Using a combined signature of multiple biomarkers can better account for patient and epidemiological heterogeneity, and provide a more accurate indication of patient health 2 . This has necessitated the development of technologies that can be used for multiplex biomarker profiling directly from biofluids such as plasma, serum, and urine. Conventional assays consume excessive quantities of precious clinical samples, time and budget when used for discovery and validation of multi-analyte biomarker signatures. To address this need, we developed the Firefly Technology platform, which allows for sensitive and accurate detection of up to 75 protein analytes or 68 miRNAs directly from a biological sample. The Firefly platform uses patented Firefly® hydrogel particles and a three-region encoding design that allows for true, in-well multiplexing, providing flexibility over which analytes can be quantified in the same well and allowing for custom panel design. For the detection of protein analytes, the Firefly immunoassays use high-performance matched antibody pairs that reduce cross reactivity between individual analytes, provide up to 5 logs dynamic range, and typically demonstrate single-digit pg/ml sensitivity, while requiring only 12.5 µl biofluid input. Similarly, the Firefly microRNA assay can reliably detect as few as 1000 microRNA copies per sample with a linear dynamic range of ~5 logs, and without the need of prior RNA purification. This assay utilizes single step RT-PCR signal amplification using universal primers, thus leveraging PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target-specific qPCR. The Firefly assays have been validated in a wide range of biological samples including plasma, serum, urine and cell culture supernatant, providing experimental flexibility. The 96well plate assay format enables high-throughput screening of samples, with readout conducted on standard flow cytometers, thereby omitting the need for complex and expensive dedicated instrumentation. Finally, the integrated Firefly Analysis Workbench software enables easy and rapid data analysis, visualization, and export in under ten minutes, and includes key features such as standard curve analysis and publication-quality heatmaps and graphs. Here we present data from several studies investigating cytokine profiling in human and rodent samples, and circulating and tumor microRNA profiles, using the Firefly Platform. Together, this novel combination of bioinformatics tools and multiplex, high-sensitivity assays enables rapid discovery and validation of biomarker signatures from fluid samples. Citation Format: Elnaz Atabakhsh, Graeme Doran, Conor Rafferty, Jennifer Heath, Sarah Albertson, Peter Gillis, Jonathan Pinney, Russell Neuner, Michael Chipchase, James Murray, Daniel Pregibon. Multiplexing done differently: flexible, sensitive, and accurate detection of miRNA and protein analytes using Firefly particle technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2756. doi:10.1158/1538-7445.AM2017-2756