New HCR Technologies: 10-Plex Quantitative Spectral Imaging of RNAs and Proteins; Multiplexed Quantitative Imaging of Protein:Protein Complexes; and Sensitive, Instrument-Free, At-Home Pathogen Detection

Signal amplification based on the mechanism of hybridization chain reaction (HCR) enables researchers to quantitatively image RNA and protein expression in highly autofluorescent biological samples. This thesis extends the capabilities of HCR to three new domains: spectral HCR imaging for quantitative 10-plex immunofluorescence and in situ hybridization in highly autofluorescent samples; imaging of protein:protein complexes using cooperative probes for logical control over HCR signal amplification; and HCR lateral flow tests for sensitive, instrument-free, at-home testing for infectious diseases. While 4- or 5-plex imaging is readily achieved using orthogonal HCR systems labeled with spectrally distinct fluorophores, higher levels of multiplexing are challenging due to overlap in the broad excitation and emission spectra of commonly used fluorophores. In Chapter 2, we simultaneously image a combination of 10 protein and RNA targets via spectral imaging with linear unmixing. A combination of 10 reference spectra for 10 fluorophores chosen for optimal unmixing, 10 orthogonal HCR systems, and 11 optimized excitation and emission settings enable robust, user-friendly performance, which is demonstrated in whole-mount zebrafish embryos and mouse brain sections. We validate that unmixed subcellular voxel intensities enable accurate and precise relative target quantitation with subcellular resolution across all 10 channels and demonstrate single-molecule sensitivity and resolution for absolute RNA quantitation. In Chapter 3, we introduce an enzyme-free method for multiplexed imaging of protein:protein complexes using split-initiator HCR signal amplification. Antibodies specific to each protein of the complex carry fractional initiators that become colocalized upon introduction of a DNA ruler strand to form a full HCR initiator and trigger growth of a tethered amplification polymer. Automatic background suppression is present throughout the protocol, as split-initiator antibody probes that bind to the sample nonspecifically or to isolated protein targets are too far apart to become colocalized by the ruler strand, precluding colocalization of a full initiator and preventing HCR signal amplification. We demonstrate the technique with high signal-to-background in adherent mammalian cells, pro-T cells, and highly autofluorescent formalin-fixed paraffin-embedded human breast tissue sections. Leveraging existing orthogonal HCR amplifiers, we design three orthogonal cooperative junctions for simultaneous 3-plex detection of protein:protein complexes. We validate that quantitative subcellular voxel intensities are generated, allowing for built-in relative quantitation of protein:protein complexes within the spatial context of the sample. Lastly, we demonstrate simultaneous detection of protein targets, RNA targets, and protein:protein complexes via a unified protocol for HCR immunofluorescence, in situ hybridization, and protein:protein complex imaging. In Chapter 4, we enhance the sensitivity of conventional unamplified lateral flow tests for at-home infectious disease testing by developing an amplified assay with isothermal, enzyme-free signal amplification based on the mechanism of HCR. Traditional lateral flow tests are amenable to at-home testing and return a result within 10–15 minutes but demonstrate a high false-negative rate (e.g., 25-50% for SARS-CoV-2) due to the absence of signal amplification. The HCR lateral flow assay we develop maintains the simplicity of the conventional lateral flow assay user experience via a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. To perform a test, the user loads the sample, closes the device, and reads the result by eye after 60 minutes. Detecting gamma-irradiated SARS-CoV-2 virions in a mixture of saliva and extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/μL using available antibodies to target the SARS-CoV-2 nucleocapsid protein. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies exhibit limits of detection of 500 copies/μL, 1000 copies/μL, 2000 copies/μL, 2000 copies/μL, and 20,000 copies/μL. By swapping out antibody probes to target different pathogens, amplified HCR lateral flow assays offer a platform for simple, rapid, and sensitive at-home testing for infectious diseases.