An efficient nonlinear hybridization chain reaction-based sensitive fluorescent assay for in situ estimation of calcium channel protein expression on bone marrow cells.

A sensitive and highly efficient approach to monitor the expression of proteins on live cells was urgently needed to demonstrate its factor and mechanism and most important for clinical diagnostics and molecular biology. Herein, we developed a simple and highly efficient strategy, nonlinear hybridization chain reaction (nonlinear HCR), for the sensitive determination of proteins on live cells with transient receptor potential vanilloid 4 (TRPV4) and RAW264.7 cells as a model. Unlike the normal hybridization chain reaction (HCR) with multiplicative amplification, an exponential amplified fluorescent response could be obtained in theory based on the proposed nonlinear HCR. As a result, the nonlinear HCR generated a significant enhancement about 3 times compared with the normal HCR and 10 times compared with the directly immunofluorescence assay. Based on the proposed nonlinear HCR, the fluorescent signals increased with the concentration of TRPV4 in the range from 10 pg/mL to 100 ng/mL with a detection limit of 2.8 pg/mL, which would be useful for the sensitive detection of proteins in cell lysis or on cell surface. At the same time, the significant improvements via nonlinear HCR were achieved in the fluorescent imaging system compared with traditional immunofluorescence staining and normal HCR, proving the significant value of nonlinear HCR-based amplification strategy. Success in the establishment of the highly efficient nonlinear HCR strategy offered a simple and sensitive approach to demonstrate the concentration of special proteins on cell and other proteins and nucleotide potentially, revealing a simple and efficient technology for research fields of clinical diagnostics and molecular biology.