In Situ Assay of Proteins Incorporated with Unnatural Amino Acids in Single Living Cells by Differenced Resonance Light Scattering Correlation Spectroscopy

Abstract

Site-specific incorporation of unnatural amino acids (UAAs) into target proteins (UAA-proteins) provides the unprecedented opportunities to study cell biology and biomedicine. However, it is a big challenge to in situ quantitatively determine the expression level of UAA-proteins due to serious interferences from autofluorescence, background scattering, and different viscosity in living cells. Here, we proposed a novel single nanoparticle spectroscopy method, differenced resonance light scattering correlation spectroscopy (D-RLSCS), to measure the UAA-proteins in single living cells. The D-RLSCS principle is based on the simultaneous measurement of the resonance scattering light fluctuation of a single gold nanoparticle (GNP) in two detection channels irradiated by two coaxial laser beams and then autocorrelation analysis on the differenced fluctuation signals between two channels. D-RLSCS can avoid the interferences from intracellular background scattering and provide the concentration and rotational and translational diffusion information of GNPs in solution or in living cells. Furthermore, we proposed a parameter, the ratiometric diffusion time and found that this parameter is proportional to the square of particle size. The theoretical and experimental results demonstrated that the ratiometric diffusion time was not influenced by the intracellular viscosity. This method was successfully applied for in situ quantification of the UAA-protein within single living cells based on the increase in the ratiometric diffusion time of nanoprobes bound with proteins. Using UAA-EGFP (enhanced green fluorescent protein) as a model, we observed the significant difference in the UAA-protein concentrations at different positions in single living cells.

Brightness Analysis per Moving Particle: In Situ Analysis of Alkaline Phosphatase in Living Cells

“…Here, a confocal dual-wavelength differenced (DWD) imaging technique was used, and the aim was to indicate the position of intracellular AuNPs. The DWD imaging method can eliminate the influence of the autofluorescence and intracellular scattering from the organelles on the imaging . Then, after the objective lens assembled on a 3D nanopositioning system was moved to these spots, the RLS intensity traces and single-particle RLSCS curves at these bright spots were obtained with the correlation spectroscopy system.…”

“…During the in situ detection of intracellular ALP activity, the cells in the dish were placed above the objective lens of the RLSCS system. The dual-wavelength differenced (DWD) RLS scanning images of cells with nanoparticles were obtained with the imaging system . Then, the objective lens was moved to the bright spots in the obtained cell image by controlling the PI nanopositioning system ( x , y -direction), and at the chosen location, the RLSCS curves and RLS intensity traces of moving particles were measured with the RLSCS system.…”

“…Due to the tiny detection volume (the lateral radius is about 300 nm), among them, FCS has been widely used for in situ analysis within single living cells. − However, as we know, during the FCS analysis or other fluorescence analyses, the fluorescence intensity of molecules is remarkably influenced by the solution viscosity . Particularly, the viscosity in the different locations within the living cells is different and unknown, which makes its effects unpredictable.…”

“…In order to overcome the influence of viscosity, autofluorescence, and scattering within cells, Xu et al proposed a differenced RLSCS (D-RLSCS) detection strategy. Utilizing the independence of the ratio of rotational diffusion time to translational diffusion time (τ RD ) on viscosity, a method employing the linear relation between τ RD and concentration of unnatural amino acid-modified proteins (UAA proteins) was established to achieve the quantitative analysis method for UAA proteins in living cells . However, in order to increase the diffusion time of probes, the target proteins have to be conjugated with probes via strong binding such as a covalent bond.…”

Photonic and Magnetic Dual-Responsive Molecularly Imprinted Sensor for Highly Specific Recognition of Enterovirus 71

“…The RLS technique, established by Pasternack et al in 1993 by using an ordinary fluorescence spectrophotometer, is widely used in recognition and detection of biomolecules such as nucleic acids, proteins and viruses, etc . − Under some conditions, the intensity of RLS is directly proportional to the concentration of the tested substance, which constitutes the basis of quantitative analysis.…”