Simple and Sensitive Method for Determination of Protein Kinase Activity Based on Surface Charge Change of Peptide-Modified Gold Nanoparticles As Substrates

Protein tyrosine kinases play a pivotal role in intracellular signal transduction pathways and oncogenic transformation. It is necessary to develop a simple, cost-effective, and sensitive kinase assay for study of protein kinases and discovery of kinase-target drugs. In this paper, we present a simple and sensitive method for homogeneous detection of protein kinase activity and screening of inhibitor by measuring surface charge change on the peptide-modified gold nanoparticles (GNPs) as kinase substrates. In this assay, Abl (Abelson murine leukemia viral oncogene) kinase was used as a model. In the presence of Abl kinase and ATP, the surface negative charge on GNPs significantly increases due to phosphorylation of the peptide-modified GNPs. The surface charge on the peptide-modified GNPs was measured by zeta potential analyzer. Under the optimum conditions, the zeta potential on the peptide-modified GNPs was linearly dependent on Abl kinase concentration, the linear range was from 1 to 40 nM and the detection limit was 1 nM. This method was used to evaluate the inhibition efficiency of inhibitors, and the obtained IC₅₀ values were well in agreement with the results reported in the references. Furthermore, this method was successfully applied to determine Abl kinase activity in the cell lysates. Compared to current methods, this new method shows simplicity, short analysis time, high sensitivity, and will become a promising platform for kinase-related fundamental research and inhibitor screening

Nonbleaching Fluorescence of Gold Nanoparticles and Its Applications in Cancer Cell Imaging

In this paper, we investigated the fluorescent properties of gold nanoparticles (GNPs) with several tens of nanometers by ensemble fluorescence spectrometry, fluorescence correlation spectroscopy (FCS), and fluorescence microscopy. We observed that GNPs synthesized by the citrate reduction of chloroauric acid possessed certain fluorescence, narrow full width at half-maximum (17 nm), and with an increase of particle sizes, the emission intensity showed a gradual increase while the emission wavelength remained almost constant (at 610 nm). Especially, the fluorescence of GNPs possessed the excellent behavior of antiphotobleaching under strong light illumination. Despite their low quantum yields, GNPs exhibited strong native fluorescence under relatively high excitation power. The fluorescence of GNPs could be characterized by fluorescence imaging and FCS at the single particle level. On the basis of this excellent antiphotobleaching of GNPs and easy photobleaching of cellular autofluorescence, we developed a new method for imaging of cells using GNPs as fluorescent probes. The principle of this method is that after cells stained with GNPs or GNPs bioconjugates are illuminated by strong light, the cellular autofluorescence are photobleached and the fluorescence of GNPs on cell membrane or inside cells can be collected for cell imaging. On the basis of this principle, we imaged living HeLa cells using GNPs as fluorescent probes and obtained good cell images by photobleaching of cellular autofluorescence. Furthermore, anti-EGFR/GNPs were successfully used as targeted probes for fluorescence imaging of cancer cells. Our preliminary results demonstrated that GNPs possessed excellent behaviors of antiphotobleaching and were good fluorescent probes in cell imaging. Our cellular imaging method described has potential applications in cancer diagnostics, studies, and immunoassays

Deubiquitination Detection of p53 Protein in Living Cells by Fluorescence Cross-Correlation Spectroscopy

Deubiquitination is a reverse post-translational modification of ubiquitination and plays significant roles in various signal transduction cascades and protein stability. The p53 is a very important tumor-suppressor protein and closely implicates more than 50% of human cancers. Although extracellular studies on the deubiquitination of p53 were reported, the process of p53 deubiquitination in living cells due to the shortage of an efficient in situ method for single living cells is still not clear. In this study, we described an in situ method for studying p53 deubiquitination in living cells by combining fluorescence cross-correlation spectroscopy with a fluorescent protein labeling technique. We first constructed the stable cell line expressing EGFP-Ub-p53-mCherry as the substrate of p53 deubiquitination. Then, we established a method for in situ monitoring of the deubiquitination of p53 in living cells. Based on the amplitudes of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy curves from living cells, we obtained the deubiquitination percentage for evaluating the level of p53 protein deubiquitination. Furthermore, we studied the effects of ubiquitin structures on p53 deubiquitination in living cells and found that the C-terminal Gly75-Gly76 motif of ubiquitin is a key location for p53 deubiquitination and the deubiquitination cannot occur when ubiquitin lacks the C-terminal Gly75-Gly76 motif. Our results documented that the developed strategy is an efficient method for in situ study of deubiquitination of proteins in living cells

Assessing the Blinking State of Fluorescent Quantum Dots in Free Solution by Combining Fluorescence Correlation Spectroscopy with Ensemble Spectroscopic Methods

The current method for investigating the blinking behavior is to immobilize quantum dots (QDs) in the matrix and then apply a fluorescent technique to monitor the fluorescent trajectories of individual QDs. So far, no method can be used to directly assess the blinking state of ensemble QDs in free solution. In this study, a new method was described to characterize the blinking state of the QDs in free solution by combining single molecule fluorescence correlation spectroscopy (FCS) with ensemble spectroscopic methods. Its principle is based on the observation that the apparent concentration of bright QDs obtained by FCS is less than its actual concentration measured by ensemble spectroscopic method due to the QDs blinking. We proposed a blinking index (<i>K</i>_blink) for characterizing the blinking state of QDs, and <i>K</i>_blink is defined as the ratio of the actual concentration (<i>C</i>_b,actual) measured by the ensemble spectroscopic method to the apparent concentration (<i>C</i>_b,app) of QDs obtained by FCS. The effects of certain factors such as laser intensity, growth process, and ligands on blinking of QDs were investigated. The <i>K</i>_blink data of QDs obtained were successfully used to characterize the blinking state of QDs and explain certain experimental results

Assessing the Blinking State of Fluorescent Quantum Dots in Free Solution by Combining Fluorescence Correlation Spectroscopy with Ensemble Spectroscopic Methods

The current method for investigating the blinking behavior is to immobilize quantum dots (QDs) in the matrix and then apply a fluorescent technique to monitor the fluorescent trajectories of individual QDs. So far, no method can be used to directly assess the blinking state of ensemble QDs in free solution. In this study, a new method was described to characterize the blinking state of the QDs in free solution by combining single molecule fluorescence correlation spectroscopy (FCS) with ensemble spectroscopic methods. Its principle is based on the observation that the apparent concentration of bright QDs obtained by FCS is less than its actual concentration measured by ensemble spectroscopic method due to the QDs blinking. We proposed a blinking index (Kblink) for characterizing the blinking state of QDs, and Kblink is defined as the ratio of the actual concentration (Cb,actual) measured by the ensemble spectroscopic method to the apparent concentration (Cb,app) of QDs obtained by FCS. The effects of certain factors such as laser intensity, growth process, and ligands on blinking of QDs were investigated. The Kblink data of QDs obtained were successfully used to characterize the blinking state of QDs and explain certain experimental results

Probing the Protein Corona of Nanoparticles in a Fluid Flow by Single-Particle Differenced Resonance Light Scattering Correlation Spectroscopy

The protein corona of nanoparticles (NPs) plays a crucial role in determining NPs’ biological fates. Here, a novel measurement strategy was proposed to in situ investigate the protein corona formed in the NPs with the home-built dual-wavelength laser-irradiated differenced resonance light scattering correlation spectroscopy (D-RLSCS) technique, combined with the modified generation method of the D-RLSCS curve. With the measurement strategy, the dissociation constants and the binding rates between proteins and gold nanoparticles (GNPs) were determined based on the binding-induced ratiometric diffusion change of NPs (the ratio of characteristic rotational diffusion time to translational one), using the formation of the protein corona of bovine serum albumin (BSA) or fibrinogen (FIB) on gold nanoparticles as a model. It was found that BSA shows a stronger binding constant and faster binding rate to gold nanospheres (GNSs) compared with those of FIB. Meanwhile, the dynamic behavior of the protein corona in a fluid flow mimicking biological vessels was further studied based on the combination of the D-RLSCS technique with a microfluidic channel. The measurement results indicated that some “loose” protein corona layers would strip off the surface of NPs within the microchannel due to the fluid sheath force. This method can provide the comprehensive information of a protein corona by averaging the diffusion behavior of many particles different from some conventional methods and overcome the shortcomings of conventional correlation spectroscopy methods

Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by <i>In Situ</i> Fluorescence Cross-Correlation Spectroscopy

Autophagy is a widely conserved and multistep cellular catabolic process and maintains cellular homeostasis and normal cellular functions via the degradation of some harmful intracellular components. It was reported that high basal autophagic activity may be closely related to tumorigenesis. So far, the fluorescence imaging technique has been widely used to study autophagic processes, but this method is only suitable for distinguishing autophagosomes and autolysosomes. Simultaneously monitoring multiple autophagic processes remains a significant challenge due to the lack of an efficient detection method. Here, we demonstrated a new method for simultaneously monitoring multiple autophagic processes and assessing autophagic flux in single cells based on in situ fluorescence cross-correlation spectroscopy (FCCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) was fused with two tandem fluorescent proteins [mCherry red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP)] to achieve the simultaneous labeling and distinguishing of multiple autophagic structures based on the differences in characteristic diffusion time (τD). Furthermore, we proposed a new parameter “delivery efficiency of autophagosome (DEAP)” to assess autophagic flux based on the cross correlation (CC) value. Our results demonstrate that FCCS can efficiently distinguish three autophagic structures, assess the induced autophagic flux, and discriminate different autophagy regulators. Compared with the commonly used fluorescence imaging technique, the resolution of FCCS remains unaffected by Brownian motion and fluorescent monomers in the cytoplasm and is well suitable to distinguishing differently colored autophagic structures and monitoring autophagy

Tempo-Spatially Resolved Scattering Correlation Spectroscopy under Dark-Field Illumination and Its Application to Investigate Dynamic Behaviors of Gold Nanoparticles in Live Cells

In this study, a new tempo-spatially resolved fluctuation spectroscopy under dark-field illumination is described, named dark-field illumination-based scattering correlation spectroscopy (DFSCS). DFSCS is a single-particle method, whose principle is similar to that of fluorescence correlation spectroscopy (FCS). DFSCS correlates the fluctuations of the scattered light from single nanoparticle under dark-field illumination. We developed a theoretical model for translational diffusion of nanoparticles in DFSCS system. The results of computer simulations documented that this model was able to well describe the diffusion behaviors of nanoparticles in uniformly illuminated field. The experimental setup of DFSCS was achieved by introducing a dark-field condenser to the frequently used bright-field microscope and an electron multiplying charge-coupled device (EMCCD) as the array detector. In the optimal condition, a stack of 500 000 frames were collected simultaneously on 64 detection channels for a single measurement with acquisition rate of 0.5 ms per frame. We systematically investigated the effect of certain factors such as particle concentration, viscosity of the solution, and heterogeneity of gold nanoparticles (GNPs) samples on DFSCS measurements. The experiment data confirmed theoretical model proposed. Furthermore, this new method was successfully used for investigating dynamic behaviors of GNPs in live cells. Our preliminary results demonstrate that DFSCS is a practical and affordable tool for ordinary laboratories to investigate the dynamic information of nanoparticles <i>in vitro</i> as well as <i>in vivo</i>

Study on Phase Separation of Fused in Sarcoma by Fluorescence Correlation Spectroscopy

Liquid–liquid phase separation (LLPS) of fused in sarcoma (FUS) has emerged as a fundamental principle underpinning cellular function and malfunction. However, we know little about the FUS phase transition process from individual molecules to nanoscale condensates, which plays important roles in neurodegenerative diseases. Here, we propose the fluorescence correlation spectroscopy (FCS) method to quantitatively study the phase separation process of FUS protein with the fluorescent tag-enhanced green fluorescent protein (EGFP), from individual molecules to nanoscale condensates. The characteristic diffusion time (τD) of the protein condensates can be obtained from the FCS curve, which increases with the growth of the protein hydration radius. The bigger the τD value of the protein condensates, the larger the condensates formed by the phase separation of FUS. By this method, we discovered that the critical concentration for FUS to phase separation was 20 nM. We then plotted FUS phase diagrams based on τD under different concentrations of NaCl and found that both low-salt and high-salt concentrations tended to promote FUS-EGFP phase separation. Our results showed that ATP has a good inhibitory effect on FUS phase separation, and its inhibition constant IC50 was 3.2 mM. Finally, we evaluated the inhibition efficiency of single-stranded DNA sequences (ssDNA) on FUS phase separation and demonstrated that ssDNA containing three copies of TCCCCGT had relatively strong inhibition efficiency. In summary, our work provides detailed insight into the FUS phase transition process from individual molecules to nanoscale condensates at nanomolar concentrations and can be exploited for drug screening of neurodegenerative diseases

Blinking Behavior of CdSe/CdS Quantum Dots Controlled by Alkylthiols as Surface Trap Modifiers

The blinking behavior of single quantum dots (QDs) is an intrinsic drawback for some biological and photoelectric applications that rely on single-dot emission. Some studies demonstrate that the blinking behavior of QDs is mainly attributed to nonradiative recombination processes associated with traps at the nanocrystal surface. In this study, we systematically investigated the effects of surface ligand alkylthiols on the blinking of CdSe/CdS QDs prepared in the organic phase and observed that the blinking of CdSe/CdS QDs was significantly dependent on the annealing time and structure and concentration of alkylthiol ligands. In the optimal conditions, we prepared thiol-modified CdSe/CdS QDs with a “nonblinking” fraction up to 83%. The mechanism of the blinking suppression was mainly attributed to modifying surface traps of QDs with alkylthiols. In this modification, the decomposition of alkylthiols can slowly release activated “S” under high temperature. Then the activated S can bind to surface traps of QDs, which induces a secondary growth of the core/shell QDs coupled with surface reconstruction and the efficient suppression of the blinking. The method described here can be used to suppress the blinking of other QDs