Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by In Situ 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.

In vivo measurement of autophagic flux by fluorescence correlation spectroscopy.

Autophagy is a fundamental and phylogenetically conserved self-degradation process and plays a very important role in the selective degradation of deleterious proteins, organelles, and other macromolecules. Although flow cytometry and fluorescence imaging techniques have been used to assess autophagic flux, we remain less able to in vivo monitor autophagic flux in a highly sensitive, robust, and well-quantified manner. Here, we reported a new method for real-time and quantitatively monitoring autophagosomes and assessing autophagic flux in living cells based on fluorescence correlation spectroscopy (FCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) fused with an enhanced green fluorescent protein (EGFP-LC3B) was used as a biomarker to label autophagosomes in living cells, and FCS was used to monitor EGFP-LC3B labeled autophagosomes by using the characteristic diffusion time (τD) value and brightness per particle (BPP) value. By analyzing the distribution frequency of the τD values in living cells stably expressing EGFP-LC3B, mutant EGFP-LC3B (EGFP-LC3BΔG) and enhanced green fluorescent protein (EGFP), we found that the τD value greater than 10 ms was attributed to the signal of EGFP-LC3B labeled autophagosomes. So, we proposed a parameter PAP as an indicator to assess the basal autophagic activity and induced autophagic flux. This new method was able to evaluate autophagy inducers, early-stage autophagy inhibitors, and late-stage autophagy inhibitors. Compared with current methods, our method shows high spatiotemporal resolution and very high sensitivity for autophagosomes in low EGFP-LC3B expressing cells and will become an attractive and alternative method for biological and medical studies, some drug screening, and disease treatment.

In situ quantitative measurements on MMP-9 activity in single living cells by single molecule fluorescence correlation spectroscopy.

Matrix metalloproteinase-9 (MMP-9) plays an important role in tumor progression. It is of great significance to establish a sensitive in situ assay strategy for MMP-9 activity in single living cells. Here a novel in situ single molecule spectroscopy method based on the fluorescence correlation spectroscopy (FCS) technique was proposed for measuring the MMP-9 activity at different locations within single living cells, using a fluorescent specific peptide and a reference dye as dual probes. The measurement principle is based on the decrease of the ratiometric translational diffusion time of dual probes in the detection volume due to the peptide cleavage caused by MMP-9. The peptide probe was designed to be composed of an MMP-9 cleavage and cell-penetrating peptide sequence that was labeled with a fluorophore and conjugated with a streptavidin (SAV) molecule. The ratiometric translational diffusion time was used as the measurement parameter to eliminate the effect of intracellular uncertain viscosity. The linear relationship between the ratiometric diffusion time and MMP-9 activity was established, and applied to the determination of enzymatic activity in cell lysates as well as the evaluation of the inhibitory effects of different inhibitors on MMP-9. More importantly, the method was successfully used to dynamically determine MMP-9 activity in single living cells or under the stimulation with phorbol 12-myristate 13-acetate (PMA) and inhibitors.

A study of protein-drug interaction based on solvent-induced protein aggregation by fluorescence correlation spectroscopy.

The identification of molecular targets for achieving beneficial effects from small-molecule drugs is a crucial and currently unsolved challenge, which leads to high costs and long development cycles. Therefore, it is urgent to develop methods for easily and quickly acquiring information about protein-drug interaction at a molecular level. In this study, we propose a novel method for the study of protein-drug interaction by fluorescence correlation spectroscopy (FCS) based on organic solvent-induced protein aggregation. We used ß-secretase (BACE-1) and dihydrofolate reductase (DHFR) as model proteins. Fluorescence-labelled proteins aggregated in aqueous solutions containing organic solvents. In the presence of drugs, the aggregation of proteins was inhibited greatly, and FCS was used to characterize protein aggregates. The decrease in the characteristic diffusion time (τD) of protein aggregates demonstrated a strong interaction between proteins and drug molecules. We presented a new parameter IC50 to assess the inhibitory effects of drugs on the basis of the changes in the τD of fluorescence-labelled proteins under different concentrations of the drugs in the presence of organic solvents. We acquired a remarkable difference in the IC50 values for different drugs and in terms of the trend, our results were consistent with those reported by other methods. Compared with current methods, our approach is simple, low-cost, and time-saving, and has the potential to become a promising and universal tool for drug screening at the molecular level.

Single-Particle Catalytic Analysis by a Photon Burst Counting Technique Combined with a Microfluidic Chip.

Single-particle catalytic analysis plays an important role to understand the catalytic mechanism of nanocatalysts. Currently, some methods are used to study the relationship between single-particle catalytic activity and morphology. However, there is still lack of a simple and rapid analysis method for evaluating the catalytic activity of an individual nanocatalyst that freely moves in solution. Here, we proposed a novel single-particle catalytic analysis method for investigating the catalytic activity of a free nanocatalyst. Its working principle is based on the photon burst counting analysis on fluorescent catalytic products of an individual nanocatalyst combined with a microfluidic chip. In this study, we used the reduction reaction of resazurin (RZ) to resorufin (RF) catalyzed by gold nanoparticles (GNPs) as a model. When nonfluorescent RZ molecules in one microchannel of the microfluidic chip mixed with the GNPs flowing in another channel under the control of flow rates, each individual photon burst from the catalytic product RF by GNPs was measured in real time with a constructed flow single-particle catalytic analysis (SPCA) system. With the method, the obtained intensity of each photon burst reflects the capacity of a particle to catalyze RZ molecules into RF(s). The number of photon burst within sampling time reflects the particle number of GNPs with catalytic activity. The experimental conditions including the mixing mode of the nanocatalyst and the substrate, the flow rate, RZ concentration, and detection time were optimized. Finally, the method was successfully used to study the catalytic activity of GNPs with different sizes and morphologies.

A dual-responsive fluorescent probe for detection of fluoride ion and hydrazine based on test strips.

Hydrazine (N2H4) and fluoride ion (F-) are regarded as environmental pollutants and potential carcinogens. A dual-functional fluorescent probe (probe 1) was developed for both F- and N2H4 with high selectivity and sensitivity. 1 was based on nucleophilic aromatic substitution reaction for N2H4 detection and selective cleavage of 4-nitrobenzenesulphonyl group for the determination of F-. The limits of detection of probe for F- and N2H4 were 77.82 nM and 29.34 nM, respectively, which are far below the threshold limit value (TLV) of United States Environmental Protection Agency (EPA). The home-made test strips of 1 provided the positive tool for F- and gaseous N2H4 in different system. And the confocal fluorescence images indicated that 1 can quantitatively detect N2H4 in living PC12 cells. Promisingly, 1 has great prospects for N2H4 imaging and determining in living system.

A highly selective fluorescent probe for the detection of hypochlorous acid in tap water and living cells.

A turn-on fluorescent probe (DAME) for sensing hypochlorous acid (HClO) with excellent selectivity was presented. The fluorescent probe was composed of coumarin derivative as the fluorophore and dimethylcarbamothioic chloride group with a sulfide moiety as modulator. Additionally, the sulfide moiety would be oxidized by HClO, and then free dye of coumarin derivate was released and exhibited significant fluorescence. In addition, the probe could respond to HClO in solutions within 60 s and the limit of detection was down to 34.75 nM. Moreover, the probe was used for the detection of HClO in tap water through the home-made test paper. And confocal images confirmed that probe DAME could be used for recognizing HClO in living cells.

Carbon Dots with Red Emission for Sensing of Pt2+, Au3+, and Pd2+ and Their Bioapplications in Vitro and in Vivo.

Red emissive carbon dots (CDs) have drawn more and more attention due to their good organ penetration depth and slight biological tissue photodamage. Herein, the fluorescent CDs with red emission were synthesized by the facile one-pot hydrothermal treatment of citric acid and neutral red and they show red fluorescence both in aqueous solution and solid state. The solution of CDs exhibits the quantum yield of 12.1%, good stability against photobleaching, and low cytotoxicity. As-prepared CDs can be used as a fluorescent probe for peculiar detection of Pt2+, Au3+, and Pd2+. Furthermore, the CDs show excellent biocompatibility, which were successfully used as hopeful bioimaging and biosensing of noble metal ions in PC12 cells and zebrafish.