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Lipid droplets (LDs), which are ubiquitous organelles existing in almost all eukaryotic cells, have attracted a lot of attention in the field of cell biology over the last decade. For the biological study of LDs via fluorescence imaging, the superior LD fluorescent probes with environmental polarity-sensitive character are highly desired and powerful but are very scarce. Herein, we have newly developed such a kind of fluorescent probe named LDs-Red which enables us to visualize LDs and to further reveal their polarity information. This fluorescent probe displays the advantages of intense red/near-infrared emission, high LD staining specificity, and good photostability; thus, it would be very useful for LD fluorescence imaging application. As a result, the three-dimensional confocal imaging to visualize spatial distribution of LDs and the multicolor confocal imaging to simultaneously observe LDs and other cellular organelles have been realized using this new LD fluorescent probe. Furthermore, the polarity-sensitive emission character of this probe enables us to quantitatively determine the LD polarity via spectral scan imaging. Consequently, the cancer cells (HepG2, HeLa, and Panc02) displaying lower polarity of LDs than the normal cells (L929, U251, and HT22) have been systematically demonstrated. In addition, this polarity-sensitive probe displaying shorter fluorescence wavelengths in cancer cells than in normal cells has an important and potential ability to distinguish them.

A cost-effective and highly reproducible capillary-based surface-enhanced Raman scattering (SERS) platform for sensitive, portable detection and identification of fentanyl is presented. Through encapsulating gold trisoctahedra (Au TOH) in the capillary tube for the first time, the SERS platform was constructed by combining the superior SERS properties of Au TOH and the advantages of capillaries in SERS signal amplification, facile sample extraction, and portable trace analysis. The effects of the size and density of Au TOH on the SERS performance were investigated by experiments and simulations, which showed that the maximum SERS enhancement was obtained for Au TOH with the size of 75 nm when particle density reached 74.54 counts/μm

On-site quantitative analysis of pesticide is of significant importance for addressing serious public health issues in clinical, food, and environmental settings. Herein, we designed a novel smartphone-assisted sensing platform for on-site monitoring of 2,4-dichlorophenoxyacetic acid (2,4-D) based on carbon dots/cobalt oxyhydroxide nanosheet (CDs/CoOOH) composite. In this work, a red emissive CDs/CoOOH composite was proposed as a signal indicator for shielding background interference, enhancing anti-interference capability. 2,4-D as an inhibitor of alkaline phosphatase could specifically suppress the production of ascorbic acid, which restrained in situ etching of the CDs/CoOOH composite and further triggered the fluorescence response of the biosensor. By employing a lab-on-smartphone based device and self-designed application software, the fluorescence image was directly captured and analyzed with a sensitive detection limit of 100 μg L

Forster resonance energy transfer (FRET) is a well-established method for studying macromolecular interactions and conformational changes within proteins. Such a method normally uses fluorescent proteins or chemical-labeling methods which are often only accessible to surface-exposed residues and risk-disturbing target protein structures. Here, we demonstrate that the genetic incorporation of a synthetic fluorescent amino acid, L-(7-hydroxycoumarin-4-yl) ethylglycine (Cou) and natural endogenous fluorophore Tryptophan (Trp) residues of a protein could serve as an efficient FRET pair to monitor protein interactions, using the signaling transducer β-arrestin-1 as a model system. We used this technology to record the dynamic spectra in both binding and competition experiments of β-arrestin-1, the contribution of each specific phosphate in ternary complex formation, in a rapid and efficient manner. The determined Kd value for the association between the active arrestin and Fab30 is 0.68 μM in the three-component interaction system. Moreover, we were able to determine the contributions of the site 3 phospho-site and the site 6 phospho-site binding, each contributing to the high affinity ternary complex assembly as 2.7 fold and 15.5 fold, respectively, which were never determined before. These results thus highlighted the potential usage of this new method in measurement of the allosteric-induced enhanced affinity with small amount proteins and in a fast manner and in a complex system. Collectively, our newly developed Trp:Cou FRET system based on genetic expansion technology has extended the molecular toolboxes available for biochemical and structural biology studies.

Highly sensitive detection of molecular tumor markers is essential for biomarker-based cancer diagnostics. In this work, we showcase the implementation of fiber light-coupled optofluidic waveguide (FLOW) immunosensor for the detection of p53 protein, a typical tumor marker. The FLOW consists of a liquid-core capillary and an accompanying optical fiber, which allows evanescent interaction between light and microfluidic sample. Molecular binding at internal surface of the capillary induces a response in wavelength shift of the transmission spectrum in the optical fiber. To enable highly sensitive molecular detection, the evanescent-wave interaction has been strengthened by enlarging shape factor R via fine geometry control. The proposed FLOW immunosensor works with flowing microfluid, which increases the surface molecular coverage and improves the detection limit. As a result, the FLOW immunosensor presents a log-linear response to the tumor protein at concentrations ranging from 10 fg/mL up to 10 ng/mL. In addition, the nonspecifically adsorbed molecules can be effectively removed by the fluid at an optimal flow rate, which benefits the accuracy of the measurement. Tested in serum samples, the FLOW successfully maintains its sensitivity and specificity on p53 protein, making it suitable for diagnostics applications.

Bacterial γ-glutamyltranspeptidases (γ-GT) is a well-known metabolic enzyme, which could cleave the γ-glutamyl amide bond of γ-glutamyl analogues. As a key metabolic enzyme of bacteria and a virulence factor for the host, bacterial γ-GT was determined to be a novel pharmaceutical target for new antibiotics development. However, there is no efficient method for the sensing of γ-GT activity in bacteria and the recognition of γ-glutamyltransferase rich-bacteria. In the present work, a dicyanoisophorone derivative (ADMG) has been designed and developed to be a sensitive and selective near-infrared fluorescent probe for the sensing of bacterial γ-GT. ADMG not only sensed bacterial γ-GT in vitro, but also imaged intestinal bacteria in vivo. More interesting, the intestinal bacteria existed in the duodenum section of mouse displayed significant fluorescence emission. Under the guidance of the sensing of γ-GT using ADMG, three intestinal bacteria strains K. pneumoniae CAV1042, K. pneumoniae XJRML-1, and E. faecalis were isolated successfully, which expressed the bacterial γ-GT. Therefore, the fluorescent probe ADMG not only sensed the endogenous bacterial γ-GT and imaged the intestinal bacteria but also guided the isolation of intestinal bacteria possessing γ-GT efficiently, which suggested a novel biological tool for the rapid isolation of special bacteria from a mixed sample.

In clinical diagnosis, accurate and reliable measurement technologies for the detection of disease biomarkers at ultralow concentrations can provide guidance for the initiation of treatment and potentially improve survival for patients. Here, we demonstrate an optical microfiber reader for enhanced analytical sensitivity in enzyme-linked immunosorbent assays (ELISA) that enables the detection of tiny changes of the refractive index (RI) induced by the catalyzed oxidation of substrate, owing to the strong interaction between the evanescent field and surrounding medium. By employing the microfiber reader for the C-reaction protein (CRP) and interleukin-6 (IL-6) assays after the enzymatic signal amplification in ELISA, we experimentally investigate the biosensing capacity of the device. As a result, log-linear relations of CRP and IL-6 detection in PBS and human serum between the concentration and spectral response were obtained at both nanogram and picogram levels, respectively, and anti-CRP/HRP detection as low as 9.75 pg/mL was achieved, which was undetectable by the conventional spectrophotometry. With a stable, accurate, and color-free detection capacity, this optical microfiber reader has a promising prospect in early disease diagnosis and clinical treatment.

β-Glucuronidase (GLU) is an important biomarker for primary cancers and intestinal metabolism of drugs or endogenous substances; however, an effective optical probe for near-infrared (NIR) monitoring in vivo is still lacking. Herein, we design an enzyme-activated off-on NIR fluorescent probe, HC-glu, based on a hemicyanine keleton, which is conjugated with a d-glucuronic acid residue via a glycosidic bond, for the fluorescent quantification and trapping of endogenous GLU activity in vitro and in vivo. The newly developed NIR probe exhibited prominent features including prominent selectivity, high sensitivity, and ultrahigh imaging resolution. It has been successfully used to detect and image endogenous GLU in various hepatoma carcinoma cells, tumor tissues, and tumor-bearing mouse models, for cancer diagnosis and therapy. Moreover, it could detect the in vivo activity of GLU in the intestinal tracts of animals including mice and zebrafish, where GLU performs a vital biological function and is mainly distributed. It could also evaluate real intestinal distribution and real-time variations of GLU in development and growth, all of which are very helpful to guide rational drug use in the clinic. Our results fully demonstrated that HC-glu may serve as a promising tool for evaluating the biological function and process of GLU in living systems.

A method to fabricate cylindrical nanopore electrodes is presented. The volume of the cavity formed in the cylindrical nanopore electrode can be as small as several hundred attoliters. It has been characterized by using scanning electron microscopy and electrochemical methods. Our results show that the radius of the cavity can affect the diffusion coefficient of a redox species in the cavity. The cylindrical nanopore electrode has also been used to study charge transfer across the interface between an aqueous phase of several hundred attoliters in volume and a bulk chloroform phase. Compared with the same charge-transfer reaction across the interface between a bulk aqueous phase and a bulk chloroform phase, the potential of the charge-transfer reaction has a negative shift. The effect of the phase ratio on the distribution of the supporting electrolyte in the aqueous and organic phases has been used to explain the shift.

This study demonstrates an improved magnetic protein microsphere-aided sandwich fluoroimmunoassay for the analysis of myoglobin and heart-type fatty acid binding protein (H-FABP), early protein markers associated with acute myocardial infarction. In preparation for the assay we constructed superparamagnetic human serum albumin (HSA)/gamma-Fe(2)O(3) microspheres, and grafted capture antibodies (monoclonal antimyoglobin 7C3 and anti-H-FABP 10E1) onto the protein microspheres using the avidin-biotin system. Then the antibody-carrying microspheres were used in a sequential sandwich fluoroimmunoassay along with detection antibodies (Alexa fluor594-labeled antimyoglobin 4E2 and FITC-labeled anti-H-FABP 9F3). The magnetic HSA/gamma-Fe(2)O(3) microspheres were characterized by scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectrophotometry, atomic absorption spectrophotometry, and vibrating sample magnetometry. Fluorescence images of the post-immunoassay microspheres recorded using an inverted fluorescence microscope showed that the average fluorescence intensity was correlated with the concentration of cardiac markers, in agreement with the results obtained by an F-4500 FL spectrophotometer; this indicated that the fluoroimmunoassay could be used to semiquantitatively detect both myoglobin and H-FABP. The detection limit was 10 ng/mL for myoglobin and 1 ng/mL for H-FABP.

Slightly recessed nanoelectrodes were prepared by controlled etching of nanometer-sized, flat Pt electrodes. By using high-frequency (e.g., 2 MHz) ac voltage, the layer of Pt as thin as greater, approximately >3 nm was removed to produce a cylindrical cavity inside the insulating glass sheath. The etched electrodes were characterized by combination of voltammetry and scanning electrochemical microscopy (SECM) to determine the radius and the effective depth of the recess. The theory was developed for current versus distance curves obtained with a recessed tip approaching either a conductive or an insulating substrate. Good agreement between the theoretical and experimental approach curves indicated that recessed nanotips are suitable for quantitative feedback mode SECM experiments.

A novel method for fabrication of nanometer-sized electrodes and tips suitable for scanning electrochemical microscopy (SECM) is reported. A fine etched Pt wire is coated with polyimide, which was produced by polymerization on the Pt surface initiated by heat. This method can prepare electrodes with effective radii varying from a few to hundreds of nanometers. Scanning electron microscopy, cyclic voltammetry, and SECM were used to characterize these electrodes. Well-defined steady-state voltammograms could be obtained in aqueous or in 1,2-dichloroethane solutions. This method produced the nanoelectrodes with exposed Pt on the apex, and they can also be employed as the nanotips for SECM investigations. Different sizes of Pt nanotips made by this method were employed to evaluate the kinetics of the redox reaction of Ru(NH3)6(3+) on the surface of a large Pt electrode by SECM, and the standard rate constant kappa0 of this system was calculated from the best fit of the SECM approach curve. This result is similar to the values obtained by analysis of the obtained voltammetric data.

As a powerful tool for biological analysis, especially protein structure and dynamic studies, nuclear magnetic resonance (NMR) spectroscopy suffers from intrinsic low signal to nose ratio (SNR) and long acquisition time required for multidimensional (nD) experiments. Nonuniform sampling (NUS) can effectively speed up the experiment but often introduces artifacts into the spectrum. In addition to the development of highly sensitive hardware and NMR pulse sequences, data postprocessing is a relative simple and cost-effective method to improve the SNR and suppress the artifacts. In this work, we propose an effective approach for simultaneously suppressing noise and artifacts based on the resampling principle. The method is named NASR for short and tested using one-, two-, and three-dimensional (1D, 2D, and 3D) NMR spectra that were acquired using ether conventional or NUS (spiral and random, for 3D) approaches. The results reveal that the NASR is fast and applicable for improving the quality of ID to nD NMR spectra with all kinds of sampling schemes.

The fabrication of polishable integrated dual submicrometer-sized electrodes has been reported. The electrode assembly consists of two closely spaced submicrometer-sized platinum disks. It has been accomplished by sealing and pulling platinum (Pt) wires in a theta glass pipet. Monitored by a microscope, the electrodes are polished on a 50 nm diamond lapping tape. Scanning electron microscopy (SEM) and cyclic voltammetry (CV) show that the electrodes are well sealed and are disks. The two submicrometer-sized Pt disk electrodes can work either independently or in the generation-collection model. The shielding effect, feedback effect, and generation-collection efficiency have been discussed.

The kinetics of several fast heterogeneous electron-transfer reactions were investigated by steady-state voltammetry at nanoelectrodes and scanning electrochemical microscopy (SECM). The disk-type, polished Pt nanoelectrodes (3.7-400-nm radius) were characterized by a combination of voltammetry, scanning electron microscopy, and SECM. A number of experimental curves were obtained at the same nanoelectrode to attain the accuracy and reproducibility similar to those reported previously for micrometer-sized probes. A new analytical approximation was developed and used for analysis of steady-state tip voltammograms. The self-consistent kinetic parameter values with the uncertainty margin of approximately 10% were obtained for electrodes of different radii and for a wide range of the SECM tip/substrate separation distances. The determined standard rate constants are compared to those previously measured at the electrodes of different dimensions, and the correlation between the heterogeneous and self-exchange rate constants is discussed.

The electrochemical behavior of pyridine distribution at the water/1,2-dichloroethane interface with variable phase volume ratios (r = VO/VW) was investigated by cyclic voltammetry. The system was composed of an aqueous droplet supported on a Ag/AgCl disk electrode covered with an organic solution or an organic droplet supported on a Ag/AgTPBCl disk electrode covered with an aqueous solution. In this way, a conventional three-electrode potentiostat can be used to study an ionizable compound transfer process at a liquid/liquid interface with a wide range of phase volume ratios (from 0.0004 to 1 and from 1 to 2500). Using this special cell we designed, only very small volumes of both phase were needed for r equal to unity, which is very useful for the investigation of the distribution of ionizable species at a biphasic system when the available amount of species is limited. The ionic partition diagrams were obtained for different phase volume ratios.