Search

Understanding the pH evolution during endocytosis is essential for our comprehension of the fundamental processes of biology as well as effective nanotherapeutic design. Herein, we constructed a plasmonic Au@PANI core-shell nanoprobe, which possessed significantly different scattering properties under acidic and basic conditions. Encouragingly, the scattering signal of Au@PANI nanoprobes displayed a positive linear correlation with the pH value not only in PBS but also in nigericin-treated cells. Ultimately, benefiting from the excellent response properties as well as the excellent biocompatibility and stability, the Au@PANI nanoprobes have successfully enabled a dynamic assessment of the evolving pH in the endosomal package as the endosome matured from early to late, and eventually to the lysosome, by reporting scattering signal changes.

Electrogenerated chemiluminescence, also known as electrochemiluminescence (ECL), is an electrochemically induced production of light by excited luminophores generated during redox reactions. It can be used to sense the charge transfer and related processes at electrodes via a simple visual readout; hence, ECL is an outstanding tool in analytical sensing. The traditional ECL approach measures averaged electrochemical quantities of a large ensemble of individual entities, including molecules, microstructures and ions. However, as a real system is usually heterogeneous, the study of single entities holds great potential in elucidating new truths of nature which are averaged out in ensemble assays or hidden in complex systems. We would like to review the development of ECL intensity and imaging based single entity detection and place emphasis on the assays of small entities including single molecules, micro/nanoparticles and cells. The current challenges for and perspectives on ECL detection of single entities are also discussed., We summarize the history and recent development that has been made in the ECL detection of single entities.

Plasmonic nanoparticles (e.g., gold, silver) have attracted much attention for biological sensing and imaging as promising nanoprobes. Practical biomedical applications demand small gold nanoparticles (Au NPs) with a comparable size to quantum dots and fluorescent proteins. Very small nanoparticles with a size below the Rayleigh limit (usually, An absorption modulated scattering microscopy technique that allows for the imaging of sub-10 nm gold nanoparticles within a cellular scattering medium is presented.

Development of simple and effective synergistic therapy by combination of different therapeutic modalities within one single nanostructure is of great importance for cancer treatment. In this study, by integrating the anticancer drug DOX and plasmonic bimetal heterostructures into zeolitic imidazolate framework-8 (ZIF-8), a stimuli-responsive multifunctional nanoplatform, DOX-Pt-tipped Au@ZIF-8, has been successfully fabricated. Pt nanocrystals with catalase-like activity were selectively grown on the ends of the Au nanorods to form Pt-tipped Au NR heterostructures. Under single 1064 nm laser irradiation, compared with Au NRs and Pt-covered Au NRs, the Pt-tipped Au nanorods exhibit outstanding photothermal and photodynamic properties owing to more efficient plasmon-induced electron–hole separation. The heat generated by laser irradiation can enhance the catalytic activity of Pt and improve the O2 level to relieve tumor hypoxia. Meanwhile, the strong absorption in the NIR-II region and high-Z elements (Au, Pt) of the DOX-Pt-tipped Au@ZIF-8 provide the possibility for photothermal (PT) and computed tomography (CT) imaging. Both in vitro and in vivo experimental results illustrated that the DOX-Pt-tipped Au@ZIF-8 exhibits remarkably synergistic plasmon-enhanced chemo-phototherapy (PTT/PDT) and successfully inhibited tumor growth. Taken together, this work contributes to designing a rational theranostic nanoplatform for PT/CT imaging-guided synergistic chemo-phototherapy under single laser activation., A plasmon-enhanced theranostic nanoplatform for synergistic chemo-phototherapy (PTT/PDT) of hypoxic tumors in the NIR-II window.

Plasmonic photocatalysts have opened up a new direction in utilization of visible light and promoting photocatalytic efficiency. An electrochemical deposition method is reported to synthesise metal@semiconductor (M@SC) core-shell nanocrystals. Due to the strong affinity of Au atoms to S2- and Se2- reduced at negative potential, CdS, CdSe and ZnS were selectively deposited on the surface of the Au core to form a uniform shell with a clear metal/semiconductor interface, which conquered the barrier caused by the large lattice mismatch between the two components. Plasmonic effects increased the photocatalytic performance, as well as provided a chance to in situ monitor the surface nucleation and growth. The structure formation process could be observed under dark-field microscopy (DFM) in real-time and precisely controlled via the scattering color, intensity and wavelength. The proof-of-concept strategy combines the electrochemical deposition and plasmonic imaging, which provides a universal approach in controllable synthesis of core-shell heterostructures, and leads to the improvement of plasmonic photocatalysts.

Uncovering the relationship between the structure, surface properties and electrochemical activity of nanoparticles is of great importance for constructing novel nanocatalysts and highly efficient electrocatalytic devices. Here we report a study of the heterogeneously distributed electrocatalytic activity on individual 2D gold nanoplates. On the basis of electrogenerated chemiluminescence (ECL) microscopy, the size, shape, and site-specific catalytic activity of 2D nanocrystals could be directly imaged at the single particle level with submicron resolution. Since the microelectrode effect with higher fluxes at the perimeter was offset by diffusion of excited species of Ru(bpy)32+, calculated by finite element simulation, the ECL distribution was supposed to be uniform on the micro-sized plates. Therefore, it is highly possible that the observed nonuniform ECL distribution at single nanoplates reflected distinct surface electrocatalytic activities at different sites. Furthermore, ECL microscopy allows continuous in situ imaging, which elucidates the time-varying changes in the spatial distribution of electrocatalytic activity on individual nanoplates, indicating that the corners and edges with more defect sites exhibit higher reactivity, but lower stability than the flat facet. We believe that real-time and high-throughput ECL microscopy may lead to more comprehensive understanding of reactivity patterns of single nanocatalysts.

Development of tumor microenvironment responsive and modulating theranostic nano-systems is of great importance for specific and efficient cancer therapy. Herein, we report a redox-sensitive nanoagent combining manganese dioxide (MnO2) and gold nanoshell coated silicon nanoparticles for synergistic chemo-photothermal therapy of hypoxia solid tumors. In highly reducing tumor tissues, the outer MnO2 nanosheet with the loaded drug would be dissociated by intracellular glutathione (GSH), resulting in on-demand drug release, as well as generating Mn2+ ions which provided high contrast magnetic resonance imaging (MRI), and fluorescence imaging (FI) in vitro and in vivo. While upon near-infrared (NIR) light irradiation, the gold nanoshell modulated the hypoxic tumor microenvironment via increasing blood flow, achieving enhanced photothermal therapy (PTT) and chemotherapy. After tail vein injection into tumor-bearing mice and monitoring in real time, the intelligent redox-activated nanoagent exhibited high tumor accumulation and powerful synergistic chemo-photothermal therapy efficiency. The proposed work developed a noninvasive strategy to modulate the tumor microenvironment and enhance the anticancer therapeutic effect. We believe that this single nano-platform exhibits promising potential as a comprehensive theranostic agent to enhance the efficacies of synergistic cancer therapy.

Hydrogen sulfide (H2S) has been recognized as an important endogenous gasotransmitter associated with biological signaling transduction. However, recent biological studies implied that the H2S-related cellular signaling might actually be mediated by hydrogen polysulfides (H2Sn, n > 1), not H2S itself. Unraveling such a mystery strongly demanded the quantification of endogenous H2Sn in living systems. However, endogenous H2Sn has been undetectable thus far, due to its extremely low concentration within cells. Herein, we demonstrated a strategy to detect ultra-trace endogenous H2Snvia a fluorescent τ-probe, through changes of fluorescence lifetime instead of fluorescence intensity. This τ-probe exhibited an ultrasensitive response to H2Sn, bringing about the lowest value of the detection limit (2 nM) and a lower limit of quantification (10 nM) to date. With such merits, we quantified and mapped endogenous H2Sn within cells and zebrafish. The quantitative information about endogenous H2Sn in cells and in vivo may have a significant implication for future research on the role of H2Sn in biology. The methodology of the τ-probe established here might provide a general insight into the design and application of any fluorescent probes, beyond the limit of utilizing fluorescence intensity.

Regulation of gene expression is highly important in the area of cell biology. In this work a novel convenient and versatile strategy is reported which permits both gene regulation and imaging in living cells. An oligonucleotide-based biomimetic probe was designed to target an RNA-induced silencing complex (RISC) and served as an agent for the modulation of c-Myc protein expression in living cells through regulating the RNA interference (RNAi) pathway. In this probe, a DNA strand (Strand1) serving as the frame was immobilized on a AuNP with a thiol group at the 5′ end. Strand2, designed to recognize the target RISC with an RNA fragment, was hybridized with the complementary sequence of Strand1. In the original state, the fluorescence of the Cy3 modifier at the 5′ end of Strand2 was quenched by both the AuNP and BHQ2, which labelled the 3′ end of Strand1. In the presence of RISC, Strand2 was cleaved, resulting in a shorter oligo part with a corresponding lower melting temperature than that of the original full-length Strand2. The shorter oligonucleotide strand containing the Cy3 fluorophore was released, accompanied by a recovered fluorescence signal. Through evaluating the fluorescence intensity, the competition for RISC was dynamically monitored in single cells. Furthermore, capturing RISC by this probe resulted not only in restored fluorescence intensity but also increased c-Myc oncogene expression. Hence, gene expression could be selectively and precisely regulated and imaged via the RISC targeting probe. The synthetic method for the biomimetic probe is universally applicable, and facilitates the fundamental study of RNAi pathways, or development of a gene regulation strategy without cytokine activation. The gene regulation and imaging strategy will accelerate the unveiling of the basic role of the RISC cleavage interaction, the mystery of RNA-silencing and therapeutic monitoring of cancer.

A FRET-based strategy is developed to image and track specific newly synthesized endogenous proteins in situ., Metabolic azide amino acid labelling followed by the use of bioorthogonal chemistry is an efficient technique for imaging newly synthesized proteins. Recently, AHA-labelling together with the proximity-ligation assay was used to identify newly synthesized proteins of interest (POI) (Tom Dieck et al., Nat. Meth. 2015, 12, 411). Here we build on this study replacing the proximity-ligation assay with FRET to improve the spatial resolution. Herein, we develop a FRET-based strategy for imaging the newly synthesized endogenous POI within cells: a FRET acceptor is installed onto the newly synthesized proteins via click chemistry, and a FRET donor onto the POI via immunocytochemistry. We found that a photobleaching based FRET efficiency imaging mode and a fluorescence lifetime imaging mode showed the distribution of newly synthesized proteins more accurately compared to the direct observation of FRET signals. We demonstrated the capability of this FRET-based imaging method by visualizing several newly synthesized proteins including TDP-43, tubulin and CaMKIIα in different cell lines. This novel analytical imaging method could be used to visualize other specific endogenous proteins of interest in situ.

Apoptosis and necrosis are distinct cell death processes related to many cellular pathways. In situ, quantitatively and dynamically monitoring such processes may provide vitally important information for cell studies. However, such a method still remains elusive, even though current immunochemical methodologies have developed extremely valuable tools. Herein, we demonstrate Raman spectroscopic metrics for validating and quantifying apoptotic and necrotic cells based on their distinct molecular vibrational fingerprints. It not only allows us to quantify apoptotic and necrotic cell populations in situ in adherent cell samples, but also to be capable of continuously monitoring the dynamical processes of apoptosis and necrosis at the same time in one sample. This method provides comparable results with the “gold standard” of flow cytometry, moreover, with several incomparable advantages. Our work offers a powerful new tool for cell apoptosis and necrosis assays and is expected to become a benchmark technology in biological and medical studies.

A proof-of-concept strategy for a circulating miRNA assay using a personal glucose meter (PGM) was proposed., The development of non-invasive techniques for the diagnosis of cancer, characterization of mutation and monitoring treatment response could greatly reduce the morbidity and mortality caused by cancer. Nevertheless, the extremely low amount of cell free nucleic acids makes liquid biopsy a very challenging task. Herein, taking advantage of the pocket size, reliable quantitative results and simple operation of the pocket-sized personal glucose meter (PGM), we report an approach of circulating microRNA-21 (miR-21) detection with high precision and low cost. Via target-induced release of invertase from the DNA–invertase conjugate, which could convert sucrose into glucose, the detection of miR-21 in serum was linked to PGM readings. Combining the DNAzyme feedback amplification (DFA) program and highly efficient enzymatic turnover, an ultralow detection limit of 7 × 10–16 M for miR-21 was achieved using a PGM as the reporter. The high sensitivity and selectivity of the proposed method meets the requirement of quantifying cell free nucleic acids in serum. In addition, this approach fills the shortage of quantitative RT-PCR and next-generation sequencing in quantifying miRNAs with a short length and greatly reduces the cost of detection. We believe that widely used personal diagnosis devices could hold an important place in the booming area of liquid biopsy.

Due to the complexity of biological systems and the ultralow concentration of analytes, improving the signal-to-noise ratio and lowering the limit of detection to allow highly sensitive detection is key to biomolecule analysis, especially intracellular analysis. Here, we present a method for highly sensitive imaging of mRNA in living cells by using novel invisible oriented probes to construct a turn-on signal generation mechanism from zero background. Two DNA probes (S1 and S2) are asymmetrically modified on two small gold nanoparticles (AuNPs) with a diameter of 20 nm. The hybridization of the two DNA probes with a single target mRNA leads to the formation of an AuNP dimer which shows a prominent plasmonic coupling effect. It generates a strong scattering signal from zero-background under a dark-field spectral analysis system. The unique design of the oriented assembly dimer has the ability to easily discriminate the target signal from the inherent cellular background noise in intracellular detection, thus making this approach a valuable technique for imaging single survivin mRNA and monitoring the distribution of survivin mRNA in tumor cells.

Fingerprints are a unique characteristic of an individual. Recently, it has been realized that fingerprints carry more information about individuals than just their identity, for example, they may identify potential addicts and terrorists carrying explosives. Therefore, the development of imaging moieties capable of both fingerprint staining and drug/explosive visualization is of significant importance for forensic chemistry. Here we developed a nanohybrid comprising green- and red-emitting QDs for simultaneous fingerprint imaging and TNT visualization in fingerprints. The red-emitting Cu-doped ZnCdS (Cu–ZnCdS) QDs were embedded into silica nanoparticles and the green-emitting ZnCdS QDs were anchored onto the surface of the silica nanoparticles and further functionalized with polyallylamine (PAA). Both components of the nanohybrid, i.e., the PAA-functionalized green QDs and red QD-doped silica nanoparticles, could be explored for fingerprint imaging. Due to the formation of a Meisenheimer complex between TNT and PAA, the green-emitting QDs could be quenched by TNT, meanwhile the red-emitting QDs were inert. Therefore, the nanohybrid exhibited a traffic light-type fluorescence color change (green-yellow-red) to TNT concentration in the range of 40–400 μM. This method is promising for potential applications in security-screening needs in public areas such as airports and train stations.

Three endogenous biothiols in single cells were simultaneously quantified by plasmonic Raman probes and quantitative principal component analysis (qPCA)., Intracellular biothiols mediate many important physiological and pathological processes. Due to their low content and competing thiol-reactivity, it is still an unmet challenge to quantify them within a complicated intracellular environment. Herein, we demonstrated a strategy to discriminate three biothiols, i.e. cysteine (Cys), homo-cysteine (Hcy) and glutathione (GSH), and quantify their concentrations within single living cells, using one platform of Raman probe. By monitoring the reaction kinetics of biothiols with Raman probes and discriminating their products with a quantitative principal component analysis (qPCA) method, these three biothiols could be simultaneously quantified in both cell lysis and single living cells. The concentrations of Cys, Hcy and GSH in single Hela cells were 158 ± 19 μM, 546 ± 67 μM and 5.07 ± 0.62 mM, respectively, which gives the precise concentrations of these three biothiols at a single cell level for the first time. This method provides a general strategy for discriminating each component from a mixed system and has potential for quantifying any biomolecules within an in vitro or in vivo biological environment.

A versatile strategy is reported which permits gene regulation and imaging in living cells via an RNA interference antagonistic probe., Regulation of gene expression is highly important in the area of cell biology. In this work a novel convenient and versatile strategy is reported which permits both gene regulation and imaging in living cells. An oligonucleotide-based biomimetic probe was designed to target an RNA-induced silencing complex (RISC) and served as an agent for the modulation of c-Myc protein expression in living cells through regulating the RNA interference (RNAi) pathway. In this probe, a DNA strand (Strand1) serving as the frame was immobilized on a AuNP with a thiol group at the 5′ end. Strand2, designed to recognize the target RISC with an RNA fragment, was hybridized with the complementary sequence of Strand1. In the original state, the fluorescence of the Cy3 modifier at the 5′ end of Strand2 was quenched by both the AuNP and BHQ2, which labelled the 3′ end of Strand1. In the presence of RISC, Strand2 was cleaved, resulting in a shorter oligo part with a corresponding lower melting temperature than that of the original full-length Strand2. The shorter oligonucleotide strand containing the Cy3 fluorophore was released, accompanied by a recovered fluorescence signal. Through evaluating the fluorescence intensity, the competition for RISC was dynamically monitored in single cells. Furthermore, capturing RISC by this probe resulted not only in restored fluorescence intensity but also increased c-Myc oncogene expression. Hence, gene expression could be selectively and precisely regulated and imaged via the RISC targeting probe. The synthetic method for the biomimetic probe is universally applicable, and facilitates the fundamental study of RNAi pathways, or development of a gene regulation strategy without cytokine activation. The gene regulation and imaging strategy will accelerate the unveiling of the basic role of the RISC cleavage interaction, the mystery of RNA-silencing and therapeutic monitoring of cancer.

Rapid and sensitive detection of tumor biomarkers plays a critical role in the early diagnosis of cancer and better understanding of disease progression. We report here a novel wireless electrochemiluminescence (ECL) strategy for visualizing prostate-specific antigen (PSA) on the basis of electrical switch control of ECL generation on bipolar electrodes (BPEs). The visual device comprises a two-channel microfluidic chip with two indium tin oxide (ITO) bands with a gap of 200 μm. The gap between the two ITO bands in one channel could be regarded as an electrical switch, the conductivity of which controls ECL generation on the ITO bands in the other channel, which are used as bipolar electrodes (BPEs). The electronic conductivity of the electrical switch could be tuned by PSA guided silver particles deposition via an immunosandwich assembly and a silver enhancement strategy. At the “on” state of the electrical switch, PSA induced deposition of silver particles forms an electronic circuit between the adjacent BPEs and makes them behave like a continuous H-shaped BPE, which results in only one ECL signal. Meanwhile, the external voltage for driving the oxidation reactions of Ru(bpy)32+ and TPA is significantly reduced compared with the “off” state. This important characterization of the electrical switch could eliminate the background signal and enable a sensitive measurement of PSA by observing the ECL lightspots on BPEs, providing a simple and sensitive visual means of detecting cancer biomarkers. Besides, this two-channel design avoids the chemical interference between sensing and reporting reactions. Combining the advantages of BPE and the high visual sensitivity of the electrical switch, it could be easily expected to achieve sensitive screening of other biomarkers.