Fluorescent Signal Transduction Research Articles

Triplex DNA-based aggregation-induced emission probe: A new platform for hybridization chain reaction-based fluorescence sensing assay

The hybridization chain reaction (HCR), as one of the nucleic acid amplification technologies, is combined with fluorescence signal output with excellent sensitivity, simplicity, and stability. However, current HCR-based fluorescence sensing methods still have some defects such as the blocking effect of the HCR combination with fluorophores and the aggregation-caused quenching (ACQ) phenomenon of traditional fluorophores. Herein, a triplex DNA-based aggregation-induced emission probe (AIE-P) was designed as the fluorescent signal transduction, which is able to provide a new platform for HCR-based sensing assay. The AIE-P was synthesized by attaching the AIE fluorophores to terminus of the oligonucleotide through amido bond, and captured the products of HCR to form triplex DNA. In this case, the AIE fluorophores were located in close proximity to generate fluorescence. This assay provided turn-on fluorescence efficiency with a high signal-to-noise ratio and excellent amplification capability to solve the shortcoming of HCR-based fluorescence sensing methods. It enabled sensitive detection of Vibrio parahaemolyticus in the range of 102–106 CFU mL−1, and with a low limit of detection down to 39 CFU mL−1. In addition, this assay expressed good specificity and practicability. The triplex DNA-based AIE probe forms a universal molecular tool for developing HCR-based fluorescence sensing methods.

TCR ligand potency differentially impacts PD-1 inhibitory effects on diverse signaling pathways.

Checkpoint blockade revolutionized cancer therapy, but we still lack a quantitative, mechanistic understanding of how inhibitory receptors affect diverse signaling pathways. To address this issue, we developed and applied a fluorescent intracellular live multiplex signal transduction activity reporter (FILMSTAR) system to analyze PD-1-induced suppressive effects. These studies identified pathways triggered solely by TCR or requiring both TCR and CD28 inputs. Using presenting cells differing in PD-L1 and CD80 expression while displaying TCR ligands of distinct potency, we found that PD-1-mediated inhibition primarily targets TCR-linked signals in a manner highly sensitive to peptide ligand quality. These findings help resolve discrepancies in existing data about the site(s) of PD-1 inhibition in T cells while emphasizing the importance of neoantigen potency in controlling the effects of checkpoint therapy.

Rapid On-Chip Isolation of Cancer-Associated Exosomes and Combined Analysis of Exosomes and Exosomal Proteins.

Exosomes are lipid bilayer extracellular vesicles secreted by various types of cells and inherit abundant molecular information from parental cells. Tumor-derived exosomes have been widely recognized as noninvasive biomarkers for early cancer diagnosis and surveillance, but the separation of intact exosomes and detection of exosomal proteins remain challenging. Herein, we proposed a microfluidic chip for specific exosome isolation, integrated with sensitive quantification by a novel PTCDI-aptamer signal switch strategy. To enhance the capture efficiency, an alternating drop-shaped micropillar array was designed to assist the capture of tumor-derived exosomes by Tim4-modified magnetic beads (Tim4 beads) on the chip. Following capture, a chelating agent can easily elute intact exosomes which were further used for profiling exosomal surface proteins by the multiplexed fluorescence turn-on approach. Profiting from the efficient on-chip enrichment of the Tim4 beads and superior fluorescence signal transduction strategy, the detection limit of the analysis platform for HepG2 exosomes is as low as 8.69 × 103 particles/mL with a wide linear range spanning 6 orders of magnitude. Meanwhile, the proposed platform could recognize subtle changes in protein levels on the exosomal surface from various cell lines. More importantly, this strategy is successfully applied to analyze exosomes in human serum to distinguish liver cancer patients from healthy individuals. Combined analysis of different types of biomarkers on the exosomal membrane surface can greatly improve the accuracy of cancer type identification and disease monitoring. We hope that this convenient, rapid, and sensitive platform may become a powerful tool in the field of exosome analysis and early cancer screening.

A target-driven DNA-based molecular machine for rapid and homogeneous detection of arginine-vasopressin.

Rapid detection of physiological changes of neuropeptides is of great importance as they are involved in a wide range of physiological processes and behaviors. Abnormalities in their expression level are correlated with various neurological diseases. However, current methods such as radioimmunoassay, enzyme-linked immunosorbent assays and liquid chromatography tandem mass spectrometry relied on cumbersome operation steps and could not rapidly provide the information of their concentration fluctuations. Thus motivated, we developed a target-driven DNA-based molecular machine that could be triggered only in the presence of a specific target neuropeptide. Using arginine-vasopressin (AVP) as a model neuropeptide, we integrated the DNA-based molecular machine with fluorescence signal transduction and amplification technology. The assay was rapid and homogeneous, which offered a linear range of 75-700 pM and a limit-of-detection as low as 75 pM. It holds great potential for further applications in real-time monitoring of the variations of the AVP level in biological samples.

Fluorescent signal transduction in a self-assembled Hg2+ chemosensor tuned by various interactions in micellar aqueous environment

A new OFF-ON fluorescent chemosensor for ions Hg2+ in water has been designed by associating fluorophore 10-methylacridinium perchlorate (MA+ClO4−) with a selective lipophilic sulfur-containing ligand, such as N,N-bis (2-hydroxyethylthio-1-ethyl)dodecyl amine (DBSO). The communication between the two subunits is only due to their spatial closeness, ensured by sodium dodecyl sulfate (SDS) micellar aggregates. The quenching of MA+ fluorescence is ascribed to a photoinduced electron-transfer from the ligand to the excited fluorophore, still the emission is quickly restored by addition of Hg2+ (as chloride salt) due to the complex formation between ligand and metal. The sensitivity of the system improves by decreasing the ligand concentration, as well as by changing the stoichiometry of the ligand-Hg2+ complex from 1:1 to 2:1. DFT calculations give interesting insights into the structures of the complexes and provide useful information on the sensorial activity experimentally observed. The replacement of MA+ClO4− with 9-cyanoanthracene, a fluorophore differently localized on the micellar aggregate, highlights the effect of Cl− coordination to the complexed Hg2+ on the operating mechanism of the sensor. These results demonstrate as the micellar aggregates provide for a straightforward means to regulate the response of the sensorial system without altering the recognition or reporter unit.

Small Molecule Fluorescent Probes for G- Quadruplex Visualization as Potential Cancer Theranostic Agents

G-quadruplexes have gained prominence over the past two decades for their role in gene regulation, control of anti-tumour activity and ageing. The physiological relevance and significance of these non-canonical structures in the context of cancer has been reviewed several times. Putative roles of G-quadruplexes in cancer prognosis and pathogenesis have spurred the search for small molecule ligands that are capable of binding and modulating the effect of such structures. On a related theme, small molecule fluorescent probes have emerged that are capable of selective recognition of G-quadruplex structures. These have opened up the possibility of direct visualization and tracking of such structures. In this review we outline recent developments on G-quadruplex specific small molecule fluorescent probes for visualizing G-quadruplexes. The molecules represent a variety of structural scaffolds, mechanism of quadruplex-recognition and fluorescence signal transduction. Quadruplex selectivity and in vivo imaging potential of these molecules places them uniquely as quadruplex-theranostic agents in the predominantly cancer therapeutic context of quadruplex-selective ligands.

Multi-stimuli-responsive high contrast fluorescence molecular controls with a far-red emitting BODIPY-based [2]rotaxane

A novel fluorescent switchable [2]rotaxane NIR4 composed of two different molecular stations and rotaxane arms terminated with far-red boron-dipyrromethene (BODIPY) fluorophores and its derivatives were synthesized by CuAAC click chemistry. The molecular shuttling motion of mechanically interlocked molecules (MIMs) could be addressed by the fluorescence signal transduction via distance dependent photo-induced electron transfer process of [2]rotaxane NIR4 triggered by external chemical stimuli of acid/base. Moreover, the flexible arms of triazolium moiety in [2]rotaxane NIR4 and its axle NIR2 exhibited impressive selectivity and sensitivity toward complementary anionic analyte (H2PO4−), where the specific mechanical molecular motion was supported by quantum mechanical calculations. The development of [2]rotaxane NIR4 with a high level of structural complexity can be utilized for novel dual sensory detections of acid-base and dihydrogen phosphate (H2PO4−) anion. Importantly, the host of NIR2 and [2]rotaxane NIR4 could be applied for the vitro imaging and clarify the distribution of H2PO4− at subcellular levels.

An 8-aminoquinoline derivative as a molecular platform for fluorescent sensors for Zn(II) and Cd(II) ions

The amide derivatives of 8-aminoquinoline with two types of amino acid pendants, i.e. glycine and β-alanine, are evaluated for fluorescence sensing of various metal ions. In Tris-HCl aqueous buffer solution, the derivative containing glycine exhibits selective fluorescence enhancement with Zn2+. The fluorescence turn-on signal at a longer wavelength is a result of the binding between N-8-aminoquinolinylglycinamide and Zn2+ which is promoted by the deprotonation of the amide proton. However, both Zn2+ and Cd2+ induce the turn-on signal in ethanol solution. X-ray crystallography of single crystals of both Zn2+ and Cd2+ complexes formed in ethanol reveals 1-D coordination polymer structures in which the metal ions are tethered with N and O of the amide groups. Simultaneous detection of Zn2+ and Cd2+ are possible by using paper chromatographic separation and the ligand as a visualizing agent. The fluorescence imaging of either Zn2+ or Cd2+ in plant tissue is demonstrated. The conjugation of the ligand with dimethylaminophenylacetylene effectively tunes the emission color of the ligand while retaining the sensing selectivity. Therefore, N-8-aminoquinolinylglycinamide is a simple yet effective binding probe for Zn2+ and Cd2+ with excellent fluorescence signal transduction.

Ratiometric NanoCluster Beacon: A Label-Free and Sensitive Fluorescent DNA Detection Platform.

Although researches until now have emphasized the influence of an oligonucleotide sequence on the fluorescence of oligonucleotide-stabilized silver nanoclusters (AgNCs), this influence has been explored as a novel ratiometric fluorescent signal transduction in this work. This study builds on our original discovery of a template-transformation phenomenon, which demonstrated that the connection of a special DNA fragment (5'-CACCGCTTT-3') with a green-emitting AgNC nucleation sequence (GNuS, 5'-TGCCTTTTGGGGACGGATA-3') creates a red-emitting AgNC nucleation sequence (RNuS, 5'-CACCGCTTTTGCCTTTTGGGGACGGATA-3'). Attempts to expand this idea and construct elegant ratiometric NanoCluster Beacons (NCBs) for DNA sequence detection are not straightforward, and, thus, we carried out a series of investigations with the goal of understanding the mechanism of this template-transformation phenomenon. Experimental results showed that the six-nucleotide fragment (5'-CACCGC-3') at the 5'-end of RNuS acts as a template convertor and takes full responsibility for the template transformation from GNuS to RNuS. Moreover, we found that the appropriate proximity of the convertor to GNuS also plays a significant role in the template transformation. We then show that the insights gained here for the template-transformation mechanism allow us to construct ratiometric NCBs by simply appending the convertor and the GNuS onto a rationally designed stem-loop probe. This new type of NCB emits intense red fluorescence without the addition of a target DNA and emerges as a new, bright green emission only when hybridized to its target DNA. By measuring the distinct variation in the fluorescence intensity ratios of green and red emission, this ratiometric NCB was demonstrated to sensitively detect Hepatitis-A virus gene sequences, a proof-of-concept target in this work, with good selectivity.

(Invited) In Vivo Carbon Nanotube Sensors

Single-walled carbon nanotubes (SWNT) are particularly attractive for biomedical applications because they exhibit unique optical properties, fluorescing in the near infrared tissue transparency window, and allow for information transfer without the need for an internal power source. Although SWNT have been used as highly-sensitive detectors for various compounds, their use as in vivo biomarkers requires the simultaneous optimization of multiple parameters, including molecular recognition, biocompatibility, high fluorescence quantum efficiency and signal transduction. This work demonstrates two SWNT delivery mechanisms for in vivo detection of an inflammatory factor, nitric oxide (NO), that is associated with numerous diseases, including cancer, but is poorly understood due to its fast degradation rate and the lack of detection devices. First, a polyethylene glycol ligated copolymer is shown to stabilize SWNT sensors in physiological solutions, enabling an intravenous injection into mice. Second, a subcutaneous implant is investigated, with multiple SWNT concentrations, hydrogel shapes and sizes, and the gel’s physical properties examined to determine the preferred material for implantation. We found that both the injected and implanted SWNT are able to detect inflammation within an animal, thus providing the first demonstration of in vivo SWNT sensors as well as the first long term in vivo NO detection modality. This work demonstrates the ability of SWNT sensors to be utilized for in vivo data collection and possibly a wide range of biomedical applications.

Comparison of agonist and antagonist binding sites of the neuropeptide Y receptor 2

e.g. appetite regulation, energy homeostasis, bone formation, circadian rhythm, memory retention, obesity, epilepsy, angiogenesis and cancer (Pedragosa-Badia et al., 2013; Walther et al., 2011). In various tumor tissues the hY2R is overexpressed and promotes tumor growth and vascularization (Lorner and Reubi, 2008). Therefore, the hY2R has great therapeutic potential and antagonists could represent promising drugs for the treatment of neuroblastoma or glioblastoma. Wewere interested in investigating the binding mode of BIIE0246, compound 40 and compound46 at the hY2R (Mittapalli et al., 2012).We generated receptor mutants and tested membrane localization by fluorescence microscopy and signal transduction by inositol phosphate accumulation assay in presence of pNPY and pNPY/antagonist. Here we report on suggested binding modes of three different antagonists on hY2R, which opens up the possibility for the development of more selective compounds.

Graphene oxide-assisted nucleic acids assays using conjugated polyelectrolytes-based fluorescent signal transduction.

In this work, we investigated the interactions between graphene oxide (GO) and conjugated polyelectrolytes (CPEs) with different backbone and side chain structures. By studying the mechanism of fluorescence quenching of CPEs by GO, we find that the charge and the molecular structure of CPEs play important roles for GO-CPEs interactions. Among them, electrostatic interaction, π-π interaction, and cation-π bonding are dominant driving forces. By using a cationic P2, we have developed a sensitive homogeneous sensor for DNA and RNA detection with a detection limit of 50 pM DNA and RNA, which increased the sensitivity by 40-fold as compared to GO-free CPE-based sensors. This GO-assisted CPE sensing strategy is also generic and shows a high potential for biosensor designs based on aptamers, proteins, peptides, and other biological probes.

Reversible fluorescent detection for sulfide with quinoline-ligated copper complexes and its application in living cells

To realized highly selective detection for sulfide anion, a simple and hydrophilic fluorescent chemosensor, sodium 2-(2-(1H-benzo[d]imidazol-2-yl)quinolin-8-yloxy)acetate (HL) based on quinoline derivative was designed and synthesized. Once combined with Cu2+ in aqueous solution, the forming quinoline-ligated copper complex displayed a rapid response with high selective and sensitive detection for sulfide anion. The fluorescent signal transduction occurs via formation-separation of complex (CuL) and CuS, which achieve reversible “ON–OFF–ON” fluorescence change, and the complex (CuL) through the combination of HL and Cu2+ was further confirmed by crystal structure. The detection limits of complex (CuL) to sulfide anion was estimated to be 1.7×10−7M. Furthermore, its potential utility for biological application was explored by fluorescence microscopic imaging in live cells.

In vivo biosensing via tissue-localizable near-infrared-fluorescent single-walled carbon nanotubes

Single-walled carbon nanotubes (SWNT) are particularly attractive for biomedical applications, because they exhibit a fluorescent signal in a spectral region where there is minimal interference from biological media. Although SWNT have been used as highly-sensitive detectors for various molecules, their use as in vivo biosensors requires the simultaneous optimization of various parameters, including biocompatibility, molecular recognition, high fluorescence quantum efficiency and signal transduction. Here we demonstrate that a polyethylene glycol ligated copolymer stabilizes near infrared fluorescent SWNT sensors in solution, enabling intravenous injection into mice and the selective detection of local nitric oxide (NO) concentration with a detection limit of 1 μM. The half-life for liver retention is 4 hours, with sensors clearing the lungs within 2 hours after injection, thus avoiding a dominant route of in vivo nanotoxicology. After localization within the liver, it is possible to follow the transient inflammation using NO as a marker and signalling molecule. To this end, we also report a spatial-spectral imaging algorithm to deconvolute fluorescence intensity and spatial information from measurements. Finally, we show that alginate encapsulated SWNT can function as an implantable inflammation sensor for in vivo NO detection, with no intrinsic immune reactivity or other adverse response, for more than 400 days. These results open new avenues for the use of such nanosensors in vivo for biomedical applications.

Multi-functional core-shell hybrid nanogels for pH-dependent magnetic manipulation, fluorescent pH-sensing, and drug delivery

Remotely optical sensing and drug delivery using an environmentally-guided magnetically-driven hybrid nanogel particle could allow for medical diagnostics and treatment. Such multifunctional hybrid nanogels (<200 nm) were prepared through the first synthesis of magnetic Ni NPs, followed by a moderate growth of fluorescent metallic Ag on the surface of Ni NPs, and then a coverage of a pH-responsive copolymer gel shell of poly(ethylene glycol- co-methacrylic acid) [p(EG-MAA)] onto the Ni-Ag bimetallic NP cores (18 ± 5 nm). The introduction of the pH-responsive p(EG-MAA) gel shell onto the magnetic and fluorescent Ni-Ag NPs makes the polymer-bound Ni-Ag NPs responsive to pH over the physiologically important range 5.0–7.4. The hybrid nanogels can adapt to surrounding pH and regulate the sensitivity in response to external magnetic field (such as a small magnet of 0.1 T), resulting in the accumulation of the hybrid nanogels within the duration from hours to a few seconds as the pH value decreases from 7.4 to 5.0. The pH-dependent magnetic response characteristic of the hybrid nanogels were further integrated with the pH change to fluorescent signal transduction and pH-regulated anticancer drug (a model drug 5-fluorouracil) delivery functions. The hybrid nanogels can overcome cellular barriers to enter the intracellular region and light up the mouse melanoma B16F10 cells. The multiple responsive hybrid nanogel that can be manipulated in tandem endogenous and exogenous activation should enhance our ability to address the complexity of biological systems.

Aptamer beacons for visualization of endogenous protein HIV-1 reverse transcriptase in living cells

Direct visualization of endogenous proteins in living cells remains a challenge. Aptamer beacon is a promising technique to resolve this problem by combining the excellent protein binding specificity of the aptamer with the sensitive signal transduction mechanism of the molecular beacon. In this study, aptamer 93del against HIV-1 reverse transcriptase (RT) was engineered into aptamer beacons to recognize and image HIV-1 RT. The constructed aptamer beacons could specifically bind to HIV-1 RT and the beacon-RT binding showed effective fluorescence signal transduction in homogeneous solution. In solutions with 1μM of the aptamer beacon, the effective fluorescence signal increased with increasing concentration of HIV-1 RT from 0.5μM to 5μM. When the aptamer beacons were delivered into the living cells that transiently expressed HIV-1 RT, HIV-1 RT could be specifically labeled and imaged. The designed aptamer beacons were further successfully applied for RT imaging in HIV-1 integrated U1 cells. The method developed here may be extended to visualize many other endogenous proteins in living cells using appropriate aptamer beacons.

Fluorescent bis-cyclen tweezer receptors for inositol (1,4,5)-trisphosphate

Herein, we report the development of two fluorescent sensors for inositol 1,4,5-trisphosphate (InsP 3), containing either two free cyclen or zinc(II)/cyclen groups preorganized into a binding cleft. Preorganization was achieved using a rigid acridine backbone, which was also exploited for fluorescence signal transduction. In addition, click chemistry was used to facilitate receptor synthesis and produce triazole moieties that add to structural rigidity and recognition. In binding studies, both receptors were found to bind InsP 3 with high affinity ( K a=0.5–1.0×10 6 M −1) in competitive media (1:1 methanol/water).

Fluorescence signal transduction mechanism for immunoassay based on zinc ion release from ZnS nanocrystals

In this work, a fluorescence signal transduction mechanism based on cation release from ZnS nanocrystals was developed for sandwich immunoassay. In this mechanism, ZnS nanocrystals as labels in immunoassay are dissolved by acid to release zinc ions. After pH adjustment of the dissolving solution using a basic solution, zinc-ion sensitive fluorescence indicator Fluozin-3 is added to bind with the released zinc ions for sensitive fluorescence measurement. Using mouse IgG as a model analyte, the immunoassay adopting this signal transduction mechanism demonstrates a low detection limit around 1 pM and a detection range with two orders of magnitude (1 pM to 0.5 nM).

Analysis of allosteric signal transduction mechanisms in an engineered fluorescent maltose biosensor

We previously reported the construction of a family of reagentless fluorescent biosensor proteins by the structure-based design of conjugation sites for a single, environmentally sensitive small molecule dye, thus providing a mechanism for the transduction of ligand-induced conformational changes into a macroscopic fluorescence observable. Here we investigate the microscopic mechanisms that may be responsible for the macroscopic fluorescent changes in such Fluorescent Allosteric Signal Transduction (FAST) proteins. As case studies, we selected three individual cysteine mutations (F92C, D95C, and S233C) of Escherichia coli maltose binding protein (MBP) covalently labeled with a single small molecule fluorescent probe, N-((2-iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), each giving rise to a robust FAST protein with a distinct maltose-dependent fluorescence response. The fluorescence emission intensity, anisotropy, lifetime, and iodide-dependent fluorescence quenching were determined for each conjugate in the presence and absence of maltose. Structure-derived solvent accessible surface areas of the three FAST proteins are consistent with experimentally observed quenching data. The D95C protein exhibits the largest fluorescence change upon maltose binding. This mutant was selected for further characterization, and residues surrounding the fluorophore coupling site were mutagenized. Analysis of the resulting mutant FAST proteins suggests that specific hydrogen-bonding interactions between the fluorophore molecule and two tyrosine side-chains, Tyr171 and Tyr176, in the open state but not the closed, are responsible for the dramatic fluorescence response of this construct. Taken together these results provide insights that can be used in future design cycles to construct fluorescent biosensors that optimize signaling by engineering specific hydrogen bonds between a fluorophore and protein.

Monitoring molecular beacon/DNA interactions using atomic force microscopy.

The molecular beacon (MB) is a new fluorescence probe containing a single-stranded oligonucleotide with a probe sequence embedded in complementary sequences that form a hairpin stem. Due to the inherent fluorescent signal transduction mechanism, an MB functions as a sensitive probe with a high signal-to-background ratio for real-time monitoring and provides a variety of exciting opportunities in DNA, RNA, and protein studies. To better understand the properties of MBs, the specific interactions between MB and target DNA (complementary and one-base mismatch) have been directly investigated by atomic force microscopy. The interaction force between a linear DNA probe and the target DNA was also detected and compared to that between MB and target DNA. The results demonstrate the high specificity of the MB/target DNA compared to the linear DNA/target DNA interaction.