Fluorophore Cy5 Research Articles

Construction of a sensitive protease sensor with DNA-peptide conjugates for single-molecule detection of multiple matrix metalloproteinases

Protease expression is closely linked to various pathological phenomena, and their accurate quantification is essential to clinical diagnosis and cancer therapy. Herein, we demonstrate for the first time the construction of a sensitive protease sensor by integrating protease-sensitive cleavage with nicking enzyme-assisted signal amplification (NESA) for single-molecule detection of multiple matrix metalloproteinases (MMPs). This protease sensor involves two DNA-peptide conjugates which contain both specific protease cleavage sites and trigger DNAs and two report DNAs which are modified with a fluorophore (Cy3 or Cy5) and a quencher (BHQ2). In the presence of specific MMPs, MMPs-mediated cleavage reactions lead to the release of specific trigger DNAs from the corresponding DNA-peptide conjugates. After the magnetic separation, the resultant trigger DNAs may hybridize with the corresponding report DNAs to initiate the cyclic NESA reaction, releasing large amounts of Cy3/Cy5 fluorescent molecules which can be simply quantified by using total internal reflection fluorescence-based single-molecule detection. Taking advantage of the high specificity of proteolytic cleavage, the high amplification efficiency of cyclic NESA, and the high sensitivity of single-molecule detection, this protease sensor can simultaneously detect multiple MMPs with a detection limit of 3.33 pM for MMP-2 and 1.71 pM for MMP-7, superior to the target peptide-based methods. Moreover, this protease sensor can be applied for the measurement of MMP-2 and MMP-7 in cancer cells and the screening of protease inhibitors, holding great promise in clinic diagnosis and drug discovery.

Evaluation of asymmetric orthogonal cyanine fluorophores

Pentamethine cyanine (Cy5) fluorophores have proven to be versatile imaging agents (i.e., tracers) for a range of micro- and macroscopic imaging applications, including image-guided surgery. In this study the relationship between the structure of asymmetric Cy5 fluorophores and their photophysical properties was studied. To this end, seven Cy5 analogues, bearing orthogonal N-indole substituents (H, SO3−, or benzene), were synthesised and evaluated. In-depth analysis revealed that introduction of sulfonates enhanced the fluorescence brightness and photostability, while reducing the lipophilicity, serum binding and stacking tendency. The addition of benzene moieties induced a bathochromic shift of 10–20 nm, increased the lipophilicity (LogP = -1.56–1.23) and serum binding (67.3–93.8% bound), as well as negatively impacted the brightness (0.74–42.9 · 103 M−1 cm−1), photostability (24.4–90.6% remaining), and stacking tendency. Chemical stability was uninfluenced by the substitution pattern. Additionally, the generation of a c[RGDyK]-based hybrid tracer based on one of these fluorophores in combination with a diethylenetriaminepentaacetic acid (DTPA) chelate and an 111In-isotope was reported. This compound was evaluated in vitro using αvβ3-overexpressing Geβ3 cells and in vivo using a 4T1 mouse tumour model. Overall, the presented results imply that alterations of the asymmetrical orthogonal Cy5 fluorophore structure have impact on the (photo)physical properties. Furthermore, the orthogonal Cy5 fluorophore framework can readily be applied in tracer development.

A split aptamer sensing platform for highly sensitive detection of theophylline based on dual-color fluorescence colocalization and single molecule photobleaching

A new split aptamer sensing platform is developed for highly sensitive and selective detection of theophylline based on single molecule photobleaching (SMPB) technique. The sensing system contains two probes. One is formed by one streptavidin and four biotinylated RNA fragments labelled with fluorescein isothiocyanate (FITC). Each biotinylated RNA fragment contains two repeating aptamer fragments. The other probe is the complementary aptamer fragment labelled with Cy5 dye. The existence of theophylline can trigger the first probe to bind as many as eight Cy5-labelled probes. The average combined number depends on the theophylline concentration and can be measured by SMPB technique. In the sensing system, the dual-color fluorescence colocalization is performed by the red fluorophore (Cy5) and green fluorophore (FITC), in which the red fluorophore is utilized for quantitative counting of photobleaching steps, while the green fluorophore serves as a counting reference to increase detection efficiency. On basis of the principle, an ultra-sensitive sensing platform of theophylline is created with a low limit of detection (LOD) of 0.092 nM. This work provides not only a highly sensitive method for theophylline detection but also a novel perspective for the applications of SMPB technology to construct biosensors.

Membrane Interactions of Cy3 and Cy5 Fluorophores and Their Effects on Membrane-Protein Dynamics

Organic fluorophores, such as Cy3 and Cy5, have been widely used as chemical labels to probe the structure and dynamics of membrane proteins. Although a number of previous studies have reported on the possibility of some of the water-soluble fluorophores to interact with lipid bilayers, detailed fluorophore-lipid interactions and, more importantly, the potential effect of such interactions on the natural dynamics of the labeled membrane proteins have not been well studied. We have performed a large set of all-atom molecular dynamics simulations employing the highly mobile membrane mimetic model to describe spontaneous partitioning of the fluorophores into lipid bilayers with different lipid compositions. Spontaneous membrane partitioning of Cy3 and Cy5 fluorophores captured in these simulations proceeds in two steps. Electrostatic interaction between the fluorophores and the lipid headgroups facilitates the initial, fast membrane association of the fluorophores, followed by slow insertion of hydrophobic moieties into the lipid bilayer core. After the conversion of the resulting membrane-bound systems to full-membrane representations, biased-exchange umbrella sampling simulations are performed for free energy calculations, revealing a higher energy barrier for partitioning into negatively charged (phosphatidylserine or phosphatidylcholine) membranes than purely zwitterionic (phosphatidylcholine or phosphatidylethanolamine) ones. Furthermore, the potential effect of fluorophore-lipid interactions on membrane proteins has been examined by covalently linking Cy5 to single- and multipass transmembrane helical proteins. Equilibrium simulations show strong position-dependent effects of Cy5-tagging on the structure and natural dynamics of membrane proteins. Interactions between the tagged protein and Cy5 were also observed. Our results suggest that fluorophore-lipid interactions can affect the structure and dynamics of membrane proteins to various extents, especially in systems with higher structural flexibility.

Profiling Glucose-Stimulated and M3 Receptor-Activated Insulin Secretion Dynamics from Islets of Langerhans Using an Extended-Lifetime Fluorescence Dye.

Pulsatile insulin from pancreatic islets is crucial for glucose homeostasis, but the mechanism behind coordinated pulsatility is still under investigation. One hypothesis suggests that cholinergic stimulation of islets by pancreatic ganglia resets these endocrine units, producing synchronization. Previously, it was shown that intracellular Ca2+ oscillations within islets can be entrained by pulses of a cholinergic agonist, carbachol (CCh). Although these proxy measurements of Ca2+ provided insight into the synchronization mechanism, measurement of insulin output would be more direct evidence. To this end, a fluorescence anisotropy competitive immunoassay for online insulin detection from single and grouped islets in a microfluidic system was developed using a piezoelectric pressure-driven fluid delivery system and a squaraine rotaxane fluorophore, SeTau-647, as the fluorescent label for insulin. Due to SeTau-647 having a longer lifetime and higher brightness compared to the previously used Cy5 fluorophore, a 45% increase in the anisotropy range was observed with enhanced signal-to-noise ratio (S/N) of the measurements. This new system was tested by measuring glucose-stimulated insulin secretion from single and groups of murine and human islets. Distinct islet entrainment of groups of murine islets by pulses of CCh was also observed, providing further evidence for the hypothesis that pulsatile output from the ganglia can synchronize islet behavior. We expect that this relatively straightforward, homogeneous assay can be widely used for examining not only insulin secretion but other secreted factors from different tissues.

A suite of polymerase chain reaction-based peptide tagging plasmids for epitope-targeted enzymatic functionalization of yeast proteins.

The budding yeast and model eukaryote Saccharomyces cerevisiae has been invaluable for purification and analysis of numerous evolutionarily conserved proteins and multisubunit complexes that cannot be readily reconstituted in Escherichia coli. For many studies, it is desirable to functionalize a particular protein or subunit of a complex with a ligand, fluorophore or other small molecule. Enzyme-catalysed site-specific modification of proteins bearing short peptide tags is a powerful strategy to overcome the limitations associated with traditional nonselective labelling chemistries. Towards this end, we developed a suite of template plasmids for C-terminal tagging with short peptide sequences that can be site-specifically functionalized with high efficiency and selectivity. We have also combined these sequences with the FLAG tag as a handle for purification or immunological detection of the modified protein. We demonstrate the utility of these plasmids by site-specifically labelling the 28-subunit core particle subcomplex of the 26S proteasome with the small molecule fluorophore Cy5. The full set of plasmids has been deposited in the non-profit plasmid repository Addgene (http://www.addgene.org).

Fluorescence immunoassay for multiplex detection of organophosphate pesticides in agro-products based on signal amplification of gold nanoparticles and oligonucleotides

Herein, we developed a multi-analyte fluorescence immunoassay for detection of three organophosphate pesticides (triazophos, parathion, and chlorpyrifos) in various agro-products (rice, wheat, cucumber, cabbage, and apple) using fluorescently labeled oligonucleotide and gold nanoparticle (AuNP) signal amplification technology. The AuNP probes for the three analytes were constructed by simultaneously modifying the corresponding antibodies and fluorescently labeled oligonucleotides on the probe surface. Three fluorophores (6-FAM, Cy3, and Texas red) with high fluorescence intensity and little overlap of excitation/emission wavelengths were selected. The method showed satisfactory linearity for triazophos, parathion, and chlorpyrifos in the ranges of 0.01–20, 0.05–50, and 0.5–1000 μg/L, respectively. For the 3 analytes, the limits of detection (LODs) were 0.007, 0.009, and 0.087 μg/L, respectively. The average recoveries were 77.7–113.6%, with relative standard deviations (RSDs) of 7.1–17.1% in various food matrices. The proposed method offers great potential in food safety surveillance, and could be used as well as a reference for multi-residue analysis of other small-molecule contaminants.

A well-designed Gold nanoparticle based fluorescence probe for assay Argonaute2 and Let-7a interaction in living cells

We have carefully designed a probe for in situ detection and imaging of Argonaute2 (Ago2), a key RNA interference (RNAi) protein in human cells and siRNA (Let-7a) interactions. The probe mainly contains two parts: a gold nanoparticle (AuNP) being quenched and a functionalized nucleic acid as a fluorescent output carrier. After the probe is endocytosed into the cell, the modified hairpin nucleic acid can be cut into two parts by the Ago2/Let-7a complex (ALC). The cut nucleic acid will replace a DNA strand modified on another duplex nucleic acid. The replaced DNA strand’s 3’-end is modified with a fluorophore (Cy3). By cascading single cells, the probe can directly achieve the fluorescence recovery of the probe in the cell. By dynamically monitoring the in-situ quantitative response of the interaction between Ago2 and Let-7a, and distinguishing between strongly RNA-expressing cells and other cells, the interaction between Ago2 and Let-7a can be monitored dynamically, proving the utility of the probe. Probes will accelerate the basic role of reducing protein-RNA interactions, the mystery of RNA silencing, and monitoring the treatment of cancer.

Switch-conversional ratiometric fluorescence biosensor for miRNA detection

MicroRNAs play a crucial role in regulating gene expression and cellular function. Reliable detection of miRNA is highly demanded in clinical diagnosis and therapy. Herein, we designed a structure-convertible DNA switch and constructed a novel switch-conversional ratiometric fluorescence biosensor (SCRF biosensor) for highly sensitive miRNA detection by the use of amplicon fragments to convert the structure of the switch. The DNA switch was a sophisticated designed single-strand DNA with a stem-loop structure and modified with two fluorophores (Cy3 and Cy5) and one quencher at specific sites of the switch. Amplicon fragments (c*) were produced by an exponential amplification reaction. When the c* hybridized to the loop of a DNA switch, the structure of the switch would convert, and fluorescence resonance energy transfer occurred between Cy5 and Cy3. Then two fluorescence signals with different trends would be observed. As a result, by the ratio of the two signals, we can quantitatively and quickly detect the target miRNA with the concentration range from 100 fM to 100 nM and the excellent detection limit down to 70.9 fM, providing this new SCRF biosensor broad application prospects.

Investigating Nucleosome Stability via FRET

DNA's susceptibility to oxidative damage leads to the formation of the mutagenic Spiroiminodihydantoin (Sp) lesion. The Sp lesion contains a chiral center that disrupts the DNA double helix. When the Sp lesion is not repaired, it may lead to permanent mutations, apoptosis, cellular aging, or cancer. Previous work in our lab suggests that the Sp lesion can cause changes in nucleosome stability and DNA positioning. A nucleosome is the fundamental level of DNA packaging into chromosomes and forms when 146 base pair DNA duplex wraps around a histone octamer. This structure regulates access to DNA by repair proteins, transcription factors, and polymerases. With the use of Florescence Resonance Energy Transfer (FRET), we can examine how the Sp lesion affects nucleosome stability. We synthesized 146-mer duplexes with Cy3 and Cy5 fluorophores with Sp lesions at three potential locations. Then, we assembled nucleosome core particles with fluorescent donor-acceptor pairs on the DNA. FRET is a function of the proximity of the donor and acceptor fluorophores. Thus, when nucleosomes are gradually titrated with NaCl, DNA ends begin peeling and leads to a loss in acceptor fluorescence. With FRET, we are quantitatively measuring the correlation between nucleosome stability and location of the Sp lesion. We hope to advance our understanding of how the Sp lesion influences DNA compaction into nucleosomes.

Nondestructive analysis of tumor-associated membrane protein MUC1 in living cells based on dual-terminal amplification of a DNA ternary complex.

Non-destructive analysis of cells at the molecular level is of critical importance for cell research. At present, immunoassay-based and aptamer-based methods can achieve non-structural destructive cell analysis, but still lead to changes in cells at the molecular level. Here, we have proposed a dual-terminal amplification (DTA) strategy, which enables nondestructive analysis of membrane protein MUC1 without the effect on protein expression and cell viability in living cells.Methods: A fluorophore (Cy5)-labeled DNA ternary complex consisting of three oligonucleotides is designed. It can recognize MUC1 through its aptamer region, and thus make the MUC1 of cells visible under a fluorescence microscope. When DNA polymerase is added, dual-terminal amplification is performed. One direction dissociates aptamer from MUC1, and the other direction, also known as rolling circle amplification (RCA), produces long linear DNA strands, which can be further adopted for quantitative analysis of MUC1. In this way, all reagents are removed from the surface of the cells after the analysis, which allows nondestructive analysis. We named this strategy dual-terminal amplification (DTA) analysis.Results: By using the DTA analysis, both in situ fluorescence imaging analysis and ex situ fluorescence quantitative analysis of MUC1 were achieved. In addition, the aptamer-containing DNA ternary complex stays on cell surface only during the analysis and leaves the cell after the analysis is complete. The cells can be maintained in a non-interfering state for the rest of the time. So after the analysis, it is found that there are no effect on the physiological activity of cells and the expression of target protein even after two rounds of repeatable imaging and quantitative analysis.Conclusion: In summary, we have successfully constructed a strategy for nondestructive analysis of membrane protein in living cells. We believe that this method provides a promising way for the analysis of the key membrane proteins of cells and the versatile utilization of precious cell samples.

Targeted Imaging Agent to HSP70 Induced In Vivo.

Heat shock protein expression can be induced by heat shock making it possible to artificially modulate their levels noninvasively in vivo in a spatially and temporally controlled manner. Here, we report the use of the major heat shock protein 70 (HSP70) as an inducible target by using the small molecule deoxyspergualin (DSG) conjugated to the near-infrared fluorophore (Cy5.5). We demonstrate that heat induction in the form of localized hyperthermia of normal tissue in living mice results in sufficient HSP70 overexpression for detection with DSG-Cy5.5 conjugate. This effect is dependent on total energy delivered and reaches maximum fluorescence signal in 6 to 8 hours post heat induction and declines over a period of up to 24 hours. These results suggest that DSG-Cy5.5 agent accumulates in tissue with elevated HSP70 by heat.

Detection of Small Subsets of CD4+ Lymphocytes with SmartFlare Nanoprobes.

SmartFlare technology allows detection of mRNA in single living cells. We studied the possibility of using SmartFlare nanoprobes for detection of small subsets of CD4+ lymphocytes. It was found that SmartFlare allows detection of transcriptional master regulators of major CD4+T helper subsets in living human lymphocytes. Nanoprobes labeled with Cy5 fluorophore were better detected by flow cytometry than nanoprobes labeled with Cy3. Appropriate time of lymphocyte incubation with SmartFlare probes was 24 h.

Size and affinity kinetics of nanobodies influence targeting and penetration of solid tumours

A compound's intratumoural distribution is an important determinant for the effectiveness of molecular therapy or imaging. Antibodies (Abs), though often used in the design of targeted compounds, struggle to achieve a homogenous distribution due to their large size and bivalent binding mechanism. In contrast, smaller compounds like nanobodies (Nbs) are expected to distribute more homogenously, though this has yet to be demonstrated in vivo at the microscopic level. We propose an intravital approach to evaluate the intratumoural distribution of different fluorescently labeled monomeric and dimeric Nb tracers and compare this with a monoclonal antibody (mAb).Monomeric and dimeric formats of the anti-HER2 (2Rb17c and 2Rb17c-2Rb17c) and control (R3B23 and R3B23-R3B23) Nb, as well as the dimeric monovalent Nb 2Rb17c-R3B23 were generated and fluorescently labeled with a Cy5 fluorophore. The mAb trastuzumab-Cy5 was also prepared. Whole-body biodistribution of all constructs was investigated in mice bearing subcutaneous xenografts (HER2+ SKOV3) using in vivo epi-fluorescence imaging. Next, for intravital experiments, GFP-expressing SKOV3 cells were grown under dorsal window chambers on athymic nude mice (n = 3/group), and imaged under a fluorescence stereo microscope immediately after intravenous injection of the tracers. Consecutive fluorescence images within the tumour were acquired over the initial 20 min after injection and later, single images were taken at 1, 3 and 24 h post-injection. Additionally, two-photon microscopy was used to investigate the colocalization of GFP (tumour cells) and Cy5 fluorescence (tracers) at higher resolution.Whole-body images showed rapid renal clearance of all Nbs, and fast tumour targeting for the specific Nbs. Specific tumour uptake of the mAb could only be clearly distinguished from background after several hours. Intravital imaging revealed that monomeric Nb tracers accumulated rapidly and distributed homogenously in the tumour mere minutes after intravenous injection. The dimeric compounds initially achieved lower fluorescence intensities than the monomeric. Furthermore, whereas the HER2-specific dimeric bivalent compound remained closely associated to the blood vessels over 24 h, the HER2-specific dimeric monovalent tracer achieved a more homogenous tumour distribution from 1 h post-injection onwards. Non-specific tracers were not retained in the tumour. Trastuzumab had the most heterogenous intratumoural distribution of all evaluated compounds, while -due to the long blood retention- achieving the highest overall tumour uptake at 24 h post-injection.In conclusion, monomeric Nbs very quickly and homogenously distribute through tumour tissue, at a rate significantly greater than dimeric Nbs and mAbs. This underlines the potential of monomeric Nb tracers and therapeutics in molecular imaging and targeted therapies.

Directing Single-Molecule Emission with DNA Origami-Assembled Optical Antennas.

We demonstrate the capability of DNA self-assembled optical antennas to direct the emission of an individual fluorophore, which is free to rotate. DNA origami is used to fabricate optical antennas composed of two colloidal gold nanoparticles separated by a predefined gap and to place a single Cy5 fluorophore near the gap center. Although the fluorophore is able to rotate, its excitation and far-field emission is mediated by the antenna, with the emission directionality following a dipolar pattern according to the antenna main resonant mode. This work is intended to set out the basis for manipulating the emission pattern of single molecules with self-assembled optical antennas based on colloidal nanoparticles.

Molecular Switching of a Self-Assembled 3D DNA Nanomachine for Spatiotemporal pH Mapping in Living Cells.

DNA nanomachines have received great interest due to their potential to mimic various natural biomolecular machines. Intracellular pH sensing and imaging are of great significance to understand cellular behaviors and disease diagnostics. In this work, we report the novel molecular switching of a self-assembled 3D DNA triangular prism nanomachine (TPN) for pH sensing and imaging in living cells. The TPN was self-assembled in quantitative yields by hybridization with two DNA triangles and three I-strands (containing i-motif sequences). At acidic conditions, the TPN was compressed due to the I-strand that formed an intramolecular i-tetraplex, which was in between the fluorophores Cy3 and Cy5, resulting in a significant fluorescence resonance energy transfer (FRET) signal. At neutral or weakly alkaline conditions, the TPN adopted an extended state due to the random coil form of the I-strand, leading to spatial separation of the two fluorophores and the FRET being blocked. The TPN was fully reversible and could rapidly respond to the pH changes, entered into living cells automatically via an endocytic pathway, monitored spatiotemporal pH changes during endocytosis, maintained its structural integrity after escape from lysosomes, still had the ability for pH sensing, and also visualized pH fluctuations under varying stimuli in living cells. We foresee that this TPN can become a generic platform for a pH-related cell biology study and in disease diagnostics.

A near infrared fluorescent probe for one-step detection of histone deacetylase activity based on an intramolecular FRET

It is urgent to implement ideal fluorophores to detect enzymatic activity of histone deacetylases (HDACs) because of their important roles in many cellular metabolisms such as transcription regulation and tumorigenesis. However, the emission wavelength of reported one-step fluorescent probes for the detection of HDACs is limited at visible region. In this paper, we construct a near infrared (NIR) fluorescent probe Lys-OBD for detecting the activity of sirtuin 1 (SIRT1), one of the most important HDACs, with NIR emission based on an intramolecular FRET effect. Lys-OBD contains a non-emissive O-aryl NBD group (OBD) and a NIR emissive Cy5 fluorophore. After reaction with SIRT1, non-fluorescence OBD in Lys-OBD was changed to green emissive nitrobenzoxadiazole (NBD) through a deacetylation reaction followed by a backbone rearrangement. An intramolecular FRET between the energy donor NBD and the energy acceptor Cy5 was thus happened and the NIR emission of Cy5 could be detected. This FRET mechanism could further decrease the interference of high-concentration amino acid that are abound in living system in the process of SIRT1 detection. Thus, this probe can not only detect SIRT1 in vitro, but also image SIRT1 in live cells that high express SIRT1 with NIR fluorescence for the first time.

Nucleus reuniens of the midline thalamus of a rat is specifically damaged after early postnatal alcohol exposure.

Individuals diagnosed with fetal alcohol spectrum disorders often show behavioral impairments in executive functioning. Mechanistic studies have implicated coordination between the prefrontal cortex and the hippocampus (through thalamic nucleus reuniens) as essential for such executive functions. This study is the first to report the long-term neuroanatomical alterations to the ventral midline thalamus after alcohol exposure on postnatal days 4-9 (a rodent model of binge drinking during the third-trimester of human pregnancy). Alcohol added to a milk formula was administered to female Long-Evans rat pups on postnatal days 4-9 (5.25 g/kg/day of ethanol, intragastric intubation). Control animals were intubated without the administration of liquid. In adulthood, brains were immunohistochemically labeled for a neuronal marker (NeuN) conjugated with Cy3 fluorophore and stained with Hoechst33342 to visualize nuclei. Total non-neuronal cell number (NeuN/Hoechst) and neuron number (NeuN/Hoechst), and total volume were estimated using unbiased stereology in two neighboring midline thalamic nuclei: reuniens and rhomboid. Estimates were analyzed using linear mixed modeling to account for animal and litter as clustering variables. A 21% reduction in the total neuron number (resulting in altered neuron-to-non-neuron ratio) and an 18% reduction in total volume were found exclusively in thalamic nucleus reuniens in rats exposed to ethanol. Non-neuronal cell number was not changed in reuniens. No ethanol-induced changes on any measures were observed in rhomboid nucleus. These specific neuroanatomical alterations provide a necessary foundation for further examination of circuit-level alterations that occur in fetal alcohol spectrum disorder.

Differential Diagnostics of Active Progressing Multiple Sclerosis Using a Fluorescent Biomarker with Resonance Energy Transfer.

Previous data showed that myelin-reactive autoantibodies found in patients with multiple sclerosis and mice with experimental autoimmune encephalomyelitis recognize and hydrolyze various fragments of myelin basic protein (MBP). Moreover, antibody-mediated cleavage of the encephalithogenic fragment MBP81-103 flanked with two fluorescent proteins can serve as a new biomarker of multiple sclerosis. Here we describe creation of the next generation of this biomarker based on antibody-dependent degradation of a new chemically synthesized fluorescent substrate with resonance energy transfer that contains fluorophore Cy5 and quencher QXL680 separated by MBP81-99 protein (Cy5-MBP81-99-QXL680). This substrate is degraded during incubation with purified antibodies and B cells from patients with multiple sclerosis, but not healthy volunteers.

Programmed Dual-Functional DNA Tweezer for Simultaneous and Recognizable Fluorescence Detection of microRNA and Protein.

A programmed dual-functional DNA tweezer (DFDT) as a signaling molecule is reported for the simultaneous and recognizable fluorescence detection of microRNA 21 (miRNA 21) and mucin 1 (MUC1). This unique DFDT is assembled from two Au-NP-attached central strands (C1 and C2) and an arm strand (A) dually ended by fluorophores Cy3 and Cy5, which are spatially separated from Au NP in the originally opened state. Through the competitive affinity interaction between targets and their complementary and aptamer sequences tethered in two recognition strands (R1 and R2), miRNA 21 and MUC1 are respectively converted into two dependently displaced fuel strands (F1 and F2). The next hybridization with two pairs of unpaired segments overhung in open DFDT leads to its conformational closure, resulting in the approach of Cy3 and Cy5 to Au NP. On the basis of the nanometal surface energy transfer scheme, the fluorescence emission of Cy3 or Cy5 is cooperatively quenched by Au NPs attached in C1 and C2. The significant variation of fluorescence intensity enables one-step, cost-effective, and specific quantization of miRNA 21 and MUC1 with high sensitivity down to 32 fM and 2.6 fg·mL-1 (8.5 pM), respectively. The novel DFDT-based assay route of multiplex analytes is promising and has the potential for rapid and reliable diagnosis and treatment of cancer-related diseases.