Simultaneous Fluorescence Detection Research Articles

Simultaneous Homogeneous Fluorescence Detection of AFP and GPC3 in Hepatocellular Carcinoma Clinical Samples Assisted by Enzyme-Free Catalytic Hairpin Assembly.

Simultaneous sensitive and cost-effective detection of multiple tumor markers has shown great potential for cancer diagnostics. Herein, we reported a simple enzyme-free parallel catalytic hairpin assembly (CHA) amplification strategy with N-methyl mesoporphyrin IX (NMM) and quantum dots (QDs) as signal reporters for the homogeneous fluorescent simultaneous detection of alpha-fetoprotein (AFP) and glypican-3 (GPC3). Upon selective binding, the released single-stranded DNA (ssDNA) from the two-aptamer double-stranded DNA (dsDNA) probes triggers CHA amplification, further releasing the G-quadruplex sequence and Ag+ from the C-Ag+-C structures at the same time. Then, NMM and CdTe QDs selectively recognize G-quadruplex and Ag+, respectively. Under optimized conditions, limits of detections (LODs) as low as 3 fg/mL for AFP and 0.25 fg/mL for GPC3 were achieved using fluorescence readout. Using color- and distance-based visual readouts, an LOD of 1 fg/mL for GPC3 was reached. This method was applied to quantitatively analyze AFP and GPC3 in 41 clinical serum samples of hepatocellular carcinoma (HCC) patients. The quantitative test results for AFP and GPC3 were consistent with those obtained using the electrochemiluminescence immunoassay (ECL-IA) clinical kit and correlated with radiological and pathological findings. The results of clinical tests demonstrated the potential of GPC3 as a tumor biomarker, and we propose a cut-off value of 2 ng/mL GPC3 for HCC.

Fluorescence lifetime-based pH mapping of tumors in vivo using genetically encoded sensor SypHerRed

Changes in intracellular pH (pHi) reflect metabolic states of cancer cells during tumor growth and dissemination. Therefore, monitoring of pHi is essential for understanding the metabolic mechanisms that support cancer progression. Genetically encoded fluorescent pH sensors have become irreplaceable tools for real-time tracking pH in particular subcellular compartments of living cells. However, ratiometric readout of most of the pH probes is poorly suitable to measure pH in thick samples ex vivo or tissues in vivo including solid tumors. Fluorescence lifetime imaging (FLIM) is a promising alternative to the conventional fluorescent microscopy. Here, we present a quantitative approach to map pHi in cancer cells and tumors in vivo, relying on fluorescence lifetime of a genetically encoded pH sensor SypHerRed. We demonstrate the utility of SypHerRed in visualizing pHi in cancer cell culture and in mouse tumor xenografts using fluorescence lifetime imaging microscopy and macroscopy. For the first time to our knowledge, the absolute pHi value is obtained for tumors in vivo by an optical technique. In addition, we demonstrate the possibility of simultaneous detection of pHi and endogenous fluorescence of metabolic cofactor NADH, which provides a complementary insight into metabolic aspects of cancer. Fluorescence lifetime-based readout and red-shifted spectra make pH sensor SypHerRed a promising instrument for multiparameter in vivo imaging applications.

An Approach to the Simultaneous Detection of Multiple Biomarkers for the Early Diagnosis of Liver Cancer Using Quantum Dot Nanoprobes

Abstract Biomarker-based early diagnosis of liver cancer is of high clinical value for reducing the mortality rate. However, it has been challenging to establish early detection methods with a single biomarker such as alpha-fetoprotein (AFP) because of limited diagnostic sensitivity and specificity. Therefore, developing multiplexed biomarker detection assays is crucially important for early diagnosis. Yet, simultaneous detection methods involving three or more biomarkers have been scarce. Here we suggest employing the serological biomarker panel of glypican-3 (GPC3), dickkopf-1 (DKK1), and AFP for liver cancer detection. We present a rapid simultaneous detection approach for the biomarker panel labeled with three fluorescent quantum dot nanoprobes (emission wavelengths at 565 nm, 605 nm, and 655 nm). As a proof-of-concept, simultaneous fluorescence detection of the biomarker panel was demonstrated using mixed reference samples containing human recombinant GPC3, DKK1, and AFP antigens. Our simultaneous detection approach conferred a linear range of 0.625-2.5 ng•mL-1 for the entire biomarker panel, which merits further clinical validation for the simultaneous and accurate determination of the biomarker panel in human serum samples.

Fluorescent carbon dots crosslinked cellulose Nanofibril/Chitosan interpenetrating hydrogel system for sensitive detection and efficient adsorption of Cu (II) and Cr (VI)

• One-pot synthesis of carbon dots cross-linked cellulose nanofibril hydrogel. • The introduction of chitosan by solvent displacement forms IPN hydrogel. • Carbon dots provide visual detection during Cu (II) and Cr (VI) adsorption. • Amphoteric biomass and carbon dots provide adsorption sites for Cu (II) and Cr (VI). • The mechanism of Cu (II) and Cr (VI) sorption and sensing was determined. To address the pollution of heavy metal ions in water resources, a novel self-assembled nitrogen-doped carbon dots (NCDs) crosslinked, cellulose nanofibril (CNF) and chitosan (CS) based interpenetrating polymer network (IPN) hydrogel (NCDs-CNF/CSgel) was prepared for simultaneous fluorescent detection and adsorption of Cu (II) cation and Cr (VI) anion. The NCDs cross-linked CNF hydrogel network was synthesized by an environmentally friendly low-temperature hydrothermal process and subsequently, solvent replacement was used to form the second layer of CS network. The morphology, chemical structure, fluorescent properties, and adsorption behaviors of NCDs-CNF/CSgel were analyzed. The results showed that NCDs-CNF/CSgel exhibited a wide linear range of fluorescence response for Cu (II) (50–1000 mg/L, detection limit of 40.3398 mg/L) and superior sensitivity and selectivity of fluorescence response for Cr (VI) (linear range of 1–50 mg/L, detection limit of 0.7093 mg/L). NCDs-CNF/CSgel showed high adsorption capacities of 148.30 mg/g and 294.46 mg/g for Cu (II) and Cr (VI), respectively, and fitted well to the pseudo-second-order model and the Langmuir model. The detection of Cu (II) and Cr (VI) and their adsorption mechanisms onto NCDs-CNF/CSgel were further determined. Hence, this work provides a green and sustainable approach for the synthesis of a functional material which can be applied for the detection and adsorption of heavy metal ions.

Simultaneous preconcentration and fluorescence detection of ATP by a hybrid nanocomposite of magnetic nanoparticles incorporated in mixed metal hydroxide.

A new approach for increasing the sensitivity of adenosine triphosphate (ATP) detection was demonstrated. The assay was based on the synergetic function of a hybrid nanocomposite (MNPs@MMH) composed of magnetic nanoparticles (MNPs) incorporated in a mixed metal hydroxide (MMH). MNPs@MMH can be utilized as an efficient green extractant and peroxidase catalyst. The trace level of ATP in the sample solution was first extracted by the MNPs@MMH hybrid nanocomposite through the ion exchange properties of MMH and adsorbed on the surface of the MNPs@MMH. The concentration of ATP was related to the fluorescence intensity of 2,3-diaminophenazine (DAP) generated from peroxidase-like activity of the MNPs in the presence of H2O2 and o-phenylenediamine (OPD). In the presence of ATP, the active surface of the MNPs was diminished, and the amount of DAP generated was reduced. Thus, the concentration of ATP was related to the degree of fluorescence decrease compared to the fluorescence intensity of the system without ATP. Based on the proposed strategy, a highly sensitive assay for ATP was achieved. This assay exhibited good selectivity for detection of ATP over derivatives and other common anions. The proposed assay allowed the detection of ATP in a concentration range of 2.5-20 μM with a detection limit of 0.41 μM.

Weaving microscale wool ball-like hollow covalent organic polymers from nanorods for efficient adsorption and sensing.

Microscale covalent organic polymers with a unique 3D hollow wool ball-like morphology have been woven from 1D nanorods by a cascade emulsion strategy with a large surface area (284 m2 g-1), which showed great potential for simultaneous removal (Qmax, 358.15 mg g-1) and fluorescent detection (detection limit, 8.0 μg L-1) of bisphenol A.

Dual-Wavelength Fluorescence Monitoring of Photodynamic Therapy: From Analytical Models to Clinical Studies.

Simple SummaryFluorescence imaging is an efficient tool in monitoring photodynamic therapy procedures allowing us to track accumulation and photobleaching of a photosensitizer (PS). Chlorin-based PSs feature high absorption in the red and blue bands of visible spectrum. Due to spectral dispersion of light penetration depth in biotissues, fluorescence signals registered upon excitation by red or blue light are formed in different measurement volumes. We present analytical and numerical models of dual-wavelength fluorescence imaging for evaluation of PS localization depth in the cases of topical administration and intravenous injection. The results of analytical and numerical simulations are in good agreement with the phantom experiments, and are translated to the in vivo imaging, which allows to interpret experimental observations in animal trials, human volunteers, and clinical studies. The proposed approach allows us to noninvasively estimate typical accumulation depths of PS localization which are consistent with the morphologically expected values.Fluorescence imaging modalities are currently a routine tool for the assessment of marker distribution within biological tissues, including monitoring of fluorescent photosensitizers (PSs) in photodynamic therapy (PDT). Conventional fluorescence imaging techniques provide en-face two-dimensional images, while depth-resolved techniques require complicated tomographic modalities. In this paper, we report on a cost-effective approach for the estimation of fluorophore localization depth based on dual-wavelength probing. Owing to significant difference in optical properties of superficial biotissues for red and blue ranges of optical spectra, simultaneous detection of fluorescence excited at different wavelengths provides complementary information from different measurement volumes. Here, we report analytical and numerical models of the dual-wavelength fluorescence imaging of PS-containing biotissues considering topical and intravenous PS administration, and demonstrate the feasibility of this approach for evaluation of the PS localization depth based on the fluorescence signal ratio. The results of analytical and numerical simulations, as well as phantom experiments, were translated to the in vivo imaging to interpret experimental observations in animal experiments, human volunteers, and clinical studies. The proposed approach allowed us to estimate typical accumulation depths of PS localization which are consistent with the morphologically expected values for both topical PS administration and intravenous injection.

Enantiomer Separation of Chiral Amines and Amino Acid Esters on Polysaccharide Phenylcarbamates Derived Chiral Stationary Phases

We have developed an efficient chiral HPLC method for enantiomeric resolution of some chiral amines and α- amino acid esters as nitrobenzoxadiazole (NBD) derivatives on amylose and cellulose phenylcarbamates derived chiral stationary phases (CSPs) under simultaneous ultraviolet (UV) and fluorescence (FL) detection. The observed enantioselectivities were affected by the type of the analyte (amines or α-amino acid esters), backbone structure (cellulose or amylose) of the CSP used, and substituents of phenyl moiety on the chiral selector of CSP. Further, for same chiral selectors, coated type CSPs showed better enantiomeric separation than those observed for the covalently bonded type CSPs. In general, for the enantiomeric resolution of chiral amines, Chiralpak IE and Chiralcel OD-H columns showed the highest enantioselectivities. However, for resolution of α-amino acid esters, the performances of Chiralpak IA and Chiralpak AD-H (or Lux Amylose-1) columns were superior to those of other CSPs used. In general, the degrees of enantioselectivity for α-amino acid ethyl esters are greater than those for chiral amines. The developed analytical method was applied to determine the enantiomeric purity of two commercially available chiral amine and α-amino acid ester; the enantiomeric impurities were observed to be 0.35 and 0.05% for (S)-α-methylbenzylamine and (S)-leucine ethyl ester, respectively. The validation data obtained from intra- and inter-day accuracy assays for (S)-3,3-dimethyl-2-butylamine and (S)-leucine ethyl ester were 99.92-101.46 and 100.61-101.78%; and 100.05-102.12 and 101.25-102.73%, respectively; the intra- and interday precision assays in terms of relative standard deviation (% RSD) were found to be 0.96-1.57 and 0.81- 1.91%; and 0.24-1.01% and 0.65-1.35%, respectively. Owing to the high accuracy and precision results, the developed analytical method can prove to be useful for the enantiomeric separation of various chiral amines and amino acid esters as NBD derivatives using polysaccharide derived CSPs.

Design and in situ synthesis of ZnInS@ZIF-8-nanofilms multifunctional nanocomposite: A case application for simultaneous fluorescent sensing and enhanced photocatalytic performance toward antibiotic

Recently emerged two-dimensional (2D) conductive metal-organic framework ( c -MOF) thin films have brought new potential applications for various fields. This study presents a novel concept of in-situ self-assembled multifunctional semiconductor@2D- c -MOF platform (ZnInS nanosheet@ZIF-8 nanofilms, ZnInS NS@ZIF-8 NFs) for simultaneous fluorescent detection and photocatalysis. Synthesized by a facile method and subsequent in-situ self-template strategy, the obtained ZnInS NS@ZIF-8 NFs exhibits much higher photocatalytic efficiency in comparison with pristine ZnInS nanosheets and allows real-time, label-free, and sensitive fluorescent detection. In such process, the 2D ZnInS nanosheets not only serve as the semiconductor photocatalyst with large specific surface areas, but also act as self-sacrificial template to offer Zn 2+ ions for the in-situ oriented growth of ZIF-8 nanofilms to achieve preconcentration and fluorescent detection for the targets. More importantly, ZIF-8 nanofilms could induce photogenerated electron transport of ZnInS. As a proof of the concept, the successful sensing and photocatalytic degradation for tetracycline have been achieved. The limit of detection is 8.6 nM in water and the photocatalytic degradation efficiency reaches 73% within 5 min under visible light, which are better than most of the previous reports. Our work could open up a new perspective for advanced photocatalyst development in the future. • A novel ZnInS NS@ZIF-8 NFs multifunctional nanomaterial was successfully fabricated by a facile solvothermal method. • The ZnInS NS@ZIF-8 NFs exhibited excellent photocatalytic efficiency and fluorescence sensing for the tetracycline. • This study manifested the great promise of semiconductor@2D-c-MOF-nanofilm nanomaterials.

Efficient screening of ligand-receptor complex formation using fluorescence labeling and size-exclusion chromatography

Evidence of a complex formation is a crucial step in the structural studies of ligand-receptor interactions. Here we presented a simple and fast approach for qualitative screening of the complex formation between the chimeric extracellular domain of the nicotinic acetylcholine receptor (α7-ECD) and three-finger proteins. Complex formation of snake toxins α-Bgtx and WTX, as well as of recombinant analogs of human proteins Lynx1 and SLURP-1, with α7-ECD was confirmed using fluorescently labeled ligands and size-exclusion chromatography with simultaneous absorbance and fluorescence detection. WTX/α7-ECD complex formation also was confirmed by cryo-EM. The proposed approach could easily be adopted to study the interaction of other receptors with their ligands.

A flexible tripod fluorescent probe for multiple cations detection and its application in living cells.

To the flexible tripod platform tren (tris(2-aminoethyl)amine), a Rhodamine and two naphthalene fluorophores were introduced. The resulting fluorescence probe named TRN was fully characterized and employed in cell imaging. Probe TRN exhibited high selectivity and excellent sensitivity for the simultaneous fluorescence detection of Zn2+/Hg2+/Al3+/Cu2+. The significant changes in the fluorescence color make naked-eye detection possible. Furthermore, fluorescence imaging experiments of Zn2+/Hg2+/Al3+/Cu2+ in living PC3 cells demonstrated its value for practical applications in biological systems.

Compact optical detector utilizing light emitting diodes, 50 nL L-shaped silica capillary cell and CCD spectrometer for simultaneous multi-wavelength monitoring of absorbance and fluorescence in microcolumn liquid chromatography

Optical detection setup utilizing light emitting diodes (LEDs), 50 nL L-shaped silica capillary detection cell (L-cell), and low-cost CCD spectrometer is described in this work. Experimental configuration can be equipped with two different LEDs for absorbance measurement and other two LEDs for fluorescence excitation. This setup is capable of simultaneous multi-wavelength monitoring of absorbance and fluorescence when light produced by the individual LEDs and light emitted by the fluorescent analytes is resolved in the spectrum outputted by the CCD spectrometer. Effective optical path of the 0.25 μm I. D. L-cell is 1 mm. Absorbance baseline noise is 1 mAU due to use of low-cost and relatively noisy CCD spectrometer and LED drivers. Nevertheless, the setup can detect adenosine 5′-monophosphate down to micromolar concentration. Performance of fluorescence monitoring allows detection of 5·10−10 M fluorescein when 23 mW 470 nm LED is used for excitation. The dynamic range of absorbance and fluorescence measurement is 867:1 and 1622:1, respectively. Separation of test mixture (alkylbenzenes and polyaromatic hydrocarbons) demonstrate the effective use of the detector for simultaneous absorbance and fluorescence detection with 0.2 × 150 mm packed capillary column. The benefits of the setup are relative simplicity, compact design and the fact that it can be operated without any optical filters, slits, and extremely precise positioning of the optical elements.

A method for the detection of hCG β in spent embryo culture medium based on multicolor fluorescence detection from microfluidic droplets.

The evaluation of embryo quality via human chorionic gonadotropin beta (hCG β) and other proteins secreted by embryos in a spent embryo culture medium (SECM) receives a close review in the field of assisted reproduction. However, accurate and quantitative detection of these trace proteins is still a challenge. In this study, a highly sensitive protein detection method using microfluidic droplets and multicolor fluorescence detection was developed and used to detect hCG β secreted by embryos in SECM. β-Galactosidase (β-Gal) was used to label hCG β and can catalyze the conversion of nonfluorescent substrate fluorescein di-β-d-galactopyranoside to produce fluorescein to amplify the signal strength. Compared with previous studies, the proposed method requires only a simple microfluidic chip and can eliminate false-positive signals generated by free β-Gal through simultaneous detection of fluorescence, which can ensure the accuracy of the results. The lower detection limit of hCG β was 0.1 pg/ml. Using the developed method, hCG β in SECM was successfully detected; the hCG β secreted by top-quality blastocysts was significantly higher than that of non-top-quality blastocysts and embryos that do not develop into blastocysts. The proposed method can be used to detect secretory proteins from embryos in SECM and has application value in the screening of other biomarkers.

A Nanofluidic Device for Multiplexed Analysis of Single Exosomes

Detailed characterization of exosomes on the single particle level is challenging because of their small size and heterogeneous nature. We have developed a nanofluidic device that enables single particle fluorescence-based tracking and characterization of nano-sized biological particles, which overcomes this problem. This device allows simultaneous three-color fluorescence detection and Brownian-motion based measurement of each particle's size, opening new possibilities for the analysis of population heterogeneity and the detection of scarce events. Our nanofluidic device is fabricated in fused silica and consists of an array of 300nm × 300nm nanochannels connecting to two microchannels (for respectively sample loading and post-analysis collection). The device is fitted onto a conventional fluorescence microscope equipped with a LED light source and a multi-bandpass filter. We have developed software to determine nanoparticle size based on their Brownian motion when passed through the nanochannel at low speed, reaching better accuracy in size determination than with commercial nanoparticle tracking instruments. The device operates with very small sample volumes (2-5 µL) and a data collection time of 2-20 minutes. We demonstrate that it is possible to accurately detect, count, and determine the size of exosomes collected directly from conditioned cell media and without need of potentially sample-altering purification steps, enabling “real-time” exosome profiling from cell culture and other live biological specimen. Additionally, we have been able to visualize the co-localization of Alzheimer peptide amyloid-β peptide with exosomes and demonstrate possibilities to detect size changes associated with protein binding to the exosome surface. Altogether, our device constitutes a new tool to analyze biological nanoparticles, allowing firm establishment of correlations between their biophysical attributes (size), contents and interactions, which is important to understand their biofunctionality.

Photon-upconversion barcode for monitoring an enzymatic reaction with a fluorescence reporter in droplet microfluidics.

We report luminescent photon-upconversion barcodes for indexing the chemical content of droplets. The barcode is compatible with the simultaneous detection of fluorescence. The encoding and decoding of the initial concentration of enzyme β-galactosidase and substrate 4-methylumbelliferyl β-d-galactopyranoside are described. The fluorescent product 4-methylumbelliferone is detected simultaneously with the barcode.

A Janus 3D DNA nanomachine for simultaneous and sensitive fluorescence detection and imaging of dual microRNAs in cancer cells.

Herein, a Janus three-dimensional (3D) DNA nanomachine was constructed for the simultaneous and sensitive fluorescence detection and imaging of dual microRNAs (miRNAs) in cancer cells, which could effectively eliminate signal interference in a homogeneous nanoparticle-based 3D DNA nanostructure caused by the proximity of the two different signal probes to achieve accurate co-location in the same position of living cancer cells. In this system, the Janus nanoparticles were synthesized as the carrier for immobilizing two different oligonucleotides on two different functionalized hemispheres of the nanoparticles to form a Janus 3D DNA nanostructure, which could convert trace amounts of miRNA-21 and miRNA-155 targets into massive FAM and Cy5-labeled duplexes to induce two remarkable fluorescence emissions by the catalytic hairpin assembly (CHA) and 3D DNA walker cascade nucleic acid amplification strategy, realizing sensitive detection and imaging of miRNA targets in cancer cells. Impressively, in comparison with current miRNA imaging methods based on nanoparticle assemblies, the proposed strategy could efficiently eliminate “false positive” results obtained in single type miRNA detection and distinctly increase the immobilization concentration of two different signal probes using Janus nanoparticles as the carrier to further enhance fluorescence intensity, resulting in accurate co-location in the same position of living cells. Meanwhile, the proposed fluorescence imaging technology makes it possible to visualize low concentrations of miRNAs with tiny change associated with some cancers, which could significantly improve the accuracy and precision compared to those of the conventional fluorescence in situ hybridization (FISH) approach. Therefore, it could serve as persuasive evidence for supplying accurate information to better understand biological processes and investigate mechanisms of various biomolecules and subcellular organelles, resulting in the further validation of their function in tumor proliferation and differentiation. This strategy provided an innovative approach to design new generations of nanomachines with ultimate applications in bioanalysis and clinical diagnoses.

Proinflammatory cytokines and colorectal cancer - the impact of the stage.

Colorectal cancer is one of the most often diagnosed malignant tumors. In Kazakhstan, high incidence of CC is registered along with other oncology diseases. Despite a significant progress in the disease treatment achieved lately, CC is still one of the major reasons of mortality due to oncologic pathologies.To study the samples MilliplexMap HumanCirculationBiomarker panel in blood serum was used. XMap-based Fluorescence immunoassay was implemented, which comprised magnetic-bead-based simultaneous fluorescence detection of IL-6, IL-8, MIF, FGF-2, SCF, TGF, TNF, TRAIL analytes. Proinflammatory biomarker concentration detection at different CC stages allows to reveal the dynamics of inflammatory response of the organism to tumor and to use them (biomarkers) in further diagnostic and forecast in particular in CC. As a result of our study, it was found that IL-6, which showed the brightest reaction, due to its range of change and considerable shift already in the I stage can be recommended as a component of a complex diagnostic panel. Such markers as FGF2 and MIF also have a role in CC early stage detection.

Simultaneous fluorescent detection of multiplexed miRNA of liver cancer based on DNA tetrahedron nanotags

The level of miRNA-21, miRNA-122, and miRNA-223 are always elevated when liver cancer is present at an early stage. In this paper, a novel assay to simultaneous detect miRNA-21, miRNA-122, and miRNA-223 was proposed based on DNA tetrahedron nanotags and fluorescence resonance energy transfer (FRET), which used a single laser stimulate wavelengh from one nucleic acid stain TOTO-1 to three diverse organic dyes (Cy3, Cy3.5, Cy5). In brief, a DNA tetrahedral nanostructure (DTN) was designed with three adaptor oligos on its vertices. TOTO-1, as a fluorescent donor, can imbed into native nucleic acid backbone of DTN. Three organic dye-functionalized strands (FRET oligos) are fluorescent receptors. In the presence of target miRNAs, they can be hybridized with FRET oligos and adaptor oligos on the vertices of DTN and the stable DNA tetrahedron nanotags are formed. As a result, the confinement of TOTO-1 is in close proximity to three fluorescence dyes, the FRET between TOTO-1 and three fluorescence dyes is generate efficiently in DNA tetrahedron nanotags. Point-of-care clinical applicability is demonstrated by sensitive multiplexed quantification of three miRNAs in 10% human serum samples.

Optimization of background electrolyte composition for simultaneous contactless conductivity and fluorescence detection in capillary electrophoresis of biological samples.

In this article, optimization of BGE for simultaneous separation of inorganic ions, organic acids, and glutathione using dual C4 D-LIF detection in capillary electrophoresis is presented. The optimized BGE consisted of 30mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, 15mM 2-amino-2-hydroxymethyl-propane-1,3-diol, and 2mM 18-crown-6 at pH 7.2 and allowed simultaneous separation of ten inorganic anions and cations, three organic acids and glutathione in 20min. The samples were injected hydrodynamically from both capillary ends using the double-opposite end injection principle. Sensitive detection of anions, cations, and organic acids with micromolar LODs using C4 D and simultaneously glutathione with nanomolar LODs using LIF was achieved in a single run. The developed BGE may be useful in analyses of biological samples containing analytes with differing concentrations of several orders of magnitude that is not possible with single detection mode.

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.