Label-Free and Simple G-quadruplex-based Turn-Off Fluorescence Assay for the Detection of Kanamycin

We have developed a label-free and turn-off fluorescence assay for the determination of kanamycin. The detection system consists of an aptamer for specifically recognizing kanamycin and two auxiliary probes functionalized with two GGG repeats at the 3′ or 5′ ends for signal reporting. Two probes both hybridize with the aptamer and then their G-rich sequences combine to form a G-quadruplex. When thioflavin T, a fluorophore, is bound to the G-quadruplex, the fluorescence intensity of the solution dramatically increases. Upon the addition of the kanamycin, the aptamer–kanamycin binding inhibits the hybridization of two probes and aptamer, and restrains the GGG repeats from getting closer to form the G-quadruplex structure, resulting a significant decrease in the fluorescence intensity. The proposed aptamer-based fluorescent sensing platform showed a linear relationship with the concentration of kanamycin from 0.6 to 20.0?nM. The detection limit was determined to be 0.33?nM. The sensing platform provides resistance to interferences from other antibiotics and can be used to efficiently recognize kanamycin in real samples.     

A Simple Turn-off Schiff Base Fluorescent Sensor for Copper (II) Ion and Its Application in Water Analysis

An aniline-functionalized naphthalene dialdehyde Schiff base fluorescent probe L with aggregation-induced enhanced emission (AIEE) characteristics was synthesized via a simple one-step condensation reaction and exhibited excellent sensitivity and selectivity towards copper(II) ions in aqueous media with a fluorescence “ turn-off ” phenomenon. The detection limit of the probe is 1.64 × 10⁻⁸ mol·L⁻¹. Furthermore, according to the results of the UV-vis/fluorescence titrations, Job’s plot method and ¹H-NMR titrations, a 1:2 stoichiometry was identified. The binding constant between L and Cu²⁺ was calculated to be K_a = 1.222 × 10³. In addition, the AIEE fluorescent probe L could be applied to detection in real water samples with satisfactory recoveries in the range 99.10–102.90% in lake water and 98.49–102.37% in tap water.     

Smartphone-Assisted Colorimetric Detection of Glutathione and Glutathione Reductase Activity in Human Serum and Mouse Liver Using Hemin/G-Quadruplex DNAzyme

Abnormal levels of reduced glutathione (GSH) and glutathione reductase (GR) are usually related to a variety of diseases, so it is of great significance to determine the GSH concentration and GR activity. We herein develop a smartphone-assisted colorimetric biosensor for the detection of GSH and GR activity in human serum and mouse liver using hemin/G-quadruplex DNAzyme. Firstly, an obvious color change from colorless to green can be observed, owing to the high peroxidase-like activity of hemin/G-quadruplex DNAzyme toward 2,2′-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS). With the addition of GSH or GR, the H₂O₂-mediated oxidation of ABTS catalyzed by hemin/G-quadruplex DNAzyme is significantly inhibited, resulting in remarkable color fading. Therefore, the detection of GSH and GR activity can be achieved by observing the color transition or measuring the absorbance at 420 nm. The detection limit was estimated to be as low as 0.1 μM and 10 μU/mL for GSH and GR, respectively. More interestingly, the RGB values of the sensing system can be identified by the smartphone application (APP, color collect), which makes it an ideal format for on-site determination and point-of-care testing (POCT). In addition, the proposed method shows excellent selectivity and acceptable applicability for the determination of GSH concentration and GR activity in human serum samples and mouse liver tissues, which might hold great application potential in clinical diagnosis and drug screening.     

G-quadruplex − based homogenous fluorescence platform for ultrasensitive DNA detection through isothermal cycling and cascade signal amplification

We describe a simple and homogenous fluorimetric method for sensitive determination of DNA. It is based on target-triggered isothermal cycling and a cascade exponential amplification reaction that generates a large amount of a G-quadruplex. This results in strong fluorescence signal when using thioflavin T as a G-quadruplex-specific light-up fluorescent probe. Tedious handling after amplification is widely eliminated by the addition of thioflavin T. No other exogenous reagent is required. This detection platform is inexpensive and rapid, and displays high sensitivity for target DNA, with a detection limit as low as 91 pM.

The addition of target DNA can trigger the isothermal exponential amplification reaction to generate a large amount of G-quadruplex sequence oligonucleotides and then employ thioflavin T (Th T) (a G-quadruplex-specific light-up dye) as signal output for sensitive DNA detection.

     

An unexpected fluorescent probe for G-quadruplex DNA based on a nitro-substituted ruthenium (II) complex

The detection of G-quadruplex is of major interest. Nitro-substituted ruthenium (II) complexes have attracted much attention due to fluorescent sensitivity to environment change. We report here a new nitro-substituted ruthenium (II) complex, [Ru (phen)₂(hnoip)]²⁺ ( 1) (hnoip = 2-(2-hydroxyl-5-nitrophenyl)imidazo[4,5-f][1,10-phenanthroline]), which displays distinct fluorescent properties in aqueous solution and non-aqueous solvents. This complex exhibits large fluorescence enhancement after binding with G-quadruplex DNA, and displays good fluorescent selectivity over other DNAs. The limit of detection is 6 nm for 22AG in Na⁺ and 43 nm for 22AG in K⁺, respectively. The results demonstrated that nitro-substituted ruthenium (II) complexes can be utilized to design as G-quadruplex fluorescent probes by protection of the nitro group on the complex from water.     

Label-free fluorescent strategy for sensitive detection of tetracycline based on triple-helix molecular switch and G-quadruplex

In this assay, a label-free fluorescent sensing platform based on triple-helix molecular switch (THMS) and G-quadruplex was developed for the detection of tetracycline. We demonstrated this approach by using THMS, which consists of a central section with a shortened 8-mer aptamer sequence with high affinity to tetracycline and flanked by two arm segments. G-rich oligonucleotide can specifically bind to thioflavin T (ThT) as a signal transduction probe (STP). In the absence of tetracycline, THMS remains stable, the fluorescence of background is low. By the addition of target tetracycline, the aptamer-target binding results in the formation of a structured aptamer-target complex, which disassembles the THMS and releases the STP. The free STP self-assembles into G-quadruplex and specifically binds to ThT which generates a obvious fluorescence enhancement. Using the triple-helix molecular switch, the developed aptamer-based fluorescent sensing platform showed a linear relationship with the concentration of tetracycline ranging from 0.2 to 20.0 nmol/L. The detection limit of tetracycline was determined to be 970.0 pmol/L. The assay avoids complicated modifications or chemical labeling, making it simple and cost-effective. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.     

A label-free and signal-on supersandwich fluorescent platform for Hg sensing

A label-free supersandwich fluorescent assay was demonstrated for the first time by taking Hg²⁺ as a detection candidate. The principle of the proposed supersandwich fluorescent platform is based on the formation of supersandwich structure by T-Hg²⁺-T coordination and the fluorescence enhancement of the intercalated Genefinder (GF) in double strand DNA (dsDNA). Such supersandwich fluorescent DNA sensor exhibits a linear range of 10–300 nM for the detection of Hg²⁺, with a detection limit of 2.5 nM on the basis of the 3σ/slope (σ represents the standard deviation of the blank samples), which is well below the permit of the U.S. Environmental Protection Agency (<10 nM). The detection can be fulfilled in less than 10 min. The proposed mix-and-detect fluorescent platform exhibits excellent sensitivity, selectivity, and convenient manipulation. The assay was successfully used to detect Hg²⁺ in the lake water samples, which suggested its potential in practical samples.     

Simple and sensitive fluorescence sensor for detection of potassium ion in the presence of high concentration of sodium ion using berberine–G-quadruplex complex as sensing element

In this work, a novel potassium ion (K⁺) sensor is presented using berberine–G-quadruplex complex as a fluorescent probe. This sensor is based on the K⁺that can induce the G-rich DNA to form G-quadruplex conformation. The G-quadruplex can bind berberine to form berberine–G-quadruplex complex, resulting in remarkable enhancement of fluorescence emission of the berberine–G-quadruplex system. In the presence of 800 mM sodium ion (Na⁺), the fluorescence of the berberine–G-quadruplex complex increased linearly with increasing K⁺ concentration in the range of 0.005–1.0 mM. The turn-on fluorescent assay is simple, inexpensive, and highly sensitive. We observed that Na⁺ in 10,000-fold molar excess does not interfere. The molecular mechanisms which produce enhanced fluorescence of berberine were discussed.     

Sensitive pseudobienzyme electrocatalytic DNA biosensor for mercury(II) ion by using the autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification

Herein, a novel sensitive pseudobienzyme electrocatalytic DNA biosensor was proposed for mercury ion (Hg²⁺) detection by using autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification. Thiol functionalized capture DNA was firstly immobilized on a nano-Au modified glass carbon electrode (GCE). In presence of Hg²⁺, the specific coordination between Hg²⁺ and T could result in the assembly of primer DNA on the electrode, which successfully triggered the HCR to form the hemin/G-quadruplex DNAzyme nanowires with substantial redox probe thionine (Thi). In the electrolyte of PBS containing NADH, the hemin/G-quadruplex nanowires firstly acted as an NADH oxidase to assist the concomitant formation of H₂O₂ in the presence of dissolved O₂. Then, with the redox probe Thi as electron mediator, the hemin/G-quadruplex nanowires acted as an HRP-mimicking DNAzyme that quickly bioelectrocatalyzed the reduction of produced H₂O₂, which finally led to a dramatically amplified electrochemical signal. This method has demonstrated a high sensitivity of Hg²⁺ detection with the dynamic concentration range spanning from 1.0 ng L⁻¹ to 10 mg L⁻¹ Hg²⁺ and a detection limit of 0.5 ng L⁻¹ (2.5 pM) at the 3S_blank level, and it also demonstrated excellent selectivity against other interferential metal ions.     

A 1-Hydroxy-2,4-Diformylnaphthalene-Based Fluorescent Probe and Its Detection of Sulfites/Bisulfite

A novel 1-hydroxy-2,4-diformylnaphthalene-based fluorescent probe L was synthesized by a Knoevenagel reaction and exhibited excellent sensitivity and selectivity towards sulfite ions (SO₃²⁻) and bisulfite ions (HSO₃⁻). The detection limits of the probe L were 0.24 μM using UV-Vis spectroscopy and 9.93 nM using fluorescence spectroscopy, respectively. Furthermore, the fluorescent probe L could be utilized for detection in real water samples with satisfactory recoveries in the range 99.20%~104.30% in lake water and 100.00%~104.80% in tap water by UV-Vis absorption spectrometry, and in the range 100.50%~108.60% in lake water and 102.70%~103.80% in tap water by fluorescence spectrophotometry.     

A self-assembled deoxyribonucleic acid concatemer for sensitive detection of single nucleotide polymorphism

Polymerase-free and label-free strategies for DNA detection have shown excellent sensitivity and specificity in various biological samples. Herein, we propose a method for single nucleotide polymorphism (SNP) detection by using self-assembled DNA concatemers. Capture probes, bound to magnetic beads, can joint mediator probes by T4 DNA ligase in the presence of target DNA that is complementary to the capture probe and mediator probe. The mediator probes trigger self-assembly of two auxiliary probes on magnetic beads to form DNA concatemers. Separated by a magnetic rack, the double-stranded concatemers on beads can recruit a great amount of SYBR Green I and eventually result in amplified fluorescent signals. In comparison with reported methods for SNP detection, the concatemer-based approach has significant advantages of low background, simplicity, and ultrasensitivity, making it as a convenient platform for clinical applications. As a proof of concept, BRAF^T1799A oncogene mutation, a SNP involved in diverse human cancers, was used as a model target. The developed approach using a fluorescent intercalator can detect as low as 0.1 fM target BRAF^T1799A DNA, which is better than those previously published methods for SNP detection. This method is robust and can be used directly to measure the BRAF^T1799A DNA in complex human serum with excellent recovery (94–103%). It is expected that this assay principle can be directed toward other SNP genes by simply changing the mediator probe and auxiliary probes.     

Highly Sensitive and Selective Fluorescence “Turn-On” Detection of Pb (II) Based on Fe3O4@Au–FITC Nanocomposite

New nanocomposites, Fe₃O₄@Au–FITC, were prepared and explored to develop a fluorescent detection of Pb²⁺. The Fe₃O₄@AuNPs–FITC nanocomposites could be etched by Pb²⁺ in the presence of Na₂S₂O₃, leading to fluorescence recovery of FITC quenched by Fe₃O₄@Au nanocomposites. With the increase of Pb²⁺ concentration, the fluorescence recovery of Fe₃O₄@AuNPs–FITC increased gradually. Under optimized conditions, a detection limit of 5.2 nmol/L of Pb²⁺ with a linear range of 0.02–2.0 µmol/L were obtained. The assay demonstrated negligible response to common metal ions. Recoveries of 98.2–106.4% were obtained when this fluorescent method was applied in detecting Pb²⁺ spiked in a lake-water sample. The above results demonstrated the high potential of ion-induced nanomaterial etching in developing robust fluorescent assays.     

A Multifunctional N-Doped Cu–MOFs (N–Cu–MOF) Nanomaterial-Driven Electrochemical Aptasensor for Sensitive Detection of Deoxynivalenol

Deoxynivalenol (DON) is one of the most common mycotoxins in grains, causing gastrointestinal inflammation, neurotoxicity, hepatotoxicity and embryotoxicity, even at a low quantity. In this study, a facile electrochemical aptasensor was established for the rapid and sensitive determination of DON based on a multifunctional N-doped Cu-metallic organic framework (N–Cu–MOF) nanomaterial. The N–Cu–MOF, with a large specific surface area and good electrical conductivity, served not only as an optimal electrical signal probe but also as an effective supporting substrate for stabilizing aptamers through the interactions of amino (-NH₂) and copper. Under the optimal conditions, the proposed sensor provided a wide linear concentration range of 0.02–20 ng mL⁻¹ (R² = 0.994), showing high sensitivity, with a lower detection limit of 0.008 ng mL⁻¹, and good selectivity. The sensor’s effectiveness was also verified in real spiked wheat samples with satisfactory recoveries of 95.6–105.9%. The current work provides a flexible approach for the rapid and sensitive analysis of highly toxic DON in food samples and may also be easily extended to detect other hazardous substances with alternative target-recognition aptamers.     

Sensitive and selective determination of glutathione in probiotic bacteria by capillary electrophoresis–laser induced fluorescence

Glutathione (GSH) is a thiol with an important function in protecting tissue against the oxidative stress which has been related to carcinogenesis in the colon. For this reason the development of probiotic species producing glutathione could be of great interest. To determine the glutathione content of some probiotic bacteria of the Bifidobacterium and Lactococcus genera, a very sensitive and selective analytical method based on capillary electrophoresis coupled to laser-induced fluorescence detection has been developed. Pretreatment of cell-lysate samples is very simple—precipitation of protein with acetonitrile in 1:2 volume ratio. The fluorophore 5-iodoacetamidofluorescein (5-IAF) was chosen for glutathione derivatisation; it reacts with thiols at pH 12.5, forming a fluorescent adduct which is excited by a laser at 488 nm for detection. The reaction conditions optimised were temperature, time, and 5-IAF/GSH molar ratio. Electrophoresis was performed with a carbonate buffer (25 mmol L⁻¹, pH 9.8) as background electrolyte and a voltage of 30 kV; an electrophoretic run was complete in less than 7 min. There was a good linear relationship between concentration and response in the range 2.5–500 ng mL⁻¹ and the LOD was 0.5 ng mL⁻¹. The glutathione content of probiotic cells was determined by using the standard additions method to reduce matrix effects. The method was fully validated and shown to be of suitable sensitivity and selectivity for determination of GSH in probiotic cell lysates.     

“Turn-off-on” fluorescent sensor for (N-methyl-4-pyridyl) porphyrin -DNA and G-quadruplex interactions based on ZnCdSe quantum dots

As a new detection model, the reversible fluorescence “turn-off-on” sensor based on quantum dots (QDs) has already been successfully employed in the detections of many biochemical materials, especially in the researches on the interactions between anticancer drugs. The previous studies, however, mainly focused on simple-structured oligonucleotides and Calf thymus DNA. G-quadruplex, an important target for anti-cancer drug with special secondary structure, has been stimulating increasing research interests. In this paper, we report a new detection method based on the fluorescence “turn-off-on” model with water-soluble ZnCdSe QDs as the fluorescent probe, to analyze the interactions between anticancer drug (N-methyl-4-pyridyl) porphyrin (TMPyP) and nucleic acid, especially the G-quadruplex. The fluorescence of QDs can be quenched by TMPyP via photo-induced electron transfer and fluorescence resonance energy transfer, while on the other hand, the combination between TMPyP and G-quadruplex releases QDs from their quenchers and thus recovers the fluorescence. Most importantly, the fluorescence “turn-off-on” model has been employed, for the first time, to analyze the impacts of special factors on the interaction between TMPyP and G-quadruplex. The excellent selectivity of the system has been verified in the studies of the interactions between TMPyP and different DNAs (double-stranded DNA, single-stranded G-quadruplex, and different types of G-quadruplexes) in Na⁺ or K⁺-containing buffer.     

Direct spectrofluorimetric determination of glutathione in biological samples using 5-maleimidyl-2-(m-methylphenyl) benzoxazole

A new thiol fluorescence probe, 5-maleimidyl-2-(m-methylphenyl)benzoxazole (MMPB) has been developed for the direct determination of reduced glutathione (GSH) in real samples. Compared to the reported N-substituted maleimide type of thiol reagents, the main advantage of MMPB is its rather high selectivity for GSH to cysteine (Cys), which often coexists with GSH in biological samples. Under mild conditions similar to the physiological environment, MMPB reacted with GSH to give a highly fluorescent derivative with the excitation and emission wavelengths of 299.2 and 355.8 nm, respectively. In the presence of 0.40-fold (molar ratio) of Cys, a linear relationship was found in the range of 0-1.62×10⁻⁷ mol l⁻¹ with the detection limit (3σ) of 3.23×10⁻¹⁰ mol l⁻¹ for GSH determination. Many other amino acids (100-fold) did not interfere with the determination. Since the molar ratio of Cys to GSH in mammalian tissues and blood does not exceed the value of 0.40:1, the proposed method has been used in the direct determination of GSH in these kinds of biological samples, such as human blood, pig’s liver and heart with the recoveries of 94.3-104.5%     

Aptamer-based biosensor for label-free detection of ethanolamine by electrochemical impedance spectroscopy

A label-free sensing assay for ethanolamine (EA) detection based on G-quadruplex-EA binding interaction is presented by using G-rich aptamer DNA (Ap-DNA) and electrochemical impedance spectroscopy (EIS). The presence of K⁺ induces the Ap-DNA to form a K⁺-stabilized G-quadruplex structure which provides binding sites for EA. The sensing mechanism was further confirmed by circular dichroism (CD) spectroscopy and EIS measurement. As a result, the charge transfer resistance (R_CT) is strongly increased as demonstrated by using the ferro/ferricyanide ([Fe(CN)₆]^3−/4−) as a redox probe. Under the optimized conditions, a linear relationship between ΔR_CT and EA concentration was obtained over the range of 0.16 nM and 16 nM EA, with a detection limit of 0.08 nM. Interference by other selected chemicals with similar structure was negligible. Analytical results of EA spiked into tap water and serum by the sensor suggested the assay could be successfully applied to real sample analysis. With the advantages of high sensitivity, selectivity and simple sensor construction, this method is potentially suitable for the on-site monitoring of EA contamination.     

Colorimetric detection of potassium ions using aptamer-functionalized gold nanoparticles

Herein, a simple and novel colorimetric method for detection of potassium ions (K⁺) was developed. The colorimetric experiments revealed that upon the addition of K⁺, the conformation of anti-K⁺ aptamer in solution changed from random coil structure to compact rigid G-quadruplex one. This compact rigid G-quadruplex structure could not protect AuNPs against K⁺-induced aggregation, and thus the visible color change from wine-red to blue-purple could be observed by the naked eye. The linear range of the colorimetric aptasensor covered a large variation of K⁺ concentration from 5 nM to 1 μM and the detection limit of 5 nM was obtained. Moreover, this assay was able to detect K⁺ with high selectivity and had great potential applications.     

Graphene–DNAzyme junctions: a platform for direct metal ion detection with ultrahigh sensitivity

Many metal ions are present in biology and in the human body in trace amounts. Despite numerous efforts, metal sensors with ultrahigh sensitivity (<a few picomolar) are rarely achieved. Here, we describe a platform method that integrates a Cu²⁺-dependent DNAzyme into graphene–molecule junctions and its application for direct detection of paramagnetic Cu²⁺ with femtomolar sensitivity and high selectivity. Since DNAzymes specific for other metal ions can be obtained through in vitro selection, the method demonstrated here can be applied to the detection of a broad range of other metal ions.     

Sensitive and selective determination of imidacloprid with magnetic molecularly imprinted polymer by using LC/Q-TOF/MS

In this paper, magnetic-molecularly imprinted polymer was used for the preconcentration of trace levels of imidacloprid in water and apple samples prior to liquid chromatography-quadrupole-time-of-flight mass spectrometric determination. The selectivity of the magnetic polymer was united with the sensitivity and the high resolving power of the chromatographic system. The developed method showed a linear range from 10.0 to 500.0 µg/L. The quantitative recoveries were obtained for water and apple samples in the range of 92.0%–99.0 %. The relative standard deviations of intra-day and inter-day tests were found to be in the range of 0.8%–1.2% and 1.2%–1.6 %, respectively. In addition, the same magnetic-molecularly imprinted polymer (MMIP) can be used at least ten cycles for the determination of imidacloprid. The preconcentration factor of the method was found to be 2.5, and the total preconcentration procedure can be completed in 1 h. Characterization of synthesised particles were executed with various techniques. Due to its suitable limit of detection, dynamic linear range, sensitivity and selectivity, the developed method seemed to be ideal for the determination and preconcentration of imidacloprid in water and fruit samples.