Immobilization of DNA on magnetic microparticles for mercury enrichment and detection with flow cytometry

Mercury detection in water has attracted a lot of research interest due to its highly toxic nature and adverse environmental impact. In particular, the recent discovery of specific binding of Hg(II) to thymine-rich (T-rich) DNA resulting in T-Hg(II)-T base pairs has led to the development of a number of sensors with different signaling mechanisms. However, the majority of such sensors were non-immobilized. Immobilization, on the other hand, allows active mercury adsorption, signal amplification, and sensor regeneration. In this work, we immobilized a thymine-rich DNA on a magnetic microparticle (MMP) surface through biotin-streptavidin interactions. In the presence of Hg(II), the DNA changes from a random coil structure into a hairpin, upon which SYBR Green I binds to emit green fluorescence. Detection was carried out by using flow cytometry where the fluorescence intensity increased ≈9-fold in the presence of mercury and the binding of mercury reached equilibrium in less than 2 min. The sensor showed a unique sample-volume-dependent fluorescence signal change where a higher fluorescence was obtained with a larger sample volume, suggesting that the particles can actively adsorb Hg(II). Detection limits of 5 nM (1 ppb) and 14 nM (2.8 ppb) were achieved in pure buffer and in mercury-spiked Lake Ontario water samples, respectively.     

Fluorescence Determination of Merucury(II) Using a Thymine Aptamer

A label-free thymine-rich sequence and a molecular beacon were synthesized to construct a highly sensitive and selective fluorescence probe for the determination of mercury(II). The aptamer of the thymine-rich sequence selectively bonded with mercury(II) with an accompanying change in the fluorescence intensity of the molecular beacon due to the higher affinity of the aptamer with mercury(II). The limit of detection was 12.7 nanomolar, and a linear relationship was obtained between the fluorescence and mercury(II) concentrations up to 1 micromolar. The assay was highly selective for the mercury(II) and not significantly affected by other metal ions.     

A novel fluorescent biosensor for sequence-specific recognition of double-stranded DNA with the platform of graphene oxide

A fluorescent biosensor for sequence-specific recognition of double-stranded DNA (dsDNA) was developed based upon the DNA hybridization between dye-labeled single-stranded DNA (ssDNA) and double-stranded DNA. The fluorescence of FAM-labeled single-stranded DNA was quenched when it adsorbed on the surface of graphene oxide (GO). Upon addition of the target dsDNA, a homopyrimidine·homopurine part of dsDNA on the Simian virus 40 (SV40) (4424-4440, gp6), hybridization occurred between the dye-labeled DNA and the target dsDNA, which induced the dye-labeled DNA desorbed from the surface of GO, and turned on the fluorescence of the dye. Under the optimum conditions, the enhanced fluorescence intensity was proportional to the concentration of target dsDNA in the range 40.0-260 nM, and the detection limit was found to be 14.3 nM alongside the good sequence selectivity.     

Photophysical properties of fluorescent DNA-dyes bound to single- and double-stranded DNA in aqueous buffered solution.

The absorption and fluorescence spectra, fluorescence quantum yields, lifetimes and time-resolved fluorescence spectra are reported for nine different fluorescent DNA-dyes. The work was initiated in search of a quantitative method to detect the ratio of single-to-double stranded DNA (ssDNA/dsDNA) in solution based on the photophysics of dye-DNA complexes; the result is a comprehensive study providing a vast amount of information for users of DNA strains. The dyes examined were the bisbenzimide or indole-derived stains (Hoechst 33342, Hoechst 33258 and 4',6-diamidino-2-phenylindole), phenanthridinium stains (ethidium bromide and propidium iodide) and cyanine dyes (PicoGreen, YOYO-1 iodide, SYBR Green I and SYBR Gold). All were evaluated under the same experimental conditions in terms of ionic strength, pH and dye-DNA ratio. Among the photophysical properties evaluated only fluorescence lifetimes for the cyanine stilbene dyes allowed a convenient differentiation between ssDNA and dsDNA. The bisbenzimide dyes showed multiexponential decays when bound to either form of DNA, making lifetime-based analysis cumbersome with inherent errors. These dyes also presented biexponential decay when free in aqueous buffered solutions at different pH. A mechanism for their deactivation is proposed based on two different conformers decaying with different kinetics. The phenanthridinium dyes showed monoexponential decays with ssDNA and dsDNA, but there was no discrimination between them. High dye-DNA ratios (e.g. 1:1) resulted in multiexponential decays for cyanine dyes, resulting from energy transfer or self-quenching deactivation. Shifts in both absorption and fluorescence maxima for both ssDNA and dsDNA DNA-cyanine dye complexes were small. Broadening of dye-ssDNA absorption and fluorescence bands for the cyanine dyes relative to dye-dsDNA bands was detected and attributed to higher degrees of rotational freedom in the former.     

Sensitive detection of cyclophosphamide using DNA-modified carbon paste, pencil graphite and hanging mercury drop electrodes

The interaction of cyclophosphamide (CP) with calf thymus double-stranded DNA (dsDNA) and thermally denatured single-stranded DNA (ssDNA) immobilized at the carbon paste (CPE) and pencil graphite electrodes (PGE), was studied electrochemically based on oxidation signals of guanine and adenine using differential pulse voltammetry (DPV).As a result of the interaction of CP with DNA, the voltammetric signals of guanine and adenine increased in the case of dsDNA while a slight increase was observed in ssDNA. The effect of experimental parameters such as the interaction time between CP and DNA forms and the concentration of CP, were studied using DPV with CPE and PGE. Additionally, reproducibility and detection limits were determined using both electrodes. A comparison of the analytical performance between CPE and PGE was done. Our results showed that these two different DNA biosensors could be used for the sensitive, rapid and cost effective detection of CP itself as well as of CP-DNA interaction.Furthermore, the interaction of CP with dsDNA and ssDNA was studied in solution and at the electrode surface by means of alternating current voltammetry (ACV) in 0.3 M NaCl and 50 mM sodium phosphate buffer (pH 8.5) supporting electrolyte, using a hanging mercury drop electrode (HMDE) as working electrode.The conclusions of this study were mainly based on tensammetric peaks I (at −1.183 V) and II (−1.419 V) of DNA. This study involved the interaction of CP with surface-confined and solution phase DNA where experimental parameters, such as the concentration of CP and the interaction time, were studied. By increasing the concentration of CP, an increase of peak II was observed in both ds and ssDNA, while an increase of peak I was observed only in the case of dsDNA. An overall conclusion of the study using HMDE was that the interaction of CP with surface-confined DNA significantly differed from that with solution phase DNA. The increase of peaks I and II was lower in the case of interaction of CP with surface-confined DNA, probably due to steric positioning of DNA at the electrode surface.     

ssDNA-QDs/GO multicolor fluorescence system for synchronous screening of hepatitis virus DNA

Viral hepatitis is a common infectious disease caused by five viruses (hepatitis virus A, B, C, D, and E). Given the diversity of hepatitis virus, rapid screening and accurate typing of viral hepatitis are the prerequisites for hepatitis therapy. Here, a multicolor fluorescence system was constructed by combining with the multi-color fluorescence properties of CdSe/ZnS quantum dots (QDs, emission wavelengths: 525 nm, 585 nm and 632 nm) and the broad-spectrum fluorescence quenching performance of GO. Taking advantage of the specific recognition of ssDNA modified CdSe/ZnS QDs to target hepatitis virus DNA, the constructed system could effectively distinguish hepatitis A virus DNA (HAV-DNA), hepatitis B virus DNA (HBV-DNA), and hepatitis C virus DNA (HCV-DNA) in a homogeneous solution. Based on the different adsorption property of GO for ssDNA and dsDNA, the fluorescence Forster resonance energy transfer (FRET) process between ssDNA modified QDs and GO could be regulated. The fluorescence signal of the constructed system presented a sensitive response to HAV-DNA, HBV-DNA, and HCV-DNA content in the range of 1.0–192 nM, 8.0–192 nM, and 1.0–128 nM, respectively. The limit of detection for HAV-DNA, HBV-DNA, and HCV-DNA is 0.46 nM, 1.53 nM, and 0.58 nM. The constructed system can be used to screen hepatitis virus DNA in real samples, which provides an alternative strategy for rapid screening and diagnosis of viral hepatitis.     

Square wave and elimination voltammetric analysis of azidothymidine in the presence of oligonucleotides and chromosomal DNA

Azidothymidine (AZT, 3'-azido-3'-deoxythymidine, Zidovudine, Retrovir) is an approved and widely used antiretroviral drug for the treatment of human immunodeficiency virus (HIV) infection. Dynamic electrochemical methods have been employed for the fast and inexpensive determination of this drug in natural samples. The electrochemical signal of AZT, resulting from the reduction of azido group, was studied by square wave voltammetry (SWV), linear sweep voltammetry (LSV) and elimination voltammetry with linear scan (EVLS) using a hanging mercury drop electrode (HMDE). This paper explores the possibility of determining AZT in the presence of native (dsDNA) or denatured calf thymus DNA (ssDNA), and/or some synthetic oligodeoxynucleotides (ODNs). The detection limit of AZT in the absence and in the presence of ssDNA (10 microg/ml) is 1 and 250 nM, respectively. It was found that the signal of AZT is not substantially affected by the presence of DNA. We can therefore assume that the electrons are transferred through the adsorption layer of nucleic acids. By using the elimination procedure, both irreversible reduction signals of AZT and DNA are augmented. Moreover, the elimination signal in the peak-counterpeak form may indicate the adsorption of the analytes on the electrode surface preceding an electron transfer.     

聚胸腺嘧啶单链DNA-CuNCs荧光增强法用于汞离子的高灵敏检测

基于Hg~(2+)与DNA中胸腺嘧啶(T)结合的高度特异性和DNA铜纳米簇的荧光增强性质,构建了一种简便、灵敏检测汞离子的新方法.当Hg~(2+)存在时,聚T单链DNA(P1)通过T-Hg~(2+)-T特异性结合形成双链DNA,Cu~(2+)经抗坏血酸钠还原后生成的中间体Cu+与双链DNA螺旋结构间的氢键部分有强的结合力,促使Cu0附着聚集在双链DNA上形成铜纳米簇,导致体系荧光增强,从而实现对汞离子的高灵敏检测.体系荧光强度与Hg~(2+)浓度的对数值成正比,对Hg~(2+)检测的线性范围为1.0 nmol/L~10μmol/L,检出限达0.4 nmol/L,对湖水样品中Hg~(2+)检测的回收率达到97.2%~106.6%.与传统方法相比,该方法具有无需标记、检出限低及选择性好等优点,可用于环境水体中汞离子的测定.     

Highly sensitive and selective detection of Hg(2+) in aqueous solution with mercury-specific DNA and Sybr Green I

Sybr Green I efficiently discriminates mercury-specific DNA and mercury-specific DNA/Hg(2+) complex, which provides a label-free, fast, fluorescence turn on assay for Hg(2+) detection with high sensitivity and selectivity.     

Cyclic voltammetry of echinomycin and its interaction with double-stranded and single-stranded DNA adsorbed at the electrode.

Interactions of echinomycin (Echi) with DNA was studied by cyclic voltammetry (CV) with hanging mercury drop electrode (HMDE). Echinomycin was electrochemically active, yielding several signals. Interaction of Echi with dsDNA attached to a hanging mercury drop electrode resulted in high Echi signals, suggesting a strong binding of Echi to dsDNA by bis-intercalation at the electrode surface. Under the same conditions, interaction of Echi with ssDNA produced almost no Echi signal. This behavior is in agreement with a strong binding of Echi to dsDNA and a very weak binding of Echi to ssDNA observed earlier in solution. Echi, thus, appears to be a good candidate for redox indicator in electrochemical DNA hybridization sensors.     

A facile graphene oxide-based DNA polymerase assay

The DNA polymerase assay is fundamental for related molecular biology investigations and drug screenings, however, the commonly used radioactive method is laborious and restricted. Herein, we report a novel, simple and cost-effective fluorometric DNA polymerase detection method by utilizing graphene oxide (GO) as a signal switch. In this strategy, in the absence of DNA polymerase, the fluorophore-labeled template ssDNA could be strongly adsorbed and almost entirely quenched by GO. However, as DNA polymerase exists, the polymerized dsDNA product might lead to a much lower quenching efficiency after addition of GO due to the much weaker interaction of dsDNA with GO than ssDNA, thus resulting in a much higher fluorescence signal detected. As proof of concept, the quantitative DNA polymerase activity assay was performed using the Klenow fragment exo(-) (KF(-)) as a model. It was confirmed that, after optimization of detection conditions, KF(-) activity could be sensitively detected through facile fluorescence measurements, with a detection limit of 0.05 U mL(-1) and a good linear correlation between 0.05-2.5 U mL(-1) (R(2) = 0.9928). In addition, this GO-based method was further inspected to evaluate the inhibitive behaviors of several drugs toward KF(-) activity, the result of which firmly demonstrated its potential application in polymerization-targeted drug screening.     

Label-Free Fluorescent Aptasensor Based on a Graphene Oxide Self-Assembled Probe for the Determination of Adenosine Triphosphate

A sensitive and selective fluorescent aptasensor for adenosine triphosphate (ATP) was fabricated, composed of unbound SYBR Green I, graphene oxide, and a label-free detection probe. When ATP and complementary DNA of a signal probe were introduced, π-stacking interactions repelled the probe from the graphene oxide and formed a DNA-SYBR Green I duplex structure, triggering an increase in the fluorescence. ATP was determined over a linear range of 10 to 700 nM with a detection limit of 1 nM. The method displayed good selectivity, and is currently the most sensitive ATP fluorescence method. Furthermore, prominent fluorescence signals were also obtained in cellular assays. Consequently, the biosensor may have significant applications in protein, pathogenic microorganisms, and small molecule detection.     

Electropolymerized surface ion imprinting films on a gold nanoparticles/single-wall carbon nanotube nanohybrids modified glassy carbon electrode for electrochemical detection of trace mercury(II) in water

Electrochemical detection of Hg(II) using a electropolymerized ion imprinting poly(2-mercaptobenzothiazole) films at the surface of gold nanoparticles/single-walled carbon nanotube nanohybrids modified glassy carbon electrode (PMBT/AuNPs/SWCNTs/GCE) is described for the first time. The Hg(II)-imprinted PMBT/AuNPs/SWCNTs/GCE sensor exhibits larger binding to functionalized capacity, larger affinity, faster binding kinetics and higher selectivity to template Hg(II). The differential pulse anodic stripping voltammetry (DPASV) response of the Hg(II)-imprinted PMBT/AuNPs/SWCNTs/GCE sensor to Hg(II) is ca. 3.7- and 10.5-fold higher than that at the non-imprinted PMBT/AuNPs/SWCNTs/GCE and the imprinted PMBT/AuNPs/GCE, respectively, and the detection limit for Hg(II) is 0.08 nM (S/N = 3, which is well below the guideline value given by the World Health Organization) and a sensitivity of 0.749 μA nM⁻¹ was obtained. Excellent wide linear range (0.4–96.0 nM) and good repeatability (relative standard deviation of 2.6%) were obtained for Hg(II). The interference experiments show that Ag(I), Pb(II), Cd(II), Zn(II) and Cu(II) had little or no influence on the Hg(II) signal. These values, particularly the high sensitivity and excellent selectivity in contrast to the values reported previously in the area of electrochemical Hg(II) detection, demonstrate the analytical performance of the Hg(II)-imprinted PMBT/AuNPs/SWCNTs/GCE toward Hg(II) is superior to the existing electrodes and could be used for efficient determination of Hg(II) in natural water samples.     

基于阳离子型共轭聚合物和核酸适体的腺苷检测新方法

建立了一种基于阳离子型共轭聚合物和核酸适体的腺苷检测新方法. 荧光素修饰的短链DNA与腺苷的核酸适体部分互补, 形成双链DNA; 阳离子型共轭聚合物通过静电作用与双链DNA结合, 发生高效率的荧光共振能量转移(FRET). 加入腺苷后, 腺苷与核酸适体发生特异性结合, 导致双链DNA分解成单链, 使静电吸引力下降, 能量转移效率降低. 通过阳离子型共轭聚合物对单双链DNA的高效识别, 可快速简易地检测出腺苷.     

A fluorescence turn-on sensor for iodide based on a thymine-Hg(II)-thymine complex

Iodide plays a vital role in many biological processes, including neurological activity and thyroid function. Due to its physiological relevance, a method for the rapid, sensitive, and selective detection of iodide in food, pharmaceutical products, and biological samples such as urine is of great importance. Herein, we demonstrate a novel and facile strategy for constructing a fluorescence turn-on sensor for iodide based on a T-Hg(II)-T complex (T=thymine). A fluorescent anthracene-thymine dyad (An-T) was synthesized, the binding of which to a mercury(II) ion lead to the formation of a An-T-Hg(II)-T-An complex, thereby quenching the fluorescent emission of this dyad. In this respect, the dyad An-T constituted a fluorescence turn-off sensor for mercury(II) ions in aqueous media. More importantly, it was found that upon addition of iodide, the mercury(II) ion was extracted from the complex due to the even stronger binding between mercury(II) ions and iodide, leading to the release of the free dyad and restoration of the fluorescence. By virtue of this fluorescence quenching and recovery process, the An-T-Hg(II)-T-An complex constitutes a fluorescence turn-on sensor for iodide with a detection limit of 126 nM. Moreover, this sensor is highly selective for iodide over other common anions, and can be used in the determination of iodide in drinking water and biological samples such as urine. This strategy may provide a new approach for sensing some other anions.     

Rolling cycle amplification based single-color quantum dots–ruthenium complex assembling dyads for homogeneous and highly selective detection of DNA

In this work, a new, label-free, homogeneous, highly sensitive, and selective fluorescent biosensor for DNA detection is developed by using rolling-circle amplification (RCA) based single-color quantum dots–ruthenium complex (QDs–Ru) assembling dyads. This strategy includes three steps: (1) the target DNA initiates RCA reaction and generates linear RCA products; (2) the complementary DNA hybridizes with the RCA products to form long double-strand DNA (dsDNA); (3) [Ru(phen)₂(dppx)]²⁺ (dppx = 7,8-dimethyldipyrido [3,2-a:2′,3′-c] phenanthroline) intercalates into the long dsDNA with strong fluorescence emission. Due to its strong binding propensity with the long dsDNA, [Ru(phen)₂(dppx)]²⁺ is removed from the surface of the QDs, resulting in restoring the fluorescence of the QDs, which has been quenched by [Ru(phen)₂(dppx)]²⁺ through a photoinduced electron transfer process and is overlaid with the fluorescence of dsDNA bonded Ru(II) polypyridyl complex (Ru-dsDNA). Thus, high fluorescence intensity is observed, and is related to the concentration of target. This sensor exhibits not only high sensitivity for hepatitis B virus (HBV) ssDNA with a low detection limit (0.5 pM), but also excellent selectivity in the complex matrix. Moreover, this strategy applies QDs–Ru assembling dyads to the detection of single-strand DNA (ssDNA) without any functionalization and separation techniques.     

Highly sensitive fluorescent sensor for mercury ion based on photoinduced charge transfer between fluorophore and π-stacked T-Hg(II)-T base pairs

A novel and simple oligodeoxyribonucleotide-based sensor with single fluorophore-labeled for mercury ion sensing was reported. An oligodeoxyribonucleotide poly(dT) was labeled with fluorescein as donor. Based on the specific binding of Hg(II) to T-T mismatch base pairs, the formation of π-stacked [T-Hg(II)-T] with “sandwich” structure on the addition of Hg(II) ions facilitates the electron transfer via photoinduced charge transfer (PCT), which creates an additional nonradiative decay channel for excited fluorophore and triggers the fluorescence to be quenched. The π-stacked [T-Hg(II)-T] functioned not only as mercury ion recognition but also as an electron acceptor to quench the donor. A linear relationship was observed over the range of 0-1.0 μM with the detection limit of 20 nM for mercury ions. The fluorescence quenching phenomenon and quenching mechanism, reliability and selectivity of the system were investigated in detail.     

Conformational change detection of DNA with the fluorogenic reagent of o-phthalaldehyde-beta-mercaptoethanol

o-Phthalaldehyde-beta-mercaptoethanol (OPAME) as a fluorogenic reagent has been found wide applications in the detection of amino acids based on its reaction with primary amino groups. In this contribution, we report our new findings concerning the reactions of OPAME with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), respectively. It has been found that ssDNA can react with OPAME easily as a result of giving rise to strong fluorescence emissions, while dsDNA, prepared by hybridizing ssDNA with its complementary target prior to the reaction, displays inert chemical activity and gives out weak fluorescence emission. Mechanism investigations have shown that the reaction activity between OPAME and DNA depends on the amino groups that are related to the conformation of uncoiled and exposed extent of DNA structure, and thus the inert chemical activity of dsDNA results from screening of the dsDNA bases in the interior of the double strands. Therefore, we could design a way to detect conformation change of DNA with OPAME and further develop a novel, simple label-free sequence detection method for complementary and single-base mismatched ssDNA in the hybridization of DNA.     

An Electrochemical Aptasensor for Detection of Thrombin based on Target Protein‐induced Strand Displacement

A sensitive electrochemical aptasensor for detection of thrombin based on target protein‐induced strand displacement is presented. For this proposed aptasensor, dsDNA which was prepared by the hybridization reaction of the immobilized probe ssDNA (IP) containing thiol group and thrombin aptamer base sequence was initially immobilized on the Au electrode by self‐assembling via Au? S bind, and a single DNA labeled with CdS nanoparticles (DP‐CdS) was used as a detection probe. When the so prepared dsDNA modified Au electrode was immersed into a solution containing target protein and DP‐CdS, the aptamer in the dsDNA preferred to form G‐quarter structure with the present target protein resulting that the dsDNA sequence released one single strand and returned to IP strand which consequently hybridized with DP‐CdS. After dissolving the captured CdS particles from the electrode, a mercury‐film electrode was used for electrochemical detection of these Cd²⁺ ions which offered sensitive electrochemical signal transduction. The peak current of Cd²⁺ ions had a good linear relationship with the thrombin concentration in the range of 2.3×10^?9–2.3×10^?12 mol/L and the detection limit was 4.3×10^?13 mol/L of thrombin. The detection was also specific for thrombin without being affected by the coexistence of other proteins, such as BSA and lysozyme.