Carbon Nanodot–Based Fluorescent Method for Virus DNA Analysis with Isothermal Strand Displacement Amplification

In this work, a fluorescent method is developed for ultrasensitive detection of virus DNA, coupling DNA‐modified carbon nanodots (CDs) and an isothermal amplification technology. The sensitivity is significantly improved with cyclic strand displacement polymerization and highly luminescent CDs. Taking the hemagglutinin7 (H7) gene and neuraminidase 9 (N9) gene as examples, the limits of detection are 4.6 × 10^?15 and 3.4 × 10^?15 m , respectively. Furthermore, the proposed method is highly selective and capable of detecting target sequences in biological samples, indicating great potential for applications in life science research and clinical diagnosis.     

A Graphene Oxide–Based Sensing Platform for The Label-free Assay of DNA Sequence and Exonuclease Activity via Long Range Resonance Energy Transfer

Graphene oxide (GO) was introduced as an efficient quencher for label-free and sensitive detection of DNA. Probe DNA (pDNA) was mixed with ethidium bromide (EB) and graphene oxide (GO). The interaction between EB and GO led to the fluorescent quenching. Upon the recognition of the target, EB was intercalated into duplex DNA and kept away from GO, which significantly hindered the long range resonance energy transfer (LrRET) from EB to GO and, thus, increased the fluorescence of EB. The changes in fluorescent intensity produced a novel method for sensitivity, and specificity detection of the target. Based on the structure-switching of aptamers, this strategy could be conveniently extended for detection of other biomolecules, which had been demonstrated by the detection of exonuclease activity.     

Facile Synthesis of Carbon Dots from Biomass Material and Multi-Purpose Applications

Selective and sensitive water content measurement in organic solvents is extremely significant for both industrial use and laboratory preparation. Carbon nanodots are promising carbon nanomaterials with unique and novel properties and thus have drawn growing attention. However, the hydrothermal approach for the preparation of carbon dots always uses water as solvent, and consequently, the development of carbon dots from biomass materials for fluorescence detection of water content remains unexplored. Here, carbon dots were prepared from gallic acid via a cheap and facile one-step method. The as-prepared carbon dots present excellent sensitivity and selectivity toward water content and exhibits good linear relationships with water content in range of 0–10%. The carbon dots demonstrated a strong antioxidation capacity and colour-reaction of Fe³⁺ like gallic acid. The carbon dots also showed solid-state lighting.

     

Study on the Interaction of the CpG Alternating DNA with CdTe Quantum Dots

A novel sensitive method for detection of DNA methylation was developed with thioglycollic acid (TGA)-capped CdTe quantum dots (QDs) as fluorescence probes. Recognition of methylated DNA sites would be useful strategy due to the important roles of methylation in disease occurrence and developmental processes. DNA methylation occurs most often at cytosine-guanine sites (CpG dinucleotides) of gene promoters. The QDs significantly interacted with hybridized unmethylated and methylated DNA. The interaction of CpG rich methylated and unmethylated DNA hybrid with quantum dots as an optical probe has been investigated by fluorescence spectroscopy and electrophoresis assay. The fluorescence intensity of QDs was highly dependent to unmethylated and methylated DNA. Specific site of CpG islands of Adenomatous polyposis coli (APC), a well-studied tumor suppressor gene, was used as the detection target. Under optimum conditions, upon the addition of unmethylated dsDNA, the fluorescence intensity increased in linear range from 1.0?×?10^??10 to 1.0?×?10^??6M with detection limit of 6.2?×?10^??11 M and on the other hand, the intensity of QDs showed no changes with addition of methylated dsDNA. We also demonstrated that the unmethylated and methylated DNA and QDs complexes showed different mobility in electrophoresis assay. This easy and reliable method could distinguish between methylated and unmethylated DNA sequences.     

A Significant Fluorescent Aptamer Sensor Based on Carbon Dots and Graphene Oxide for Highly Selective Detection of Progesterone

In this paper, a fluorescent aptamer sensor was constructed based on the carbon dots (CDs) and graphene oxide (GO). This sensor combines the excellent fluorescence performance of CDs with the high specificity of aptamer, which can detect progesterone (P₄) with high sensitivity and selectivity. In the absence of P₄, the CDs-aptamer system and GO form a fluorescence resonance energy transfer process (FRET), which quenches the fluorescence of the CDs. When P₄ is added, the aptamer specifically binds to it, resulting the fluorescence of the CDs is recovered. At optimal conditions, the fluorescence intensity recovered by the CDs has a linear relationship with the concentration of P₄ in the range of 0.1–120 nM and the detection limit is 3.3?×?10^–11 M. Besides, the sensor has satisfactory detection results of P₄ in milk, indicating that constructed method has enormous potential for application in food safety.

     

Two-Photon Excited Fluorescence Energy Transfer: A Study Based on Oligonucleotide Rulers

The use of two-photon excitation of fluorescence for detection of fluorescence resonance energy transfer (FRET) was studied for a selected fluorescent donor–acceptor pair. A method based on labeled DNA was developed for controlling the distance between the donor and the acceptor molecules. The method consists of hybridization of fluorescent oligonucleotides to a complementary single-stranded target DNA. As the efficiency of FRET is strongly distance dependent, energy transfer does not occur unless the fluorescent oligonucleotides and the target DNA are hybridized. A high degree of DNA hybridization and an excellent FRET efficiency were verified with one-photon excited fluorescence studies. Excitation spectra of fluorophores are usually wider in case of two-photon excitation than in the case of one-photon excitation [1]. This makes the selective excitation of donor difficult and might cause errors in detection of FRET with two-photon excited fluorescence. Different techniques to analyze the FRET efficiency from two-photon excited fluorescence data are discussed. The quenching of the donor fluorescence intensity turned to be the most consistent way to detect the FRET efficiency. The two-photon excited FRET is shown to give a good response to the distance between the donor and the acceptor molecules.     

DNA accumulation on single-anode microelectrode structures and its application in active microarray layout design

The present work investigates, using simulation computer tools and fluorescence microscopy experimental assays, the local distribution of short single-stranded oligonucleotide fragments accumulated on a single microelectrode after the switching of an external dc electrical stimulus for some seconds (up to 75 s). It contributes to fill the existing gap in active microelectronic DNA arrays, where due to the large number of microelectrodes contained in the devices, no or little attention has been given to the final coverage of the individual electrodes by the DNA probes transported onto them, and how it is influenced by the device geometry and other variables like the current magnitude and the transport time. All these parameters play a fundamental role in both the accumulation rate of DNA strands on a polarized electrode and the degree of symmetry of their final distribution, which is convenient to know prior to any active microarray layout design in order to optimize the device performance.     

2019年5期 电子期刊

碳纳米点（碳点）是一种新型的纳米发光材料,具有优异的发光性能、良好的生物相容性、低毒性、水溶性好和表面易功能化等特性,在光电器件、生物成像、光热治疗等领域展现了潜在应用价值。然而,合成碳点的前驱体材料多种多样,合成方法各有不同,导致其发光机理复杂多样。本文主要针对使用柠檬酸作为碳源、尿素或氨水作为氮源,采用微波和溶剂热的合成方法制备的氮掺杂碳点,探索碳点的发光机理和抑制碳点聚集诱导荧光猝灭的方法,并进一步研究碳点在固态照明、可见光光通讯、生物成像和光热治疗等领域的应用前景。     

基于氧化石墨烯模拟酶比色法检测汞离子

基于富T碱基序列能特异性识别Hg2+、氧化石墨烯(GO)对单链DNA(ssDNA)和T-Hg2+-T复合物的亲和力不同以及GO自身具有的模拟酶催化性能,构建了一种可视化检测水样中痕量Hg2+的新方法。在pH 4.0的NaAc-HAc缓冲溶液中,通过π—π堆积作用力,ssDNA可以吸附在GO表面,致使GO的类过氧化物酶活性减弱,从而催化H₂O₂氧化3,3’,5,5’-四甲基联苯胺(TMB)产生的蓝色产物减少,体系位于波长652 nm处的吸光度值降低;当待测体系中存在Hg2+时,ssDNA上的胸腺嘧啶碱基(T)与Hg2+发生特异性结合作用,形成T-Hg2+-T类似双链结构的稳定复合物,该复合物与GO的作用力较弱,不易吸附于其表面,因此不影响GO的模拟酶活性,体系吸光度值增强。在一定条件下Hg2+浓度越大,覆盖在GO表面的ssDNA越少,体系吸光度越强,据此建立检测Hg2+的新方法。当汞离子浓度在3.26×10-8~9.0×10-7 mol·L-1范围内时,体系的ΔA值与汞离子浓度呈现良好的线性关系。其线性方程为ΔA=41.75c(nmol·L-1)+0.048 7,相关系数r=0.997 3,检出限为9.79×10-9mol·L-1。该方法简单、直观,抗干扰能力强、无需昂贵仪器设备,可用于检测环境水样中Hg2+的含量。     

Ratiometric FRET-based detection of DNA and micro-RNA on the surface using TIRF detection

A new FRET-based method for the ratiometric detection of DNA oligomers on a surface using TIRF detection mode is reported. The dual-labeled system consisting of two hybridized oligomers, Cy3oligoY:Cy5oligoX was immobilized on the surface, and the total internal reflection fluorescence (TIRF) was used to detect emission signals from the surface. Two signals, green and red, which originated from the green donor Cy3 and the red acceptor Cy5, have been simultaneously detected. When the target single-stranded complimentary oligomer was present in the solution, this oligomer replaced the Cy3oligoY in the donor:acceptor complex on the surface and the ratio of red-to-green signal was dramatically changed. This detection scheme is generally applicable to the detection of DNA or RNA on a surface.     

Characterization of DNA chips on the molecular scale before and after hybridization with an atomic force microscope

Using two different 25-mer oligonucleotide probes covalently grafted on a silicon substrate, we demonstrate how efficient atomic force microscopy (AFM) can be for monitoring each step of DNA chip preparation: from probe immobilization to hybridization on the molecular scale. We observed the probe-molecule organization on the chip after immobilization, and the target molecules, which hybridized with probes could be individually identified. This article presents a method of straightforwardly identifying not only single and double DNA strands, but also, and more significantly, the hybridized part on them.     

基于无标记荧光共振能量转移的microRNA检测方法研究

利用以阳离子共轭聚合物为能量供体的荧光共振能量转移(FRET)策略和滚环扩增放大技术,建立了一种新型的microRNA(miRNA)检测方法。阳离子共轭聚合物采用聚[(9,9-双(6’-N,N,N-三乙基铵)己基)亚芴基亚苯基二溴化物](PFP)。PFP是一种由大量吸光单元共轭而成的阳离子聚合物,具有独特的光捕获和荧光增强性能,可以和带有负电荷的DNA通过静电作用相互结合。SG是一种能够结合于所有双链DNA双螺旋小沟区域的染料,其在游离状态下,荧光微弱,但一旦与双链DNA结合后,荧光会大大的增强。首先,设计了一条可与目标分子特异性杂交的锁式探针和与RCA产物序列互补的DNA链。当体系中存在miRNA时,在T4 DNA连接酶作用下,锁式探针连接成环;随后,在phi29 DNA聚合酶和dNTPs共同作用下,在miRNA的3’端滚环扩增出一条与锁式探针序列互补的长单链DNA,所得产物与互补DNA链杂交形成双链DNA(dsDNA)。此时SG作为FRET受体掺入其中,形成SG-dsDNA共同体。随后, SG-dsDNA与PFP因静电相互作用而紧密接近,由于PFP的发射光谱与SG的激发光谱有重叠,因此二者之间可以发生FRET现象。反之,当体系中不存在miRNA时,挂锁探针则无法连接成环,阻止了扩增反应的进行及其产物与互补DNA链的杂交反应。加入SG后,由于SG与单链DNA的结合能力很弱, SG则游离于溶液中,不会与PFP发生有效的FRET。因此目标分子的浓度与体系的FRET效率直接相关。以let 7a作为待测miRNA分子,在0.05~5 nmol·L-1的范围内, let 7a的浓度与从反应体系测得的FRET效率(I520/I423)成正比。同时以无PFP参加的检测方案作为对比实验,证明了PFP确实具有提高灵敏度的作用。另外,以四种同族miRNA分子及两种其他miRNA分子作为干扰物质对方法的特异性进行了考察,发现除了两种与目标分子序列高度相似的物质存在干扰外,其他物质几乎不产生信号。利用该方法对细胞总RNA提取液中let 7a的含量及其加标含量进行了检测,测量所得回收率基本令人满意。所建立的方案不需要荧光标记探针,有效降低了检测成本,简化了操作步骤,在与miRNA相关的疾病诊断领域具有一定的应用前景。     

Studies on the Fluorescence Fiber-Optic DNA Biosensor Using <Emphasis Type="Italic">p</Emphasis>-Hydroxyphenylimidazo[f]1,10-phenanthroline Ferrum(III) as Indicator

A complex Fe(phen)₂·PHPIP·3ClO₄·2H₂O, where phen = 1,10-phenanthroline and PHPIP = p-hydroxyphenylimidazo[f]1,10-phenanthroline, was synthesized and acted as a good fluorescence indicator based on its interaction with double-duplex DNA. Then a fiber-optic DNA biosensor of fluorimetric detection was developed based on the recognition of target DNA in DNA hybridization assays. A probe ssDNA was covalently immobilized onto the surface of quartz optical fibers and then the probe ssDNA hybridized with complementary ssDNA introduced into the local environment of the sensor. The hybridization with complementary strands was monitored in real time by fluorimetric detection. Several factors affecting the probe immobilization, target DNA hybridization, and indicator binding reactions were optimized to maximize the sensitivity and shorten the assay time. Using this method, a sequence of the 16-mer oligonucleotides could be quantified over the range from 4.98 × 10⁻⁷ to 4.88 × 10⁻⁶ M and a detection limit of 1.08 × 10⁻⁷ M. And the designed optic-fiber biosensor could be conveniently regenerated by thermal denature. The utility of the novel hybridization indicator could provide a simple, rapid, low toxicity and reusable detection.     

Template synthesis of monodisperse carbon nanodots

Monodisperse carbon nanodots in pores of mesoporous silica particles are obtained by template synthesis. This method is based on introducing a precursor (organosilane) into pores, its thermal decomposition with formation of carbon nanodots, and the template removal. Structural analysis of the nanomaterial has been performed, which showed that carbon nanodots have an approximately spherical form and a graphite-like structure. According to dynamic light scattering data, the size of carbon nanodots is 3.3 ± 0.9 nm.

     

Investigation and Development of Quantum Dot-Encoded Microsphere Bioconjugates for DNA Detection by Flow Cytometry

The development of screening assays continues to be an active area of research in molecular diagnostics. Fluorescent microspheres conjugated to biomarkers (nucleic acids, proteins, lipids, carbohydrates) and analyzed on flow cytometer instruments offered a new approach for multiplexed detection platform in a suspension format. Quantum dots encoded into synthetic microspheres have the potentials to improve current screening bioassays and specifically suspension array technology. In this paper, commercialized quantum dot-encoded microsphere were evaluated and optimized as fluorescent probes to address some of the limitations of suspension array technologies. A comprehensive study was undertaken to adapt the bioconjugation procedure to the quantum dot-encoded microsphere structural and optical properties. Both the leaching-out of quantum dots and microspheres degradation under bioconjugation experimental conditions were minimized. A rapid, efficient and reproducible conjugation method was developed for the detection of single-stranded DNA with the commercialized quantum dot-encoded microsphere. Approximately ten thousand microspheres were conjugated to short amino-modified DNA sequences in one hour with high efficiency. The bioconjugated microspheres acting as fluorescent probes successfully detected a DNA target in suspension with high specificity. Quantum dot-encoded microsphere commercial products are limited which strongly prevents reproducible and comparative studies between laboratories. The method developed here contributes to the understanding of quantum dot-encoded microsphere reactivity, and to the optimization of adapted experimental procedure. This step is essential in the development of this new fluorescent probe technology for multiplex genotyping assay and molecular diagnostic applications.     

Trace determination of uranium preconcentrated using graphene oxide by total reflection X-ray fluorescence spectrometry

A new method based on dispersive microsolid phase extraction using graphene oxide (GO) as a solid adsorbent and total reflection X-ray fluorescence (TXRF) spectrometry is proposed for trace determination of uranium. In the developed methodology, a suspension of GO was injected into uranium-spiked multielement solutions including rubidium; after filtration, the membrane filter with collected GO was placed in a small volume of internal standard acid solution and the eluent containing uranium was deposited onto a fluorine resin-coated slide glass, which is a disposable sample stage. Using GO was effective for removal of rubidium from the measurement solution to avoid interference between Rb Kα peak and U Lα peak. The high enrichment factor of 150 enables obtaining uranium detection limits of 0.042, 0.087, and 0.12 μg L⁻¹ for ionic strength of 0.01, 0.1, and 1 mol L⁻¹, respectively. Such low detection limits were obtained by using a benchtop TXRF spectrometer with 5-min measurement. The proposed method is suitable for trace uranium determination in water, including high salinity samples.     

Porous Magnetic Nanoparticles-Based Electrochemical Biosensor for Determination of Mercury in the Aquatic Environment

Fabrication of sensitive and convenient methods for the detection of heavy metal ions is an important task due to their high toxicity to environment and human health. Divalent mercury ions (Hg²⁺) are a kind of significant public health hazard, which generate harmful toxic effects. In this work, a novel self-powered biosensor for the quantification of Hg²⁺ in aqueous solutions is developed. Porous magnetic nanoparticles are synthesized to load electrochemical species and a Hg²⁺ responsive single-stranded DNA probe is used to seal them. After specific interaction with target ions, the DNA probe forms a rigid duplex, which can no longer block the pores of the magnetic nanoparticles. The leakage of electrochemical species can then increase the electrochemical response, which is used to indicate the initial concentration of Hg²⁺. This method shows high sensitivity and selectivity, and can be applied in real water samples with excellent recoveries. Therefore, a novel approach is provided for ultrasensitive and reliable detection of Hg²⁺ in practical applications.     

新基激复合物探针检测指定序列DNA

研究目的是拓展基激复合物荧光探针方法在检测指定序列DNA中的应用。实验选择细胞色素P450 CYP2C9基因中容易发生突变的包含24个碱基的基因片段作为靶点DNA,选择两个分开的与靶点碱基相对应的包含12碱基的寡核苷酸作为探针,将荧光集团连接到探针末端3′或5′磷酸基上形成荧光探针,两个荧光探针与靶点DNA杂交后自动装备基激复合物荧光系统,实验考察芘的新衍生物与不同荧光团配对、荧光团连接不同位置、不同激发波长对基激复合物形成及发射光谱的影响,芘的新衍生物探针形成的基激复合物在505 nm的特征发射光谱最强,伴随着单体荧光的猝灭和大约120～130 nm的Stokes位移,大大减少了DNA检测过程中的背景干扰,灵敏度高,而且这对荧光物质形成的基激复合物对所处的空间位置及微环境非常敏感,可尝试用于基因遗传多态性的识别。     

Colorimetric DNA detection of transgenic plants using gold nanoparticles functionalized with L-shaped DNA probes

In this study, a DNA colorimetric detection system based on gold nanoparticles functionalized with L-shaped DNA probes was prepared and evaluated. We investigated the hybridization efficiency of the L-shaped probes and studied the effect of nanoparticle size and the L-shaped DNA probe length on the performance of the as-prepared system. Probes were attached to the surface of gold nanoparticles using an adenine sequence. An optimal sequence of 35S rRNA gene promoter from the cauliflower mosaic virus, which is frequently used in the development of transgenic plants, and the two complementary ends of this gene were employed as model target strands and probe molecules, respectively. The spectrophotometric properties of the as-prepared systems indicated that the large NPs show better changes in the absorption spectrum and consequently present a better performance. The results of this study revealed that the probe/Au-NPs prepared using a vertical spacer containing 5 thymine oligonucleotides exhibited a stronger spectrophotometric response in comparison to that of larger probes. These results in general indicate the suitable performance of the L-shaped DNA probe-functionalized Au-NPs, and in particular emphasize the important role of the gold nanoparticle size and length of the DNA probes in enhancing the performance of such a system.