基于磁性分离-硫化铜纳米粒子标记的DNA流动注射化学发光检测

本文基于磁性粒子(MB)良好的分离、富集能力,研究了硫化铜纳米粒子标记的流动注射-化学发光(FI-CL)DNA检测体系.通过硫化铜标记的探针1与目标DNA及连有磁球的探针2形成三明治结构,实现对目标DNA的捕获、分离与标记;通过其溶解释放出CuS标记颗粒的铜离子,引起化学发光信号增强,实现了目标DNA序列的定性定量检测.该方法对完全互补单链DNA(ssDNA)检测的线性范围为1.0×10-11～1.6×10-9 mol/L,检出限为3.0×10-12 mol/L,对1.0×10-9 mol/L目标DNA测定的相对标准偏差为3.2%(n=11),对目标碱基序列具有良好的识别能力.     

Nucleic acid functionalized graphene for biosensing

There is immense demand for complex nanoarchitectures based on graphene nanostructures in the fields of biosensing or nanoelectronics. DNA molecules represent the most versatile and programmable recognition element and can provide a unique massive parallel assembly strategy with graphene nanomaterials. Here we demonstrate a facile strategy for covalent linking of single stranded DNA (ssDNA) to graphene using carbodiimide chemistry and apply it to genosensing. Since graphenes can be prepared by different methods and can contain various oxygen containing groups, we thoroughly investigated the utility of four different chemically modified graphenes for functionalization by ssDNA. The materials were characterized in detail and the different DNA functionalized graphene platforms were then employed for the detection of DNA hybridization and DNA polymorphism by using impedimetric methods. We believe that our findings are very important for the development of novel devices that can be used as alternatives to classical techniques for sensitive and fast DNA analysis. In addition, covalent functionalization of graphene with ssDNA is expected to have broad implications, from biosensing to nanoelectronics and directed, DNA programmable, self-assembly.     

Covalent attachment of biotinylated molecular beacons via thiol-ene coupling. A study on conformational changes upon hybridization and streptavidin binding

The authors describe a method for the detection of DNA by using immobilized molecular beacons (MBs) on the surface of silicon, with a view on possible application in biosensing. MB hybridization and protein recognition were interrogated on silicon-on-insulator (SOI) surfaces by using fluorescently tagged probes. In order to better understand the conformational changes that occur to MBs upon hybridization, the process was studied by using dual polarization interferometry (DPI). A model system was developed that matches thickness, mass, and density parameters. The results experimentally demonstrate that hybridization promotes the displacement of a protein away from the surface. This finding may be further exploited in techniques such as photonic sensors, thereby paving the way to the design of more sensitive biosensors based on the use of MBs.

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Graphical abstract Schematic of a new DNA/RNA detection scheme by using immobilized molecular beacons (MBs) on silicon, with a view on possible application in biosensing. A study was performed on the conformational changes that occur to MBs upon hybridization by Dual Polarization Interferometry (DPI).

     

Molecular Engineering of DNA: Molecular Beacons

Molecular beacons (MBs) are specifically designed DNA hairpin structures that are widely used as fluorescent probes. Applications of MBs range from genetic screening, biosensor development, biochip construction, and the detection of single‐nucleotide polymorphisms to mRNA monitoring in living cells. The inherent signal‐transduction mechanism of MBs enables the analysis of target oligonucleotides without the separation of unbound probes. The MB stem–loop structure holds the fluorescence‐donor and fluorescence‐acceptor moieties in close proximity to one another, which results in resonant energy transfer. A spontaneous conformation change occurs upon hybridization to separate the two moieties and restore the fluorescence of the donor. Recent research has focused on the improvement of probe composition, intracellular gene quantitation, protein–DNA interaction studies, and protein recognition.     

Aptamer‐Based SERS Assay of ATP and Lysozyme by Using Primer Self‐Generation

A simple bifunctional surface‐enhanced Raman scattering (SERS) assay based on primer self‐generation strand‐displacement polymerization (PS‐SDP) is developed to detect small molecules or proteins in parallel. Triphosphate (ATP) and lysozyme are used as the models of small molecules and proteins. Compared to traditional bifunctional methods, the method possesses some remarkable features as follows: 1) by virtue of the simple PS‐SDP reaction, a bifunctional aptamer assembly binding of trigger 1 and trigger 2 was used as a functional structure for the simultaneous sensing of ATP or lysozyme. 2) The concept of isothermal amplification bifunctional detection has been first introduced into SERS biosensing applications as a signal‐amplification tool. 3) The problem of high background induced by excess bio‐barcodes is circumvented by using magnetic beads (MBs) as the carrier of signal‐output products and massive of hairpin DNA binding with SERS active bio‐barcodes relied on Au nanoparticles (Au NPs), SERS signal is significantly enhanced. Overall, with multiple amplification steps and one magnetic‐separation procedure, this flexible biosensing system exhibited not only high sensitivity and specificity, with the detection limits of ATP and lysozyme of 0.05 nM and 10 fM , respectively.     

Electrochemical enzyme-linked immunoassay in a DNA hybridization sensor

In most of the currently developed electrochemical DNA hybridization sensors short single-stranded probe DNA is immobilized on an electrode and both the hybridization and detection steps are carried out on the electrode surface. Here we use a new technology in which DNA hybridization is performed on commercially available magnetic beads and detection on solid electrodes. Paramagnetic Dynabeads Oligo(dT)₂₅ (DBT) with covalently bound (dT)₂₅ probe are used for the hybridization with target DNA containing adenine stretches. Target DNA is modified with osmium tetroxide,2,2′-bipyridine (Os,bipy) and the immunogenic DNA-Os,bipy adduct is determined by the enzyme-linked immunoassay with electrochemical detection. Electroinactive 1-naphthyl phosphate is used as a substrate and the electroactive product (1-naphthol) is measured on the carbon electrodes. Alternatively Os,bipy-modified target DNA can be determined directly by measuring the osmium signal on the pyrolytic graphite electrode (PGE). A comparison between determinations of the 67-mer oligodeoxynucleotide on carbon electrodes using (a) the guanine oxidation signal, (b) direct determination of the DNA-Os,bipy adduct and (c) its electrochemical immunoassay showed immunoassay to be the most sensitive method. In combination with DBT, the DNA hybridization of long target deoxyoligonucleotides (such as 67- and 97-mers) and a DNA PCR product (226-base pairs) have been detected by immunoassay at high sensitivity and specificity.     

Impedance DNA Biosensor Using Electropolymerized Polypyrrole/Multiwalled Carbon Nanotubes Modified Electrode

In this paper, we present an electrochemical impedance‐based DNA biosensor by using a composite material of polypyrrole (PPy) and multiwalled carbon nanotubes (MWNTs) to modify glassy carbon electrode (GCE). The polymer film was electropolymerized onto GCE by cyclic voltammetry (CV) in the presence of carboxylic groups ended MWNTs (MWNTs‐COOH). Such electrode modification method is new for DNA hybridization sensor. Amino group ended single‐stranded DNA (NH₂‐ssDNA) probe was linked onto the PPy/MWNTs‐COOH/GCE by using EDAC, a widely used water‐soluble carbodiimide for crosslinking amine and carboxylic acid group. The hybridization reaction of this ssDNA/PPy/MWNTs‐COOH/GCE resulted in a decreased impedance, which was attributed to the lower electronic transfer resistance of double‐stranded DNA than single‐stranded DNA. As the result of the PPy/MWNTs modification, the electrode obtained a good electronic transfer property and a large specific surface area. Consequently, the sensitivity and selectivity of this sensor for biosensing DNA hybridization were improved. Complementary DNA sequence as low as 5.0×10^?12 mol L^?1 can be detected without using hybridization marker or intercalator. Additionally, it was found that the electropolymerization scan rate was an important factor for DNA biosensor fabrication. It has been optimized at 20 mV s^?1.     

电化学脱氧核糖核酸传感器

本文对电化学脱氧核糖核酸（ＤＮＡ）传感器的原理，ＤＮＡ在电极表面的固定化，杂交指示剂的研究和电化学ＤＮＡ传感器的性能、分析应用及电化学石英晶体微天平ＤＮＡ传感器等方面的研究进展作了评述。提出了今后研究工作的方向。     

Multiplexed Immunosensing Platform Coupled to Hybridization Chain Reaction for Electrochemical Determination of MicroRNAs in Clinical Samples

There is an urgent need for development of rapid and inexpensive techniques for detection of microRNAs (miRNAs), which are potential biomarkers of various types of cancer. In this paper, we describe a multiplexed electrochemical platform for determination of three cancer‐relevant miRNAs: miR‐21, let‐7a and miR‐31. The strategy combines the use of magnetic beads (MBs) modified with a commercial antibody for the efficient capture of the heteroduplexes formed by hybridization of the target miRNA with DNA probe. Free non‐hybridized region of the DNA probe was thereafter hybridized with two biotin‐labeled auxiliary DNA probes in a process of hybridization chain reaction (HCR), resulting in a long hybrid bearing a large number of biotin molecules. Labeling of these multiple biotin units with streptavidin‐peroxidase conjugates allowed an amplification of the amperometric signal measured after capturing the modified MBs at a screen‐printed carbon electrode array of eight electrodes. The combined strategy demonstrated in a similar assay time significantly higher sensitivity than those previously described using modified MBs with the same capture antibody (without amplification by HCR) or a HCR strategy implemented on the surface of MBs, respectively. The methodology exhibits a good selectivity for discriminating single mismatches and was applied to the determination of the three target miRNAs in total RNA (RNA_t) extracted from various cancer cell lines and from cervical precancerous lesions.     

商品化血糖仪用于乙型肝炎病毒的便携式体外分子诊断

为解决当前乙型肝炎病毒(HBV)基因检测技术在一定程度上存在成本高、操作繁琐、误诊率高等问题,在此提出了一种便携式生物传感平台用于HBV基因的超灵敏检测,可检出低至2 copies/μL的HBV基因.首次通过设计针对HBV检测序列的环介导等温扩增(LAMP)反应,实现了在40 min之内对HBV基因型单拷贝基因的检测....     

DNA银纳米簇在功能核酸荧光生物传感器中的应用

DNA银纳米簇(DNA-AgNCs)是以DNA为模板, 通过碱基杂环上的N原子与Ag⁺结合, 用NaBH₄将Ag⁺还原得到的具有荧光性质的新兴纳米探针. 由于DNA-AgNCs具有合成方法简单、 生物相容性好和荧光发射波长可调等优点, 使其在分析检测等领域具有广泛的应用. 本文对DNA-AgNCs的合成和荧光性质两个方面进行了综述, 分类总结了以DNA-AgNCs为无标记荧光探针在功能核酸荧光生物传感器方面的应用, 对其不足与应用潜力进行展望, 以期为未来的研究与应用提供借鉴.     

Nanoparticle-based electrochemical DNA detection

Nanoscale architectures of DNA-linked particle networks are attractive for electrical detection of DNA hybridization. This article reviews a variety of new nanoparticle/polynucleotide assemblies for advanced electrical detection of DNA sequences. Recent activity has led to innovative and powerful nanoparticle-based electrochemical DNA hybridization assays based on a variety of detection schemes. Such protocols rely on the use of colloidal gold tags, semiconductor quantum dot tracers, polymeric carrier (amplification) beads, or magnetic (separation) beads. Particularly useful have been protocols based on capturing of metal nanoparticle tracers followed by dissolution and anodic-stripping voltammetric measurement of the metal tag. Remarkable sensitivity is achieved by coupling particle-based amplification units and various amplification processes. The use of nanoparticle tracers for designing multi-target electrochemical coding protocols will also be documented.     

Indicator-based and indicator-free magnetic assays connected with disposable electrochemical nucleic acid sensor system

An indicator-based and indicator-free magnetic assays connected with a disposable pencil graphite electrode (PGE) were successfully developed, and also compared for the electrochemical detection of DNA hybridization. The oxidation signals of echinomycin (ECHI) and electroactive DNA bases, guanine and adenine, respectively were monitored in the presence of DNA hybridization by using differential pulse voltammetry (DPV) technique. The biotinylated probe was immobilized onto the magnetic beads (magnetic particles, microspheres) and hybridization with its complementary target at the surface of particles within the medium was exhibited successfully using electrochemical sensor system. For the selectivity studies, the results represent that both indicator-based and indicator-free magnetic assays provide a better discrimination for DNA hybridization compared to duplex with one-base or more mismatches. The detection limits (S/N = 3) of the magnetic assays based on indicator or indicator-free were found in nM concentration level of target using disposable sensor technology with good reproducibility. The characterization and advantages of both proposed magnetic assays connected with a disposable electrochemical sensor are also discussed and compared with those methods previously reported in the literature.     

Applications of Carbon Nanotubes in Electrochemical DNA Biosensors

The discovery of carbon nanotubes (CNTs) about a decade ago has brought fascinating evolutions in electronics, material industry, as well as bio-techniques for DNA analysis, gene therapy, drug delivery etc. It has also dramatically promoted the development of DNA biosensing techniques, especially electrochemical DNA biosensor. The application of CNTs in electrochemical DNA biosensors includes two main aspects: on one hand, using CNTs as a novel substrate not only enables immobilization of DNA molecules but also serves as a powerful amplifier to amplify signal transduction event of DNA hybridization. On the other hand, CNTs can also be employed as a powerful carrier to pre-concentrate enzymes or electroactive molecules for electrochemical sensing of DNA hybridization as a novel indicator. In this review, we place emphasis on recent studies of CNTs-based electrochemical DNA biosensors based on these two aspects, with advantages and disadvantages of each aspect introduced herein.     

Recent trends in molecular beacon design and applications

A molecular beacon (MB) is a hairpin-structured oligonucleotide probe containing a photoluminescent species (PLS) and a quencher at different ends of the strand. In a recognition and detection process, the hybridization of MBs with target DNA sequences restores the strong photoluminescence, which is quenched before hybridization. Making better MBs involves reducing the background photoluminescence and increasing the brightness of the PLS, which therefore involves the development of new PLS and quenchers, as well as innovative PLS–quencher systems. Heavy-metal complexes, nanocrystals, pyrene compounds, and other materials with excellent photophysical properties have been applied as PLS of MBs. Nanoparticles, nanowires, graphene, metal films, and many other media have also been introduced to quench photoluminescence. On the basis of their high specificity, selectivity, and sensitivity, MBs are developed as a general platform for sensing, producing, and carrying molecules other than oligonucleotides.     

Nanocatalyst-based assay using DNA-conjugated Au nanoparticles for electrochemical DNA detection

Compared to enzymes, Au nanocatalysts show better long-term stability and are more easily prepared. Au nanoparticles (AuNPs) are used as catalytic labels to achieve ultrasensitive DNA detection via fast catalytic reactions. In addition, magnetic beads (MBs) are employed to permit low nonspecific binding of DNA-conjugated AuNPs and to minimize the electrocatalytic current of AuNPs as well as to take advantage of easy magnetic separation. In a sandwich-type electrochemical sensor, capture-probe-conjugated MBs and an indium-tin oxide electrode modified with a partially ferrocene-modified dendrimer act as the target-binding surface and the signal-generating surface, respectively. A thiolated detection-probe-conjugated AuNP exhibits a high level of unblocked active sites and permits the easy access of p-nitrophenol and NaBH 4 to these sites. Electroactive p-aminophenol is generated at these sites and is then electrooxidized to p-quinoneimine at the electrode. The p-aminophenol redox cycling by NaBH 4 offers large signal amplification. The nonspecific binding of detection-probe-conjugated AuNPs is lowered by washing DNA-linked MB-AuNP assemblies with a formamide-containing solution, and the electrocatalytic oxidation of NaBH 4 by AuNPs is minimized because long-range electron transfer between the electrode and the AuNPs bound to MBs is not feasible. The high signal amplification and low background current enable the detection of 1 fM target DNA.     

Analytical applications of Raman spectroscopy

In this article interaction of transition metal complexes with DNA and its applications in electrochemical DNA biosensors as hybridization indicator or electroactive marker of DNA are reviewed. Special emphasis has been given to the efforts for the development of new transition metal complexes and their interaction to DNA. DNA and polymers covalently conjugated with transition metal complexes were also reviewed.     

Rapid Electrochemical Assessment of Tumor Suppressor Gene Methylations in Raw Human Serum and Tumor Cells and Tissues Using Immunomagnetic Beads and Selective DNA Hybridization

We report a rapid and sensitive electrochemical strategy for the detection of gene‐specific 5‐methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5‐methylcytosines (5‐mC) are used for the capture of any 5‐mC methylated single‐stranded (ss)DNA sequence. A flanking region next to the 5‐mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen‐printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 pm and a limit of detection of 1.2 pm for the methylated synthetic sequence of the tumor suppressor gene O‐6‐methylguanine‐DNA methyltransferase (MGMT) promoter region. The method is applied in the 45‐min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U‐87 glioblastoma cells and paraffin‐embedded brain tumor tissues without any amplification and pretreatment step.     

Detecting DNA Damage with a Silver Solid Amalgam Electrode

Mercury electrodes modified with supercoiled (sc) DNA have been used as highly sensitive tools for the detection of DNA strand breaks or as sensors for DNA cleaving substances. In this paper we show that silver solid amalgam electrode (AgSAE), in connection with alternating current voltammetry, provides similar information about DNA damage as the hanging mercury drop electrode. The AgSAE can be used for the detection of enzymatic or chemical DNA cleavage in solution or at the electrode surface. AgSAE modified with scDNA can be utilized as a sensor for DNA nicking substances.     

Voltammetric Behavior of Osmium‐Labeled DNA at Mercury Meniscus‐Modified Solid Amalgam Electrodes. Detecting DNA Hybridization

The complex of osmium tetroxide with 2,2′‐bipyridine has been utilized as a probe of DNA structure and an electroactive marker of DNA in DNA hybridization sensors. It produces several voltammetric signals, the most negative of them has been observed only at mercury electrodes. This signal is of catalytic nature affording a high sensitivity of DNA determination. The catalytic current due to evolution of hydrogen in voltammetry of DNA modified by complex of osmium tetroxide with 2,2′‐bipyridine (DNA‐Os,bipy) was studied. Solid amalgam electrodes (modified with mercury menisci) of silver (m‐AgSAE), copper (m‐CuSAE), gold, and of combined bismuth and silver, were used as possible substitutes for mercury electrodes. Besides the hanging mercury drop electrode (HMDE), the catalytic current was observed only on m‐AgSAE and m‐CuSAE. Electrodes of gold and bismuth amalgams did not give the catalytic current. The detection limit of DNA‐Os,bipy on HMDE was 0.1 ng mL^?1 (RSD=2.3 %, N=11), and on m‐AgSAE 0.2 ng mL^?1 (RSD=3.1%, N=11). The m‐AgSAE was successfully applied as a detection electrode in double‐surface DNA hybridization experiments offering highly specific discrimination between complementary (target) and nonspecific DNAs, as well as determination of the length of a repetitive DNA sequence. The m‐AgSAE has proved a convenient alternative to the HMDE or carbon electrodes used for similar purposes in previous work.