Toehold integrated molecular beacon system for a versatile non-enzymatic application

Analytical and Bioanalytical Chemistry - A molecular beacon (MB) is an oligonucleotide hybridization probe with a hairpin-shaped structure that leads to specific and instantaneous nucleic acid...     

TaqMan-分子灯标：一种新型的荧光基因检测探针

在TaqMan及分子灯标的基础上开发了一类新型的均相荧光检测探针—— TaqMan-分子灯标(TaqMan-MB)，该探针集合了分子灯标的发夹结构及TaqMan探针降 解作用的工作原理，使检测效果更好．与实时PCR仪联用，可用于靶基因的定量检 测．     

基于分子信标末端现场标记三聚氰胺-Cu~(2+)电活性分子的高灵敏电化学传感器

本文构建了一种基于分子信标自由末端现场标记电活性信号分子的新型DNA传感器.首先将3′修饰巯基的分子信标通过Au–S键自组装到金电极表面,然后在修饰有羧基的5′自由末端通过共价偶合和配位作用依次组装上三聚氰胺(Mel)和铜离子(Cu2+),得到以Mel-Cu2+配合物为电活性信号源的分子信标.该方法简单实现了电活性分子信标的标记、分离和纯化.以[Fe(CN)6]3-/4-为电化学探针,采用循环伏安和电化学阻抗法对层层自组装过程进行了表征.杂交实验表明,Mel-Cu2+信号源所对应的峰电流强度随着杂交液浓度的增大逐渐降低,且氧化峰电流与互补序列浓度对数在1.0×10-15~1.0×10-9 mol/L范围内呈良好的线性关系.根据3σ计算得到检测限为2.4×10-16 mol/L.另外,由于分子信标特殊的茎环结构特征和Mel-Cu2+信号源稳定的无机配位组成,传感器显示了很高的特异性、再生性和稳定性.     

A versatile molecular beacon-like probe for multiplexed detection based on fluorescence polarization and its application for a resettable logic gate

A versatile molecular beacon (MB)-like probe was developed for multiplexed detection based on fluorescence polarization by target-induced allosteric effect and furthermore for resettable logic gate operation.     

Assistant deoxyribonucleic acid recycling with Zn and molecular beacon for electrochemical detection of deoxyribonucleic acid via target-triggered assembly of mutated DNAzyme

A novel enzyme-free amplification strategy was designed for sensitive electrochemical detection of deoxyribonucleic acid (DNA) based on Zn²⁺ assistant DNA recycling via target-triggered assembly of mutated DNAzyme. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme first hybridized and then cleaved the MB in the presence of cofactor Zn²⁺. After cleavage, the MB was cleaved into two pieces and the ferrocene (Fc) labeled piece dissociated from the gold electrode, thus obviously decreasing the Fc signal and forming a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles to trigger the cleavage of many MB substrates. Therefore, the peak current of Fc dramatically decreased to approximately zero. The strategy showed a detection limit at 35 fM levels, which was about 2 orders of magnitude lower than that of the conventional hybridization without Zn²⁺-based amplification. The Zn²⁺ assistant DNA recycling offers a versatile platform for DNA detection in a cost-effective manner, and has a promising application in clinical diagnosis.     

Monitoring molecular beacon DNA probe hybridization at the single-molecule level

We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.     

Bulk and single-molecule characterization of an improved molecular beacon utilizing H-dimer excitonic behavior

Pairs of fluorophores in close proximity often show self-quenching of fluorescence by the well-known H-dimer mechanism. We use a pair of fluorophores in the new dicyanomethylenedihydrofuran (DCDHF) dye family in the design and characterization of a new fluorescent probe for nucleic acid detection, which we refer to as a self-quenched intramolecular dimer (SQuID) molecular beacon (MB). We obtain a quenching efficiency of 97.2%, higher than the only other reported value for a MB employing fluorophore self-quenching by H-dimer formation. Furthermore, the excellent single-molecule (SM) emitter characteristics of the DCDHF dyes allow observation of individual SQuID MB-target complexes immobilized on a surface, where the doubled SM emission intensity of our target-bound beacon ensures a higher signal-to-background ratio than conventional fluorophore-quencher MBs. Additional advantages of the SQuID MB are single-pot labeling, visible colorimetric detection of the target, and intrinsic single-molecule two-step photobleaching behavior, which offers a specific means of discriminating between functional MBs and spurious fluorescence.     

Impedance‐Based DNA Biosensor Employing Molecular Beacon DNA as Probe and Thionine as Charge Neutralizer

In this paper, we present an impedance‐based DNA biosensor using thionine intercalation to amplify DNA hybridization signal. Beacon single‐stranded DNA (ssDNA) probe and mercaptoacetic acid were self‐assembled onto a Au electrode by forming Au? S bonds. These beacon ssDNAs were hybridized with the complementary sequences around the loop structure. Then thionine was intercalated into the double‐stranded DNA (dsDNA) immobilized on the Au electrode surface. Due to the neutralization of the negative charges of dsDNA by the intercalated thionine, the electronic transfer resistance (R_et) of the DNA modified Au electrode was significantly diminished. Herein, the decreased value of R_et resulted from the thionine intercalating into dsDNA was employed as the hybridization signal. SDS was used to reduce the unspecific adsorption between ssDNA and thionine. Several experimental conditions, including the surface coverage of ssDNA probe on Au electrode, the hybridization temperature and time were all optimized. Moreover, the hybridization reactions of the unstructured linear ssDNA probe and the structured beacon ssDNA probe with their complementary sequences were compared in this work. The sensitivity of the presented DNA biosensor highlighted that the intercalation of thionine into dsDNA was an efficient approach to amplify the hybridization signal using impedance detection technique. Additionally, in this DNA biosensing protocol, beacon ssDNA has a good ability to distinguish target DNA sequences. This results in a higher specificity than using traditional unstructured DNA probe.     

Voltammetric Determination of Folic Acid Using Adsorption of Methylene Blue onto Electrodeposited of Reduced Graphene Oxide Film Modified Glassy Carbon Electrode

This work presents a sensitive voltammetric method for determination of folic acid by adsorbing methylene blue onto electrodeposited reduced graphene oxide film modified glassy carbon electrode (MB/ERGO/GCE) in 100 mM KCl‐10 mM sodium phosphate buffer solution (pH 7.40). The surface morphology of the MB/ERGO/GCE modified electrode was characterized using scanning electron microscopy, displays that both MB and ERGO distributed homogeneously on the surface of GCE. The MB/ERGO/GCE modified electrode shows more favorable electron transfer kinetics for potassium ferricyanide and potassium ferrocyanide probe molecules, which are important electroactive compounds, compared with bare GCE, MB/GCE, and ERGO/GCE. The electrochemical behaviors of folic acid at MB/ERGO/GCE were investigated by cyclic voltammetry, suggesting that the modified electrode exhibited excellent electrocatalytic activity towards folic acid compared with other electrodes. Under physiological condition, the MB/ERGO/GCE modified electrode showed a linear voltammetric response from 4.0 μM to 167 μM for folic acid, and with the detection limit of 0.5 μM (S/N=3). The stability, reproducibility and anti‐interference ability of the modified electrode were examined. The developed method has been successfully applied to determination of FA in tablets with a satisfactory recovery from 96 % to 100 %. The work demonstrated that the electroactive MB adsorbing onto graphene modified electrode showed an enhanced electron transfer property and a high resolution capacity to FA.     

A disulfide bound-molecular beacon as a fluorescent probe for the detection of reduced glutathione and its application in cells

A disulfide-bound molecular beacon (MB) is reported to respond sensitively to changing levels of glutathione in vitro. Importantly, this successful application of a MB has exciting potential for monitoring cellular thiol.     

Ultrasensitive optical DNA biosensor based on surface immobilization of molecular beacon by a bridge structure.

A novel biotinylated molecular beacon (MB) probe was developed to prepare a DNA biosensor using a bridge structure. MB was biotinylated at the quencher side of the stem and linked on a biotinylated glass cover slip through streptavidin, which acted as a bridge between MB and glass matrix. An efficient fluorescence microscope system was constructed to detect the fluorescence change caused by the conformation change of MB in the presence of complementary DNA target. The proposed biosensor was used to directly detect, in real-time, the target DNA molecules. The bridge immobilization method caused the proposed DNA biosensor to have a faster and more stable response. Under the optimal conditions, the newly developed DNA biosensor showed a linear response toward ssDNA in the range of 5-100 nM with a detection limit of 2 nM. It was interesting to note that the described biosensor was reproducible after being regenerated by urea.     

Coupling molecular beacons to barcoded metal nanowires for multiplexed, sealed chamber DNA bioassays

We have combined molecular beacon (MB) probes with barcoded metal nanowires to enable no-wash, sealed chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based MB assays are discussed. Loop regions of 24 bases and 5 base pair stem regions in the beacon probes gave optimal performance. Our results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. Dengue virus-specific probes with predicted solution-phase DeltaG of folding in 500 mM buffered NaCl of approximately -4 kcal/mol performed better than those with DeltaG > -2 or < -6 kcal/mol. Buffered 300-500 mM NaCl was selected after comparison of several buffers previously reported for similar types of assays, and 200-500 mM NaCl was found to be the optimal ionic strength for the hybridization temperatures (25 and 50 degrees C) and probe designs used here. Target binding to the surface as a function of solution concentration fit a Sips isotherm with Kd = 1.7 +/- 0.3 nM. The detection limit was approximately 100 pM, limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for human immunodeficiency, hepatitis C, and severe acute respiratory viruses were assayed simultaneously in a no-wash, sealed chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, we demonstrated that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over 3 months.     

Hybrid molecular probe for nucleic acid analysis in biological samples

The ability to detect changes in gene expression, especially in real-time and with sensitivity sufficient enough to monitor small variations in a single-cell, will have considerable value in biomedical research and applications. Out of the many available molecular probes for intracellular monitoring of nucleic acids, molecular beacon (MB) is the most frequently used probe with the advantages of high sensitivity and selectivity. However, any processes in which the MB stem-loop structure is broken will result in a restoration of the fluorescence in MB. This brings in a few possibilities for false positive signal such as nuclease degradation, protein binding, thermodynamic fluctuation, solution composition variations (such as pH, salt concentration) and sticky-end pairing. These unwanted processes do exist inside living cells, making nucleic acid monitoring inside living cells difficult. We have designed and synthesized a hybrid molecular probe (HMP) for intracellular nucleic acid monitoring to overcome these problems. HMP has two DNA probes, one labeled with a donor and the other an acceptor. The two DNA probes are linked by a poly(ethylene glycol) (PEG) linker, with each DNA being complementary to adjacent areas of a target sequence. Target binding event brings the donor and acceptor in proximity, resulting in quenching of the donor fluorescence and enhancement of the acceptor emission. The newly designed HMP has high sensitivity, selectivity, and fast hybridization kinetics. The probe is easy to design and synthesize. HMP does not generate any false positive signal upon digestion by nuclease, binding by proteins, forming complexes by sticky-end pairing, or by other molecular interaction processes. HMP is capable of selectively detecting nucleic acid targets from cellular samples.     

DNA and PNA sensing on mercury and carbon electrodes by using methylene blue as an electrochemical label

Described here are the electrochemical parameters for MB on binding to DNA at hanging mercury drop electrode (HMDE), glassy carbon electrode (GCE), and carbon paste electrode (CPE) in the solution and at the electrode surface. MB, which interacts with the immobilized calf thymus DNA, was detected by using single-stranded DNA-modified HMDE or CPE (ssDNA-modified HMDE or CPE), bare HMDE or CPE, and double-stranded DNA-modified HMDE or CPE (dsDNA-modified HMDE or CPE) in combination with adsorptive transfer stripping voltammetry (AdTSV), differential pulse voltammetry (DPV), and alternating current voltammetry (ACV) techniques. The structural conformation of DNA and hybridization between synthetic peptide nucleic acid (PNA) and DNA oligonucleotides were determined by the changes in the voltammetric peak of MB. The PNA and DNA probes were also challenged with excessive and equal amount of noncomplementary DNA and a mixture that contained one-base mismatched and target DNA. The partition coefficient was also obtained from the signal of MB with probe, hybrid, and ssDNA-modified GCEs. The effect of probe, target, and ssDNA concentration upon the MB signal was investigated. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.     

Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids

Hybridization probes are often inefficient in the analysis of single‐stranded DNA or RNA that are folded in stable secondary structures. A molecular beacon (MB) probe is a short DNA hairpin with a fluorophore and a quencher attached to opposite sides of the oligonucleotide. The probe is widely used in real‐time analysis of specific DNA and RNA sequences. This study demonstrates how a conventional MB probe can be used for the analysis of nucleic acids that form very stable (T_m>80 °C) hairpin structures. Here we demonstrate that the MB probe is not efficient in direct analysis of secondary structure‐folded analytes, whereas a MB‐based tricomponent probe is suitable for these purposes. The tricomponent probe takes advantage of two oligonucleotide adaptor strands f and m. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to a MB probe. In the presence of a specific analyte, the two adaptor strands hybridize to the analyte and the MB probe, thus forming a quadripartite complex. DNA strand f binds to the analyte with high affinity and unwinds its secondary structure. Strand m forms a stable complex only with the fully complementary analyte. The MB probe fluorescently reports the formation of the quadripartite associate. It was demonstrated that the DNA analytes folded in hairpin structures with stems containing 5, 6, 7, 8, 9, 11, or 13 base pairs can be detected in real time with the limit of detection (LOD) lying in the nanomolar range. The stability of the stem region in the DNA analyte did not affect the LOD. Analytes containing single base substitutions in the stem or in the loop positions were discriminated from the fully complementary DNA at room temperature. The tricomponent probe promises to simplify nucleic acid analysis at ambient temperatures in such applications as in vivo RNA monitoring, detection of pathogens, and single nucleotide polymorphism (SNP) genotyping by DNA microarrays.     

Label-Free Fluorescence Molecular Beacon Probes Based on G-Triplex DNA and Thioflavin T for Protein Detection

Protein detection plays an important role in biological and biomedical sciences. The immunoassay based on fluorescence labeling has good specificity but a high labeling cost. Herein, on the basis of G-triplex molecular beacon (G3MB) and thioflavin T (ThT), we developed a simple and label-free biosensor for protein detection. The biotin and streptavidin were used as model enzymes. In the presence of target streptavidin (SA), the streptavidin hybridized with G3MB-b (biotin-linked-G-triplex molecular beacon) perfectly and formed larger steric hindrance, which hindered the hydrolysis of probes by exonuclease III (Exo III). In the absence of target streptavidin, the exonuclease III successively cleaved the stem of G3MB-b and released the G-rich sequences which self-assembled into a G-triplex and subsequently activated the fluorescence signal of thioflavin T. Compared with the traditional G-quadruplex molecular beacon (G4MB), the G3MB only needed a lower dosage of exonuclease III and a shorter reaction time to reach the optimal detection performance, because the concise sequence of G-triplex was good for the molecular beacon design. Moreover, fluorescence experiment results exhibited that the G3MB-b had good sensitivity and specificity for streptavidin detection. The developed label-free biosensor provides a valuable and general platform for protein detection.     

分子信标探针荧光法测定慢性粒细胞性白血病相关基因

根据慢性粒细胞性白血病(CML)相关基因b3a2序列设计了一种带有荧光基团和淬灭基团的凸环结构探针(分子信标,MB),研究其与互补目标DNA杂交前后的荧光变化行为,建立了b3a2基因检测的新方法.在最适条件下,得到杂交后溶液荧光强度与本底荧光强度的比值(S/B)和目标DNA 的浓度呈线性关系,r=0.9973,线性范围4.0×10-9～3.2×10-8 mol/L.该方法为实际样品的检测奠定了基础.     

Inhibited aptazyme-based catalytic molecular beacon for amplified detection of adenosine

Combining the inhibited aptazyme and molecular beacon(MB),we developed a versatile sensing strategy for amplified detection of adenosine.In this strategy,the adenosine aptamer links to the 8-17 DNAzyme to form an aptazyme.A short sequence,denoted as inhibitor,is designed to form a duplex spanning the aptamer–DNAzyme junction,which blocks the catalytic function of the DNAzyme.Only in the presence of target adenosine,the aptamer binds to adenosine,thus the inhibitor dissociates from the aptamer portion of the aptazyme and can no longer form the stable duplex required to inhibit the catalytic activity of the aptazyme.The released DNAzyme domain will hybridize to the MB and catalyze the cleavage in the presence of Zn2+,making the fluorophore separate from the quencher and resulting in fluorescence signal.The results showed that the detection method has a dynamic range from 10 nmol/L to 1 nmol/L,with a detection limit of 10 nmol/L.     

A tumor mRNA-mediated bi-photosensitizer molecular beacon as an efficient imaging and photosensitizing agent

A bi-photosensitizer molecular beacon (bi-PS MB) is assembled by coupling two PS molecules, respectively, onto the opposite ends of a single MB. The MB can be triggered by a tumor marker-survivin mRNA. Fluorescence and cytotoxic (1)O(2) generation occur effectively in breast cancer cells, but not in normal cells. Compared with a single-PS MB, a bi-PS MB exhibits much-enhanced properties in the signal-to-background ratio and (1)O(2) generation simultaneously.     

A controllable solid-state Ru(bpy)(3)(2+) electrochemiluminescence film based on conformation change of ferrocene-labeled DNA molecular beacon

A controllable solid-state electrochemiluminescence (ECL) film based on efficient and stable quenching of ECL of ruthenium(II) tris-(bipyridine) (Ru(bpy)32+) by oxidizing ferrocene (Fc) at the electrode is developed. The ECL intensity is correlated to the distance which is controlled by the conformation of the ferrocene-labeled DNA molecular beacon (Fc-MB) between the Fc and Ru(bpy)32+ immobilized on the electrode. The conformation adjustment is conducted via complementary DNA hybridizing with the bases in the loop of the Fc-MB and changing the temperature of the Fc-MB and the resultant double-stranded DNA (dsDNA). Those events all result in change of the ECL intensity. With such characteristics, the solid-state Ru(bpy)32+-ECL film has the potential to be applied to reagentless DNA ECL biosensors and to calculate thermodynamic parameters of equilibrium constants of MB binding and the stem-loop formation.