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.     

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.     

Detection of PCR products amplified from DNA of epizootic pathogens using magnetic nanoparticles and SERS

Here we present a novel approach using surface‐enhanced Raman scattering (SERS) spectroscopy for the sequence‐specific detection of DNA utilizing magnetic nanoparticles (MNPs) for the enrichment of the target molecules. To achieve fast and efficient binding of longer DNA strands, e.g. PCR products, the hybridization procedure is performed in solution. To further purify and enrich the DNA strands of interest, MNPs are used for their separation. Following the binding of the target DNA, a dye‐modified, short synthetic ssDNA is hybridized, which serves as label for the SERS detection. The SERS spectra are used to identify the bound molecules. The applicability of this approach was first tested with short synthetic oligonucleotides to evaluate its specificity. Afterward, the system was applied to detect PCR products amplified from DNA of specific agents of epizootic diseases. Sequences of the bacterium Mycoplasma mycoides subspecies mycoides small colony type (MmmSC), causing contagious bovine pleuropneumonia (CBPP) were used as PCR targets. To demonstrate the multiplexing capability of SERS, the simultaneous detection of three different PCR products labeled with three dyes was performed. Copyright © 2010 John Wiley & Sons, Ltd.     

Magnetic nanoparticles for high-sensitivity detection on nucleic acids via superconducting-quantum-interference-device-based immunomagnetic reduction assay

In this work, we investigate the feasibility of detecting quantitatively DNA molecules utilizing the technology named after the immunomagnetic reduction (IMR) assay. Magnetic nanoparticles dispersed in a phosphate buffer saline solution were bio-functionalized with probing single-strand DNA. A superconducting quantum interference device (SQUID) ac magnetosusceptometer was employed to detect IMR signals related to the concentration of the target DNA. The results reveal that use of IMR assay had merits such as a high convenience level, e.g. wash-free processes and high sensitivity, down to pM, for DNA detection.     

Direct immobilization and hybridization of DNA on group III nitride semiconductors

A key concern for group III-nitride high electron mobility transistor (HEMT) biosensors is the anchoring of specific capture molecules onto the gate surface. To this end, a direct immobilization strategy was developed to attach single-stranded DNA (ssDNA) to AlGaN surfaces using simple printing techniques without the need for cross-linking agents or complex surface pre-functionalization procedures. Immobilized DNA molecules were stably attached to the AlGaN surfaces and were able to withstand a range of pH and ionic strength conditions. The biological activity of surface-immobilized probe DNA was also retained, as demonstrated by sequence-specific hybridization experiments. Probe hybridization with target ssDNA could be detected by PicoGreen fluorescent dye labeling with a minimum detection limit of 2 nM. These experiments demonstrate a simple and effective immobilization approach for attaching nucleic acids to AlGaN surfaces which can further be used for the development of HEMT-based DNA biosensors.     

Fluorescence Turn-On Analysis of Trace Protein Based on Carbon Nanodots and Hybridization Chain Reaction

In this work, an ultrasensitive method for trace protein detection based on fluorescent carbon nanodots and hybridization chain reaction (HCR) is designed. Generally, the synthesized bright carbon nanodots are conjugated with two hairpin-structured DNA probes, respectively, which act as subsequent HCR fuel strands. Since single-stranded parts of DNA probes could be easily absorbed on graphene oxide (GO) nanosheets, fluorescence emission of carbon nanodots is effectively quenched via fluorescence resonance energy transfer. However, in the presence of target protein, the aptamer sequence in another hairpin-structured DNA probe specially interacts with target and the hairpin is opened. A single-stranded region is thus exposed, which initiates HCR by coupling with the DNA fuel strands on carbon nanodots. The formed HCR product displays a rigid, long double-stranded structure, which facilitates the release of carbon nanodots from GO surface. As a result, fluorescence of carbon nanodots is recovered and initial concentration of target protein can be estimated. This protein detection method shows a favorable linear response with a low limit of detection (2.3 fg mL⁻¹). Furthermore, it is highly selective and capable of detecting target in biological fluids like serum samples, which demonstrates the promising applications of this method.     

Widely Frequency-Tunable Terahertz Wave Generation and Spectroscopic Application

The THz-wave generator based on the resonance of phonon-polaritons in GaP enabled THz spectral measurements of organic molecules like DNA/RNA-related molecules in wide frequency range (0.6–5.8 THz). High spectral purity of the generated THz-wave enables even the detection of defect structures of organic compounds, and will give a new tool for molecular sciences. We demonstrated THz imaging of a liver cancer. The THz-wave generator can be made small-size, light-weight system.     

Enhanced Raman scattering based on fabry‐perot like resonance in a metal‐cladding waveguide

A liquid‐core metal‐cladding waveguide structure of millimeter scale is designed to enhance Raman signal via the excitation of Fabry–Perot‐like resonance. Theoretically, an oscillating field distribution covering the whole guiding layer is generated by the multireflection at the two metal interfaces. The large detection area covers the whole sample chamber because of the oscillating nature of excited high order modes with concentrated intensity. By adding metal nanoparticles, the Fabry–Perot‐like resonance can be combined with local surface plasmons resonance to further enhance the light‐matter interaction with the target molecules, which is also confirmed by the experimental results. Copyright © 2015 John Wiley & Sons, Ltd.     

Single-Pair Fluorescence Resonance Energy Transfer (spFRET) for the High Sensitivity Analysis of Low-Abundance Proteins Using Aptamers as Molecular Recognition Elements

We have developed a strategy for the detection of single protein molecules, which uses single-pair fluorescence resonance energy transfer (spFRET) as the readout modality and provides exquisite analytical sensitivity and reduced assay turn-around-time by eliminating various sample pre-processing steps. The single-protein detection assay uses two independent aptamer recognition events to form an assembly conducive to intramolecular hybridization of oligonucleotide complements that are tethered to the aptamers. This hybridization brings a donor-acceptor pair within the Förster distance to create a fluorescence signature indicative of the presence of the protein-aptamer(s) association complex. As an example of spFRET, we demonstrate the technique for the analysis of serum thrombin. The assay requires co-association of two distinct epitope-binding aptamers, each of which is labeled with a donor or acceptor fluorescent dye (Cy3 or Cy5, respectively) to produce a FRET response. The FRET response between Cy3 and Cy5 was monitored by single-molecule photon-burst detection, which provides high analytical sensitivity when the number of single-molecule events is plotted versus the target concentration. We are able to identify thrombin with high efficiency based on photon burst events transduced in the Cy5 detection channel. We also demonstrate that the technique can discriminate thrombin molecules from its analogue prothrombin. The analytical sensitivity was >200-fold better than an ensemble measurement.     

Detection of <Emphasis Type="Italic">Bifidobacterium</Emphasis> Species-specific 16S rDNA Based on QD FRET Bioprobe

Fluorescence resonance energy transfer (FRET) that consists of quantum dot as donors and organic fluorophore dyes as acceptors has been a very important method to detect biomolecules such as nucleic acids. In this work, we established a new FRET detection system of Bifidobacterium species-specific 16S rDNA using QD—ROX FRET bioprobe, in which 525 nm QD-DNA conjugation consisted of the carboxyl-modified QD and the amino-modified DNA in the presence of EDC. Both ROX-DNA and the conjugation above could hybridize with the target DNA after forming the QD—ROX bioprobe. When the hybridization made the distance between the QD and ROX to meet FRET effect needed, 525 nm QD fluorescence intensity decreased and ROX fluorescence intensity increased. In the control, there was no notable change of fluorescence intensities without target DNA. It is very clear that the change of the QD and ROX fluorescence intensities provide the good base and guaranty for this rapid and simple detection system.     

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。     

SERS activity and stability of the most frequently used silver colloids

Single‐molecule detection by surface‐enhanced resonance Raman scattering (SERRS) spectroscopy has been demonstrated for a variety of molecules. The detection of single molecules that do not have a resonance contribution, SERS, has been shown in the case of adenine. However, when colloidal particles isolated on planar substrates are used as the enhancing medium, the presence of anomalous signals significantly complicates the analysis of the spectra. Selection of a silver colloid that minimizes these spurious signals should improve the ultra‐sensitive detection of non‐resonant single molecules by SERS. A range of silver colloids, prepared by different methods, were investigated with respect to their activity and stability. Minimal anomalous signals were obtained from hydroxylamine‐reduced silver colloids, which suggests that this colloid will be better for ultra‐sensitive SE(R)RS experiments compared to the more common citrate‐ and borohydride‐reduced silver colloids. Copyright © 2011 John Wiley & Sons, Ltd.     

Influence of the Aggregation of Homo-N-Mer Cyanine Dyes on the Nucleic Acids Detection Sensitivity

Novel benzothiazolopyridinium homo-n-mer cyanine dyes are proposed for nucleic acid fluorescent detection. Dependence of the sensitivity of detection in solution from the dye molecules/DNA base pairs ratio was studied. It was shown that the presence of the dye excess could significantly decrease the detection limit. We believe this could be explained by the formation of the dye aggregates on DNA surface.     

Effects of molecular orientation of fluorescently-labeled duplex DNA on emission anisotropy

The molecular orientation of Cy5-labeled target DNA hybridized on a probe DNA layer was studied on a planar waveguide in the Kretchmann configuration at various target DNA bulk concentration. Optical waveguide fluorescence spectroscopy (OWFS) was employed to evaluate the fluorescence emission anisotropy by analyzing two polarized modes, i.e., TM and TE modes propagating in a waveguide. In this way, the orientational order parameter S could be determined. It was found that the orientation of DNA duplex molecules is disordered at lower concentrations, while relatively ordered at higher concentrations.     

基于InP@ZnS QDs/Dured纳米荧光探针的DNA检测

利用巯基丙酸包覆的In P@Zn S量子点(QDs)与Dured构建了一种检测DNA的荧光探针。在该探针中,以环境友好型带负电的In P@Zn S量子点为荧光团,与带正电的Dured通过静电结合,构建了In P@Zn S QDs/Dured纳米荧光探针。通过荧光共振能量转移(FRET)机理,量子点荧光被猝灭;当DNA存在时,Dured与DNA的特异性结合使Dured从In P@Zn S QDs表面脱附,FRET过程被打断,In P@Zn S QDs荧光恢复,以荧光"关-开"方式检测DNA。该探针检测DNA的线性范围为2.0~275.0 ng·L-1,检测限为1.0 ng·L-1,并可用于模拟生物生理条件下的DNA检测。     

Detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Carrying the Gene for Toxic Shock Syndrome Toxin 1 by Quantum-Dot-Probe Complexes

In this study, a high-sensitive and high-specific method to detect the toxic shock syndrome toxin-1 (TSST-1)-producing Staphylococcus aureus was developed based on quantum dot (QD) and oligonucleotide probe complexes. S. aureus carrying tst gene which is responsible for the production of TSST-1 were detected based on fluorescence resonance energy transfer (FRET) occurring between CdSe/ZnS QD donors and black hole quencher (BHQ) acceptors. QD-DNA probe was prepared by conjugating the carboxyl-modified QD and the amino-modified DNA with the EDC. Photoluminescence (PL) quenching was achieved through FRET after the addition of BHQ-DNA which was attached to tst gene probe by match sequence hybridization. The PL recovery was detected in the presence of target DNA by BHQ-DNA detached from QD-DNA probe because of the different affinities. In contrast, mismatch oligonucleotides and DNAs of other bacteria did not contribute to fluorescence intensity recovery, which exhibits the higher selectivity of the biosensor. The experimental results showed clearly that the intensity of recovered QD PL is linear to the concentration of target DNA within the range of 0.2–1.2 μM and the detection limit was 0.2 μM.     

Observation of molecules produced from a Bose-Einstein condensate

Molecules are created from a Bose-Einstein condensate of atomic 87Rb using a Feshbach resonance. A Stern-Gerlach field is applied, in order to spatially separate the molecules from the remaining atoms. For detection, the molecules are converted back into atoms, again using the Feshbach resonance. The measured position of the molecules yields their magnetic moment. This quantity strongly depends on the magnetic field, thus revealing an avoided crossing of two bound states at a field value slightly below the Feshbach resonance. This avoided crossing is exploited to trap the molecules in one dimension.     

On the origin of the additional bands observed in the photoelectron spectra obtained with Ne or Ar resonance lines

Photoelectron spectra of benzene, fluorobenzene and pyridine ionized by the Ne (16.85 and 16.67 eV) or by the Ar (11.83 eV and 11.62 eV) resonance lines are reinvestigated with a spherical-grid kinetic energy analyzer. Some other hydrogenated and non-hydrogenated molecules are also studied. In the spectra of the hydrogenated molecules a band not observed with the He I (21.22 eV) resonance line is generally visible. It is shown that this additional band can be interpreted as being due to the Lyman α line (10.195 eV). Hydrogen coming from the target molecules is probably responsible for the occurrence of this impurity line.     

Image resolution and the properties of optical-wave target detection and imaging by using the NR-PC flat lens

We used the method of two-dimensional (2D) finite-difference time-domain (FDTD) to study the characteristics of optical-wave target detection and imaging from negative-refraction photonic crystal (NR-PC) flat lens in the paper. The theory of image resolution was introduced. The results show that there exists a transmission peak, with a value far greater than unit, resulting from the influence of the mini-forbidden bands and resonance excitation effect at resonance frequency. And, the refocusing of backscattered optical wave from the target leads to its image with a sub-wavelength lateral resolution and great amplitude as long as the target is placed at the focus of the NR-PC flat lens. And the application of NR-PC flat lens can be extended in optical area, especially in the detection and imaging of small target.