Goldview标记的DNA荧光毛细生物传感器的研究

对Goldview(GV)作为荧光标记物的DNA荧光毛细生物传感器进行了研究。以荧光毛细分析法(fluorescence capillary analysis, FCA)为基础, 在毛细管内壁通过Poly-l-lysine将20-mer-ssDNA探针固定, 制成DNA荧光毛细生物传感器(DNA fluorescence capillary biosensor, DNA-FCB),DNA-FCB与互补靶DNA杂交,通过GV染色后,检测杂交产物的荧光强度,实现对靶DNA的定性和定量分析。样品用量12 μL, 靶DNA的浓度在0.4～4 μmol·L-1(2.4～24 mg·L-1)范围内和荧光强度有良好的线性关系(y=65.911x+3.994 4,r=0.998 9);RSD<3.5%,检出限0.39 μmol·L-1(2.2 mg·L-1),能达到定量检测靶DNA的目的。用DNA-FCB测定靶DNA操作简便, 试样、试剂用量少,测定成本极低, 能大大减少环境污染。     

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.     

表面等离子体-微腔激元对顶入射有机薄膜太阳能电池光吸收效率的增强

为了提高顶入射有机薄膜太阳能电池(TOSCs)的光吸收效率,我们将周期性矩形光栅结构引入到TOSCs中,分析了具有光栅结构的空气/Ag_1/有源层/Ag_2/空气(IMIMI)结构理想模型中复合表面等离子激元(SPPs)与微腔模式的耦合机制。通过调节光栅周期和有源层厚度,实现了复合SPPs、微腔模式以及有机材料本征吸收3个区域的重合。由于复合SPPs与微腔模式的反交叉耦合作用形成了表面等离子体-微腔激元,其局域场增强作用有效地提高了有源层的光吸收效率,提高了近19%。     

Electrochemical DNA biosensor based on avidin-biotin conjugation for influenza virus (type A) detection

An electrochemical DNA biosensor (E-DNA biosensor) was fabricated by avidin-biotin conjugation of a biotinylated probe DNA, 5′-biotin-ATG AGT CTT CTA ACC GAG GTC GAA-3′, and an avidin-modified glassy carbon electrode (GCE) to detect the influenza virus (type A). An avidin-modified GCE was prepared by the reaction of avidin and a carboxylic acid-modified GCE, which was synthesized by the electrochemical reduction of 4-carboxyphenyl diazonium salt. The current value of the E-DNA biosensor was evaluated after hybridization of the probe DNA and target DNA using cyclic voltammetry (CV). The current value decreased after the hybridization of the probe DNA and target DNA. The DNA that was used follows: complementary target DNA, 5′-TTC GAC CTC GGT TAG AAG ACT CAT-3′ and two-base mismatched DNA, 5′-TTC GAC AGC GGT TAT AAG ACT CAT-3′.     

Label-free biosensor by protein grating coupler on planar optical waveguides

We report a diffraction-based, label-free biosensor on the surface of a planar optical waveguide. By selectively immobilizing proteins on an optical waveguide, the proteins can form the ridge/valley of a latent image optical grating. Binding events with patterned surface proteins dynamically increase grating height and cause an increase in light coupling out of the waveguide. We demonstrate the efficacy of this strategy for protein detection and present the design and fabrication of a device.     

Mass spectrometric characterization of DNA microarrays as a function of primary ion species

Recent studies have shown TOF-SIMS to be an appropriate method for the detailed examination of the immobilization process of PNA and its ability to hybridize to unlabeled complementary DNA fragments. Unlabeled single-stranded DNA was hybridized to Si wafer biosensor chips containing both complementary and non-complementary immobilized PNA sequences. The hybridization of complementary DNA could readily be identified by detecting phosphate-containing molecules from the DNA backbone. An unambiguous discrimination was achieved between complementary and non-complementary sequences.In order to optimize detection parameters, different primary ions were applied, including monoatomic ions (Bi⁺) as well as cluster ions (Bi₂⁺, Bi₃⁺, Bi₄⁺, Bi₃⁺⁺, Bi₅⁺⁺), and secondary ion yield behavior and formation efficiencies were studied. It was found that cluster primary ions resulted in a significantly increased yield of DNA-correlated fragments, enabling higher signal intensities and better secondary ion efficiencies.TOF-SIMS is undoubtedly a highly useful technique for identifying hybridized DNA on PNA biosensor chips. It is suitable for studying the complexity of the immobilization and hybridization processes and may provide a rapid method for DNA diagnostics. With the absence of the labeling procedure and the simultaneous increase of the phosphate signal as a result of increasing DNA sequence length, this technique comes to be especially useful for the direct analysis of genomic DNA.     

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.     

Transmission characteristics of a microscale dielectric slab waveguide device with a deep groove and an embedded metallodielectric grating at low terahertz frequency

We discuss the transmission characteristics of a microscale dielectric waveguide device with a deep groove and an embedded metallodielectric grating illuminated by a continuous wave of TM and TE modes at low terahertz frequency. To study its performance we solve numerically the corresponding Maxwell equations by means of finite difference time domain method with uniaxial perfectly match layer as its boundary condition. By varying the angle of incident, grating filling factor and refractive index of analyte in the deep groove, it is found that the device exhibits a significant transmission enhancement for the TM mode due to the existence of surface plasmon interaction. We also demonstrate its potential application as a biosensor device.     

等离子体光栅靶的表面粗糙度对高次谐波产生的影响

极紫外光和软X射线由于其波长和脉冲持续时间极短,可用于超快物理过程和物质微观结构的探测.最近几年,研究人员发现激光和等离子体相互作用可以产生持续时间极短(阿秒)且相干性较好的高次谐波辐射,其波长可接近甚至达到水窗波段.然而,实验研究指出,理论上应出现的一些谐波在实验中并没有出现.本文针对超短超强激光与非理想条件下的等离子体光栅靶相互作用产生高次谐波的物理过程进行了理论分析和粒子模拟.研究结果表明,等离子体光栅的周期性结构对于高次谐波的频谱和辐射角分布存在显著调制效果.光栅靶表面粗糙度直接影响光栅的光学调制效果,改变高次谐波的频谱分布和辐射角分布.理想光栅条件下,满足光栅匹配条件的特定阶数谐波明显获得增强,且辐射张角集中在平行靶面的方向.靶表面粗糙度的出现,导致光栅匹配条件失效,高次谐波能量向各阶分散且辐射张角逐渐偏离靶表面方向.研究结果较好地解释了实验中观测到的谐波频谱分布,为进一步的研究提供了一定参考.     

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.     

Surface layer reflective index changes of Au nanoparticle functionalized porous silicon microcavity for DNA detection

A technique is demonstrated to detect DNA hybridization based on surface layer of Au/porous silicon microcavity (Au/PSM) substrate for very small amount of biomolecules. Simulations show that the increase of effective refractive index for the first layer of PSM will cause a blue shift for its reflectance spectrum, and the blue shift becomes less with the increase of refractive index for one more layers. In experiments, such a blue shift of reflectance spectrum of PSM comes from the increase of refractive index by DNA hybridization on the surface. The detection limit of Au/PSM biosensor is 15.15 nM for 19-base pair DNA, which is comparable to that of reported biosensors based on porous silicon (PS). Therefore such an Au/PSM could be very useful to develop simple, rapid and sensitive optical biosensors when the amount of target is very small.     

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.     

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.     

ZrO<Subscript>2</Subscript>/DNA-derivated polyion hybrid complex membrane for the determination of hydrogen peroxide in milk

An efficient biosensing substrate based on ZrO₂/DNA-derivated polyion complex (PIC) membrane has been developed for the determination of hydrogen peroxide (H₂O₂) in this study. To fabricate such a PIC membrane, ZrO₂ nanoparticles were initially electrodeposited on the bare gold electrode (ZrO₂/Au), and deoxyribonucleic acid (DNA)-doped hemoglobin mixture was then assembled onto the ZrO₂/Au surface. The double-strand DNA provided a biocompatible microenvironment for the immobilization of biomolecules, greatly amplified the surface coverage of biomolecules on the electrode surface, and improved the sensitivity of the biosensor. The fabricated procedure of the proposed biosensor was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy. The performance and factors influencing the performance of the biosensor were also evaluated. Under optimal conditions, the developed biosensor exhibited a well-defined electrochemical behavior toward the reduction of H₂O₂ ranging from 1.1 μM to 2.3 mM with a detection limit of 0.5 μM (S/N = 3). The biosensor was applied to the determination of H₂O₂ in milk with satisfactory results. It is important to note that the PIC membrane provided an alternative substrate for the immobilization of other proteins.     

DNA Biosensor with High Sensitivity Amplified by Gold Nanoparticles

A biosensor based on quartz crystal microbalance (QCM) using 50 nm gold nanoparticles as the amplification probe for DNA detection was reported in this paper. It had been found that a DNA detection sensitivity of 10^–14 M could be obtained, which was higher than what has been ever reported using the same method. In the curve of weight increase (m) against the target DNA in solution, a plateau appeared as the concentration of analyte DNA increasing, implying the existence of monolayer of gold nanoparticles binding on the top of target DNA. The high sensitivity was explained not only by the weight of the larger particles, but also the larger area occupied by the larger particles which need less target DNA for their binding.     

Field Effect Transistor Using Carbon Nanotubes and DNA as Electrical Gate

We present an electronic sensor in the molecular scale, which is very sensitive for detection and sensing of DNA characteristics and DNA activities in particular activities between DNA duplex and any protein. Here, the device shows that DNA is electronically inserted to be on the same time as an electrical device transducer and as a biological target in a carbon nanotube-DNA-carbon nanotube electronic sensor. We have performed a DNA binding through an amide group by the electron transfer through amide group. The presented device has shown an efficient and rapid procedure to bind the electrical vulnerability of DNA with the detection of enzymatic effectiveness leading to high efficient biosensor.     

生物分子膜门电极AlGaN/GaN高电子迁移率晶体管(HEMT)生物传感器研究

设计并制作了结构尺寸为毫米量级的AlGaN/GaN高电子迁移率晶体管(HEMT)生物传感器,采用数值分析的方法分析了器件传感区域长度与宽度比值及待测物调控二维电子气(2DEG)距离与感测信号之间的关系,给出了结构尺寸为毫米量级的AlGaN/GaN HEMT生物传感器的设计依据,以不同浓度的前列腺特异性抗原(PSA)为待测物,对制作的AlGaN/GaN HEMT生物传感器进行了初步测量,测试结果表明,在50 mV的电压下,毫米量级的AlGaN/GaN HEMT生物传感器的对PSA的探测极限低于0.1 pg/ml.实验表明毫米量级的AlGaN/GaN HEMT生物传感器具有灵敏度高,易于集成等优点,具备良好的应用前景.     

Simultaneous measurement of refractive index and temperature using dual-period grapefruit microstructured fiber grating

By cascading the long period fiber grating (LPFG) and fiber Bragg grating (FBG) in grapefruit microstructured fiber, a novel dual-period fiber grating sensor is proposed. The refractive index and temperature are measured simultaneously by using the different sensitivity of FBG and LPFG. The relationship between dual-period fiber grating transmission spectrum and refractive index, resonant wavelengths and temperature are analyzed theoretically, respectively. The simulation results show that the accuracy of the sensor in measuring refractive index and temperature is estimated to be 2319.6 nm/RIU in a range from 1.33 to 1.36 and 0.017 nm/°C from 0 °C to 100 °C, respectively. Thus, the sensor has high refractive index sensitivity, and can provide the theoretical foundation for the optical fiber biosensor.     

High-sensitivity Bloch surface wave sensor with Fano resonance in grating-coupled multilayer structures

A new type of device consisting of a lithium niobate film coupled with a distributed Bragg reflector (DBR) was theoretically proposed to explore and release Bloch surface waves for applications in sensing and detection. The film and grating made of lithium niobate (LiNbO₃) were placed on both sides of the DBR and a concentrated electromagnetic field was formed at the film layer. By adjusting the spatial incidence angle of the incident light, two detection and analysis modes were obtained, including surface diffraction detection and guided Bloch detection. Surface diffraction detection was used to detect the gas molecule concentrations, while guided Bloch detection was applied for the concentration detection of biomolecule-modulated biological solutions. According to the drift of the Fano curve, the average sensor sensitivities from the analysis of the two modes were 1560 °/RIU and 1161 °/RIU, and the maximum detection sensitivity reached 2320 °/RIU and 2200 °/RIU, respectively. This study revealed the potential application of LiNbO₃ as a tunable material when combined with DBR to construct a new type of biosensor, which offered broad application prospects in Bloch surface wave biosensors.     

PDMS梯度光栅结构制备技术研究

由于传统方法制作的梯度光栅,工艺条件苛刻,制作过程复杂,难以控制,制作成本高,周期较长,提出了一种成本低、工艺简单、可大量制备梯度光栅的工艺方法,采用基于刚性薄膜/柔性衬底的自组装工艺和氧等离子体(Plasma)的方法制备了微米尺度的梯度光栅,利用Plasma时间的可控性和聚二甲基硅氧烷(PDMS)优异的弹性制得所需要尺寸的光栅。首先在聚乙烯对苯二酸脂(PET)薄膜上旋涂一层PDMS薄膜,待PDMS薄膜固化后将双层薄膜弯曲并用Plasma处理,在其表面生成一层刚性氧化层,借助柔性的PET对刚性层施加均匀应力,当应力超过临界值时,在PDMS基底上自组装形成光栅褶皱结构。由于弯曲时预应力的变化,所以在PDMS薄膜上会形成周期和高度呈阶梯状的的光栅褶皱,也就是梯度光栅。采用可见光作为梯度光栅的性能测试光源,选用一级衍射光作为检测对象,从图谱中可以看出以PDMS为基底制备的光栅具有很好的衍射效应,并可实现很好的分光效果。实验表明：梯度光栅具有明显的衍射现象,并且衍射角变化显著,可广泛用于应力测量。这种方法制备的柔性梯度光栅也可以作为微型应变装置来检测应力的变化,未来有望用于微型光谱仪、扫描仪、光通讯等领域中。