Aptasensor based on triplex switch for SERS detection of cytochrome c.

We report a sensitive approach for SERS detection of cytochrome c using target binding-induced conformational changes of signal transduction probe (STP). STP labeled with a SERS-active molecule, carboxy-X-rhodamine (ROX), is immobilized on the substrate where the formation of a rigid triplex switching structure with aptamers does not allow SERS amplification to take place. The target binding event leads to an enhancement in SERS intensity of ROX adsorbed on the gold surface. Meanwhile, we found that an appropriate STP surface density could shield the SERS signal produced by protein adsorption which would foul the sensing surface. In addition, cytochrome c aptamers used were not the original sequence but reorganized in the nonspecific binding site to adapt to our design. This method provides a low detection limit of 2 nM (10 fmol within 5 µL sample solution), and shows good selectivity toward cytochrome c compared to interfering proteins such as hemoglobin and immunoglobulin G. The general strategy of the method can also be extended to aptamer or DNA based sensors.

A DNA biosensor based on graphene paste electrode modified with Prussian blue and chitosan.

A chemically modified graphene paste electrode was prepared by incorporating appropriate amounts of graphene in a paste mixture, followed by electrodepositing Prussian blue (PB) and coating chitosan on the electrode surface. The electrode was able to bind ssDNA, and gave a better voltammetric response for complement DNA than did ordinary carbon paste electrodes. The response of the electrode was characterized with respect to the paste composition, immobilization time of probe DNA on the chitosan and PB modified graphene paste electrode, and the effect of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). The electrochemical behavior of PB assembled on the graphene paste electrode was investigated. The combination of graphene and PB can enhance the current response of the graphene paste electrode. As a consequence of DNA hybridization, a significant change in the current due to daunomycin intercalated with double-stranded DNA (ds-DNA) on the surface of the graphene paste electrode was observed.

A sandwiched biological fluorescent probe for the diagnosis of human ovarian tumor based on TiO2 nanoparticles.

In this paper, we report a novel biological fluorescent probe for the diagnosis of human ovarian tumor based on sandwiched TiO(2) nanoparticles. The fluorescence nanoparticles consist of a fluorescent molecule, tetramethyl rhodamine isothiocyanate (TRITC), sandwiched between titanium dioxide (TiO(2)) nanoparticles and nano-gold via reacting with each other. The antibodies HER2, labeled on the surface of the biofluorescence nanoparticles, have granted nanoparticles the privilege of aiming at peculiar tumor antigen. The specificity of antibody-nanoparticles interacting with cells was characterized by Laser Scanning Confocal Microscope. The results showed that these sandwiched nanoparticles were innocuous and stable, and the method offered potential advantages of sensitivity and simplicity due to high combing efficiency between nanoparticles and cells and provided an alternative method for the diagnosis of human ovarian tumor (HOT).

Based on Gold Nanoparticles-L-Tyr-Amino Functionalized Mesoporous Materials-Polyphenol Oxidase Modified Biosensor for the Detection of Resorcinol.

Nowadays, resorcinol (RC) has been widely applied in the chemical and pharmaceutical industries. However, the electrochemical detection technique of RC still features some significant drawbacks, for instance, a low sensitivity. Hence, in the present work, a glass carbon electrode was developed for the electrochemical detection of RC with good specificity and stability, through modifying the glass carbon electrode (GCE) by polyphenol oxidase (PPO), an NH2-SBA-15 mesoporous material (NH2-SBA-15), L-tyrosine (L-Tyr) and gold nano-particles (AuNPs). After being successively modified by AuNPs, L-Tyr, NH2-SBA-15 and PPO, the constructed PPO/NH2-SBA-15/L-Tyr/AuNPs/GCE was used to discriminate RC from ions and other common micromolecules, which showed a fairly good specificity and stability. The proposed electrochemical detection method features a linear range of from 0.5 to 21.0 µM with a LOD down to 0.15 µM, revealing a better sensitivity than the existing methods. It is worth mentioning that the proposed PPO/NH2-SBA-15/L-Tyr/AuNPs/GCE has been successfully used as an electrochemical probe for the RC assay in domestic sewage.

Fluorescent biological label for recognizing human ovarian tumor cells based on fluorescent nanoparticles.

In this paper, we report a method for recognizing human ovarian tumor (HOT) cells using fluorescent biological label based on core-shell nanoparticles. The luminescent nanoparticles were synthesized with a water-in-oil (W/O) microemulsion technique. The fluorescent silica core-shell nanoparticles modified with anti-HER2 antibody using bifunctional cross-linker glutaraldehyde targeted the corresponding tumor antigen in the cell surface of the SKOV3 ovarian cancer cells. The specific immunoreactivity of antibody-nanoparticles with cells was characterized by laser scanning microscopy (LSM) and scanning electron microscope (SEM). The results showed that the method offered potential advantages of sensitivity and simplicity due to high binding efficiency between nanoparticles and cells and provided an alternative method for the detection of HOT.

Polyphenol oxidase/gold nanoparticles/mesoporous carbon-modified electrode as an electrochemical sensing platform for rutin in dark teas.

In the communication, by virtue of the excellent conductivity and great surface area of mesoporous carbon (FDU-15), the enhanced conductivity of Au NPs, and the good electrochemical response of polyphenol oxidase (PPO) to rutin, a PPO/AuNPs/FDU-15-modified electrode was used as a candidate for the determination of rutin in dark teas with satisfactory results.

Real-time monitoring of nucleic acid ligation in homogenous solutions using molecular beacons.

Nucleic acids ligation is a vital process in the repair, replication and recombination of nucleic acids. Traditionally, it is assayed by denatured gel electrophoresis and autoradiography, which are not sensitive, and are complex and discontinuous. Here we report a new approach for ligation monitoring using molecular beacon DNA probes. The molecular beacon, designed in such a way that its sequence is complementary with the product of the ligation process, is used to monitor the nucleic acid ligation in a homogeneous solution and in real-time. Our method is fast and simple. We are able to study nucleic acids ligation kinetics conveniently and to determine the activity of DNA ligase accurately. We have studied different factors that influence DNA ligation catalyzed by T4 DNA ligase. The major advantages of our method are its ultrasensitivity, excellent specificity, convenience and real-time monitoring in homogeneous solution. This method will be widely useful for studying nucleic acids ligation process and other nucleic acid interactions.

Transport and separation of small organic molecules through nanotubules.

An electroless plating method was applied to deposit Au onto the surfaces and the walls of pores of polycarbonate membranes to prepare gold nanotubules. The nanotubules were modified with cysteine (Cys) or with carbamidine thiocyante (Gua). The effects of modifiers and of the fine structure of organic molecules on the transport properties of those molecules through the gold nanotubules were investigated. Studies show that the hydrophilicity of modifiers and the planar structure of permeating molecules clearly affect the transport of small organic molecules in gold nanotubules. Tryptophan (Try) and vitamin B(2) (VB(2)) was cleanly separated at pH 6.8.

Influence of anions on the formation and properties of chitosan-DNA nanoparticles.

Chitosan-DNA nanoparticles were prepared by using different anions (such as chloride, sulfate, citrate, and tripolyphosphate) as mediation agents. The research suggested that the formation and morphological characteristics of chitosan-DNA nanoparticles largely depended on concentration, molecular size, charge number, and chemical structure of anions, as well as chitosan/DNA ratio. The observation by atom force microscopy showed that chitosan-DNA nanoparticles mediated by four anions (in their appropriate range of concentration) had a spherical shape, narrow size distribution, and good monodispersivity. Especially, nanoparticles mediated by sulfate and TPP had a size distribution of 40-50 nm. Additionally, the nanoparticles presented high encapsulation efficiency and good protection of DNA from DNasel digestion. The zeta-potential of nanoparticles could be adjusted moderately by adding different anions and controlling their concentrations, and DNA encapsulation efficiency was not influenced, which would reduce nonspecific interactions with the cell membrane and nanoparticle toxicity. Smaller size and lower zeta-potential will be beneficial for improving gene therapy. In addition, the anion mediation method has potential for the preparation of cationic polymer nanoparticles as drug or gene vectors.

Photostable luminescent nanoparticles as biological label for cell recognition of system lupus erythematosus patients.

In this article, we report a method for cell recognition of system lupus erythematosus (SLE) patients that uses photostable luminescent nanoparticles as biological labels. The luminescent silica nanoparticles are prepared with a water-in-oil microemulsion (W/O) technique. The silica network is produced by the controlled hydrolysis of tetraethylorthosilicate (TEOS) in water nanodroplets with the initiation of ammonia (NH3.H2O). A luminescent compound, tris(2,2'-bipyridyl)dichlororuthenium(II)hexahydrate [Ru(II)(bpy)3]2+, is doped inside as a luminescent signaling element, and the most appropriate dye concentration for the preparation of the nanoparticles with a size of 28 +/- 4 nm has been determined. The luminescent silica nanoparticles are covalently immobilized with goat anti-human immunoglobulin G (IgG), which can recognize SmIgG+ B lymphocytes. We have used antibody-labeled nanoparticles to recognize target SmIgG+ B lymphocytes isolated from the circulating blood of SLE patients. It has been observed that a bioassay based on fluorescent nanoparticles can identify target cells selectively and efficiently. And fluorescent nanoparticle labels also exhibit high photostability. The experiment results have shown that this cell recognition method was an effective one as further proof of the diagnosis of SLE.

A sensitive optode membrane for berberine using conjugated polymer as sensing material.

A new optode membrane for the sensitive determination of berberine based on fluorescence quenching of a conjugated polymer, poly(2,5-dimethoxy-phenyldiacetylene) (PDPA), is proposed. Incorporated in a membrane composed of plasticized poly(vinyl chloride) (PVC), the conjugated polymer exhibits better stability than those small sensing molecules regarding its excellent optical properties and lipophilic characteristics. Moreover, upon the introduction of a negatively charged lipophilic additive (tetraphenylborate salt) into a PVC membrane, the optode displayed enhanced sensitivity. In addition, satisfactory analytical sensing characteristics for determining beberine were obtained in terms of the selectivity, reversibility and reproducibility with a detecting range of between 7.5 x 10(-7) mol l(-1) and 7.5 x 10(-4) mol l(-1). The optode membrane has been applied to determine berberine in commercial tablets. The results showed a good agreement with those obtained by the pharmacopoeial method.

Interaction of bis(ethylene)tin(bis(salicylidene)ethylenediamine) with DNA.

The fluorescence spectral characteristics and interaction of bis(ethylene)tin(bis(salicylidene)ethylenediamine) [Et2Sn(salen)] with DNA are described. The polarity of the solvent has a strong effect on the fluorescence characteristics of Et2Sn(salen). Et2Sn(salen) bound to DNA showed a marked decrease in the fluorescence intensity with a bathochromic shift of the excitation and emission peaks. A hypochromism in the UV absorption spectra was also observed. KI quenching and competitive binding to DNA between Et2Sn(salen) and ethidium bromide (EB) were studied in connection with other experimental observations to show that the interactive model between Et2Sn(salen) and DNA is an intercalative one. The pH and salt effect on the fluorescence properties was also investigated. The intrinsic binding constant was estimated to be 1.071 x 10(5) mol L(-1) in base pairs and the binding site number is 1.98, respectively. A linear relationship between F/F0 and the concentration of calf thymus DNA covers 5.1 x 10(-6) - 2.41 x 10(-4) mol L(-1), which can be utilized for determining traces of calf thymus DNA with a detection limit of 1.1 x 10(-7) mol L(-1) in base pairs.

A three-way junction aptasensor for lysozyme detection.

A well-designed three-way junction (TWJ) aptasensor for lysozyme detection was developed based on target-binding-induced conformational change of aptamer-complementary DNA (cDNA) as probe. A ferrocene (Fc)-tagged cDNA is partially hybridized with an anti-lysozyme aptamer to form a folded structure where there is a coaxial stacking of two helices and the third one at an acute angle. In addition, the fabrication of the sensor was achieved via the single-step method, which offered a good condition for sensing. In the absence of lysozyme, electron transfer (eT), through the coaxial two helices called "conductive path", is allowed between Fc-labeled moiety and the electrode. The binding of lysozyme to the aptamer blocks eT, leading to diminished redox signal. This aptasensor with an instinct signal attenuation factor shows a high sensitivity to lysozyme, and the response data is fitted by nonlinear least-squares to Hill equation. Detection limit is 0.2nM with a dynamic range extending to 100nM. Compared with existing electrochemical impedance spectroscopy (EIS)-based approaches, TWJ-DNA aptasensor was demonstrated to be more specific for detection and simpler for regeneration procedure.

Separation and determination of four ganoderic acids from dried fermentation mycelia powder of Ganoderma lucidum by capillary zone electrophoresis.

Ganoderic acids (GAs) were bioactive secondary metabolites produced by a traditional mushroom Ganoderma lucidum. We describe a simple and efficient method for the separation and quantitative determination of four GAs, namely Ganoderic acid T (GA-T), Ganoderic acid Mk (GA-Mk), Ganoderic acid Me (GA-Me) and Ganoderic acid S (GA-S) from dried triterpene-enriched extracts of G. lucidum mycelia powder by capillary zone electrophoresis (CZE). Under the optimum conditions, the four GAs reached the baseline separation in 9 min with Glycyrrhetinic acid (GTA) as internal standard. The four GAs and internal standard (GTA) were detected at a wavelength 245 nm. All calibration curves showed good linearity (r(2)>0.9958) within test ranges. Limit of detection (LOD) and limit of quantification (LOQ) were less than 0.6 and 1.8 microg/mL, respectively. The relative standard deviation (R.S.D.) values of precision and recoveries were less than 5% and recoveries ranged from 91.4% to 103.6%. This was the first report on simultaneous determination of the four GAs and the results provided a firm basis for the trace analysis of GAs in dried fermentation mycelia powder of G. lucidum with high accuracy.

Separation and determination synchronously by multichannel mode-filtered light capillary electrochromatography.

Mode-filtered light capillary electrophoresis (CE) was developed for capillary electrochromatography (CEC) by applying annular columns of different size (250, 320, and 530 micro m) and a bovine serum albumin (BSA)-modified chiral stationary phase. BSA was encapsulated on the pretreated surface of an optic fiber and the inner wall of a capillary using sol-gel technique with a resultant larger active stationary phase area. The coating was examined by atomic force microscopy (AFM) and Fourier transform infrared (FT-IR). The separation of DL-tryptophan (10 mM) was investigated under different voltage/current conditions.

Mixed C18 and C1 modification on an optical fiber for chromatographic sensing.

An optical fiber-chromatographic sensor, aiming at simultaneous and selective response to multiple components following a chromatographic separation, is described. We report an improved approach for immobilization of octadecyl (C(18)) and methyl (C(1)) moieties as stationary phase on an optical fiber suitable as a sensing phase for organic solutes. By this approach, the stability and lifetime of the sensing layer as well as the detectability and retention behavior of the chromatographic sensor could be improved. Infrared spectroscopy was employed to confirm the presence of C(18) and C(1) moieties on the modified surface of the optical fiber. The chromatographic sensor was applied, with good sensitivity and chemical selectivity, to the simultaneous separation and detection of bromobenzene and toluene, using water as the mobile phase.

Monitoring molecular beacon/DNA interactions using atomic force microscopy.

The molecular beacon (MB) is a new fluorescence probe containing a single-stranded oligonucleotide with a probe sequence embedded in complementary sequences that form a hairpin stem. Due to the inherent fluorescent signal transduction mechanism, an MB functions as a sensitive probe with a high signal-to-background ratio for real-time monitoring and provides a variety of exciting opportunities in DNA, RNA, and protein studies. To better understand the properties of MBs, the specific interactions between MB and target DNA (complementary and one-base mismatch) have been directly investigated by atomic force microscopy. The interaction force between a linear DNA probe and the target DNA was also detected and compared to that between MB and target DNA. The results demonstrate the high specificity of the MB/target DNA compared to the linear DNA/target DNA interaction.

Bioconjugated nanoparticles for DNA protection from cleavage.

We have developed a novel method to protect DNA from cleavage using bioconjugated nanoparticles. Positively charged amino-modified silica nanoparticles have been directly prepared using water-in-oil microemulsion. Plasmid DNA can be easily enriched onto the positively charged nanoparticle surface, and the DNA strands are well protected from enzymatic cleavage. When incubated with nuclease enzyme for enzymatic cleavage, free plasmid DNA strands are completely cleaved, while those on the nanoparticle surfaces are intact. Our results clearly demonstrate unique properties of nanomaterials when combined with biomolecules. Our simple bionanotechnology will be highly useful in DNA separation, manipulation, and detection, and possibly in genetic engineering and gene therapy, as plasmid DNA can be protected in cellular environments without any change in its property.