In situ synthesis of DNA micro-arrays using typography technique

A novel typography technique was developed to in situ synthesize oligonucleotide arrays on glass slide,which has the celerity,high spatial resolution,lower cost,reliable operation,and high synthetic efficiency.The principle and process of the typography technique for fabricating gene-chips have been described in detail.A suit of poly(terafluoroethylene)devices for synthesizing oligonucleotide arrays were designed and prepared,and the fiber tubes with a number of nano-or micron-channels were em- ployed.The oligonucleotide arrays of 16 and 160 features with four different probes were synthesized using the typography technique.The four specific oligonucleotide probes including the matched and the mismatched by the fluorescent target sequence gave obviously different hybridization fluorescent signals.It was indicated that the gene-chip fabricated by the typography method could be used to rapidly screen single-nucleotide polymorphisms(SNP)and to detect mutations.     

High efficient probes with Schiff base functional receptors for hypochlorite sensing under physiological conditions

A series of novel and convenient fluorescent probes with Schiff base functionality were presented for direct detection of OCI via the irreversible OCl-promoted oxidation and hydrolyzation reaction in formation of the ring-opened product, fluorescein. Prominent high sensitivity, selectivity and antiinterference OCl-induced fluorescence and color change over a wide range of tested metal ions performance were observed for each probe under physiological conditions, thus making the probes well suitable for sensing of OCl in living cells.     

Critical assessment of a new in situ spectroelectrochemical cell designed for the study of interfacial reactions between a porous graphite anode and alkyl carbonate solution

This article critically evaluates the characteristics of a new in situ spectroelectrochemical cell with an optimized path of the IR beam, designed in our laboratory for study of the solid electrolyte interphase (SEI) layer formed between a porous graphite anode and alkyl carbonate solution for lithium-ion batteries. The cell was designed in view of the optical principles underlying the way the in situ cell works, to give depth of penetration of the evanescent IR beam through the attenuated total internal reflectance crystal into the electrolyte at such a small value, ranging from 0.277 to 2.77 μm, that it was possible to minimize the "masking effect" of the ethylene carbonate/diethyl carbonate solvent. Moreover, the "local compositional change" which may arise significantly from the "thin layer electrolyte configuration" cell also could be fairly avoided, since only the electrolyte in the vicinity of the electrode composed of graphite particles is reduced to form the SEI layer to a thickness of at most 0.1 μm during the application of potentials. Thus, it was possible to measure the in situ FT-IR spectra in the cell, which represents the real chemical composition and structure of the SEI layer. Taking the application of the designed in situ cell as an example, this article reports the effect of salt type and electrolyte temperature on the chemical composition and structure of the SEI layer between graphite particles and alkyl carbonate solution with the help of various measured in situ FT-IR spectra. Electronic Publication     

POROUS MEMBRANE TEMPLATED SYNTHESIS OF POLYMER PILLARED LAYER

The anodic porous alumina membranes with a definite pore diameter and aspect ratio were used as templates tosynthesize polymer pillared layer structures. The pillared polymer was produced in the template membrane pores, and thelayer on the template surfaces. Rigid cured epoxy resin, polystyrene and soft hydrogel were chosen to confirm themethodology. The pillars were in the form of either tubes or fibers, which were controlled by the alumina membrane pore surface wettability. The structural features were confirmed by scanning electron microscopy results.     

Target-triggered hairpin-free chain-branching growth of DNA dendrimers for contrast-enhanced imaging in living cells by avoiding signal dispersion

The development of amplification strategies is one of the central challenges for detection of lowabundance targets. One-to-many(1:M) amplification strategies in which one target lights many signal probes, has improved the detection sensitivity in bulk solution, but with discounted contrast in cell imaging, because the lighted probes are dissociative and dispersible. In this work, a one-to-large(1:L) signaling mechanism, in which the lighted probes were orderly connected to each other, was concep...     

电渗流与缓冲液浓度之间数学关系的线性表达

It was believed that electroosmotic mobility μeo is inversely proportional to the square root of the ionic strength L But the linear relationship for regression analysis was expressed differently in different papers. The paper studied the linear expression of the mathematical relationship between μeo and c （background buffer concentration） by mathematical transform and real experimental data.μeo values of fused silica capillary were determined in four buffer systems. Their experimental conditions were controlled carefully for decreasing temperature difference AT and pH difference ApH in 50 μm ID capillary, in which no double layer overlap existed. The linear relationship between the reciprocal of electroosmotic mobility and the square root of concentration （or ionic strength） was derived by mathematical method. The regression analysis of experimental data was shown to well correspond to the relationship. The constants in regression equation could be well defined and the calculated results were acceptable.     

Bioactivated in vivo assembly(BIVA) peptide-tetraphenylethylene(TPE) probe with controllable assembled nanostructure for cell imaging

The emergence of fluorescent light-up molecular probe, which can specifically turn on their fluorescent in the presence of stimulation factors, has open up a new opportunity to advance biosensing and bioimaging. In this work, we designed and synthesized a peptide-AIE conjugate probe for cell imaging with controlled in situ assembled nanostructures. The modular designed probe is consisted of a selfassembled peptide-tetraphenylethene(TPE) motif, a fibroblast activation protein alpha(FAP-α)responsive motif, a hydrophilic motif and a targeting motif. The probe exhibits typically turn-on fluorescence property specifically triggered by FAP-α, which is a significant overexpressed membrane protein on pancreatic tumor cells. Interestingly, the peptide modified the TPE dramatically impacts the assembled nanostructure, which can be modulated by peptide sequences. As a result, the peptide FF(PhePhe) modification of TPE as the self-assembled motif provides a suitable balance of the probe with lightup property and nanofiber assembled structure in situ. Finally, our probe could effectively detect the FAP-α on tumor cells with high specificity. Meantime, the nanofibers in situ assembled on the surface of CAFs enhanced the probe accumulation and prolonged the retention for cell imaging. We envision that this study may inspire new insights into the design of nanostructure controlled AIE light-up bio-probe.     

Imaging Array SPR Biosensor Immunoassays for Sulfamethoxazole and Sulfamethazine

A homemade array surface plasmon resonance (SPR)-based imaging biosensor was used to develop sensitive and fast immunoassays to determine sulfamethoxazole (SMOZ) and sulfamethazine (SMT) in buffer. Two conjugations of sulfonamide-bovine serum albumin (BSA) were separately immobilized on two different rows of the array chip with one row as reference. The immobilization was carried out in the instrument to monitor the quantity of the conjugations immobilized. The antibody mixed with the sulfonamide in the buffer was injected over the surface of the chip to get a relative response which was inversely proportional to the concentration of the sulfonamide in the PBS buffer. Two calibration curves were constructed and the limit of detection for sufamethoxazole in buffer was 3.5 ng/mL and for sulfamethazine 0.6 ng/mL. The stability and specificity of the antibody were also studied. The monoclonal antibody did not bind with BSA.     

Preparation of electrolyte membranes for micro tubular solid oxide fuel cells

Yttria-stabilized zirconia (YSZ) micro tubular electrolyte membranes for solid oxide fuel cells (SOFCs) were prepared via the combined wet phase inversion and sintering technique. The as-derived YSZ mi- cro tubes consist of a thin dense skin layer and a thick porous layer that can serve as the electrode of fuel cells. The dense and the porous electrolyte layers have the thickness of 3-5 μm and 70-90 μm, respectively, while the inner surface porosity of the porous layer is higher than 28.1%. The two layers are perfectly integrated together to preclude the crack or flake of electrolyte film from the electrode. The presented method possesses distinct advantages such as technological simplicity, low cost and high reliability, and thus provides a new route for the preparation of micro tubular SOFCs.     

Fabrication and characterization of chitosan-silk fibroin/hydroxyapatite composites via in situ precipitation for bone tissue engineering

Homogeneous chitosan-silk fibroin/hydroxyapatite(CS-SF/HA) composites were prepared by in situ precipitation method driven by a multiple-order template. The morphology of the composites was investigated by scanning electron microscope(SEM) and transmission electron microscope(TEM). The compositional analysis was carried out by X-ray diffraction analysis(XRD) and Fourier transformed infrared spectroscopy(FTIR). The mechanical properties and biocompatibility of the composites were also determined. The results indicated that the inorganic particles of uniform size(50 nm) were well-dispersed among the CS-SF matrices. The compressive modulus of the CS-SF/HA composites was enhanced with the increasing amount of SF. The in vitro results suggested that the MC3T3-E1 osteoblast-like cells on CS-SF/HA composite disks displayed strong bonding and spreading, and the cell proliferation cultured on each composite disk increased throughout the culture period for up to 7 days. Especially, the samples with higher content of SF had much better biological properties. The evidences proved that the CS-SF/HA composites possessed excellent biocompatibility. By using the freeze-drying technique, hierarchical porous scaffolds with pores ranging from 50 μm to 200 μm were obtained. This work presented the advantages of in situ precipitation method to prepare the organic/inorganic composites, and a multiple-order template was introduced in the system to improve the properties of the composites by combining the merits of each organic template.     

In situ synthesis of DNA micro-arrays using typography technique

A novel typography technique was developed to in situ synthesize oligonucleotide arrays on glass slide, which has the celerity, high spatial resolution, lower cost, reliable operation, and high synthetic efficiency. The principle and process of the typography technique for fabricating gene-chips have been described in detail. A suit of poly(terafluoroethylene) devices for synthesizing oligonucleotide arrays were designed and prepared, and the fiber tubes with a number of nano-or micron-channels were employed. The oligonucleotide arrays of 16 and 160 features with four different probes were synthesized using the typography technique. The four specific oligonucleotide probes including the matched and the mismatched by the fluorescent target sequence gave obviously different hybridization fluorescent signals. It was indicated that the gene-chip fabricated by the typography method could be used to rapidly screen single-nucleotide polymorphisms (SNP) and to detect mutations. Supported by the National Natural Science Foundation of China (Grant Nos. 60571032, 60571001, 90606027 and 60121101), the National Hi-Tech Research and Development Program of China (Grant No. 2006AA03Z357), the Natural Science Foundation of Hunan Province (Grant Nos. 04jj40023 and 06JJ4012) and the Natural Science Foundation of Guangdong Province (Grant No. 04008782)     

Fabrication of DNA polymer brush arrays by destructive micropatterning and rolling-circle amplification

A method for fabricating DNA polymer brush arrays using photolithography and plasma etching followed by solid-phase enzymatic DNA amplification is reported. After attaching oligonucleotide primers to the surface of a glass coverslip, a thin layer of photoresist is spin-coated on the glass and patterned via photolithography to generate an array of posts in the resist. An oxygen-based plasma is then used to destroy the exposed oligonucleotide primers. The glass coverslip with the primer array is assembled into a microfluidic chip and DNA polymer brushes are synthesized on the oligonucleotide array by rolling-circle DNA amplification. We have demonstrated that the linear polymers can be rapidly synthesized in situ with a high degree of control over their density and length.     

Combinatorial decoding: an approach for universal DNA array fabrication

A fiber optic microsphere-based oligonucleotide array is described that employs the sequence of the oligonucleotide probe attached to each microsphere as positional identifiers. Each microsphere serves as an immobilized array feature, functionalized with a unique single-stranded oligonucleotide sequence and randomly distributed into an array of microwells. To determine the sequences attached to individual microspheres, a series of fluorescently labeled combinatorial-pooled oligonucleotide target solutions was designed. Each combinatorial decoding solution is intended to identify the nucleotide at a particular position on every microsphere in the array. The combinatorial target solutions were synthesized by linking the four possible nucleotides at each position to four different fluorescent reporter dyes. As such, when the solutions were hybridized to the array, one of four possible fluorescent responses was generated for each position on a microsphere probe sequence. Adjusting the stringency of hybridization enabled single-base mismatch discrimination, and the signal with the highest intensity corresponded to the perfect nucleotide match. By consecutively exposing the array to a series of combinatorial decoding pool solutions, it was possible to simultaneously determine the sequence of every randomly positioned oligonucleotide-functionalized microsphere in the array. Once mapped, the microsphere array can be used for any typical genomic microarray experiment.     

Combinational synthesis of oligonucleotides and assembly fabrication of oligonucleotide array

In this paper, a simple, reliable and flexible method, which integrated in situ synthesis with the spotting technique, was reported to fabricate oligonucleotide array. Different oligonucleotide sequences are synthesized on their relative code glass slides through combinational chemistry, thus the slides are broken into smaller pieces, in which the same code pieces have the same probe sequences. An oligonucleotide array is fabricated by arbitrarily assembling these different code pieces onto another solid substrate. In principle experimentation, four different sequences of P16 gene were synthesized and a 5 × 5 array including these four sequences and the control black was fabricated. The analysis results indicated that the hybridization fluorescence intensity of the same sequences locating different sets on the array gave the approximate values, and the fluorescence intensity ratio of matched sequence to one middle location base mismatched, two base mismatched, three middle base mismatched is (1.000 ± 0.080):(0.4991 ± 0.0671):(0.2360 ± 0.0044):(0.0493 ± 0.0033). Their relative accuracies were from 6.64 to 10.2%. This result might be used to rapidly screen single-nucleotide polymorphisms (SNPs).     

Detection and identification of IHN and ISA viruses by isothermal DNA amplification in microcapillary tubes

Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia virus (ISAV) were detected and identified using a combination of surface-associated molecular padlock DNA probes (MPPs) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV or ISAV were recognized by MPPs. Circularized MPPs were then captured on the inner surfaces of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA produced DNA concatamers, which were labeled with fluorescent SYBR Green II nucleic acid stain, and measured by microfluorimetry. Molecular padlock probes, combined with this method of surface-associated isothermal RCA, exhibited high selectivity without the need for thermal cycling. This method is applicable to the design of low-power field sensors capable of multiplex detection of viral, bacterial, and protozoan pathogens within localized regions of microcapillary tubes.      

Parylene insulated probes for scanning electrochemical-atomic force microscopy

Scanning electrochemical-atomic force microscopy (SECM-AFM) is a powerful technique that can be used to obtain in situ information related to electrochemical phenomena at interfaces. Fabrication of probes to perform SECM-AFM experiments remains a challenge. Herein, we describe a method for formation of microelectrodes at the tip of commercial conductive AFM probes and demonstrate application of these probes to SECM-AFM. Probes were first insulated with a thin parylene layer, followed by subsequent exposure of active electrodes at the probe tips by mechanical abrasion of the insulating layer. Characterization of probes was performed by electron microscopy and cyclic voltammetry. In situ measurement of localized electrochemical activity with parylene-coated probes was demonstrated through measurement of the diffusion of Ru(NH)(6)(3+) across a porous membrane.     

A bipolar electrochemical approach to constructive lithography: metal/monolayer patterns via consecutive site-defined oxidation and reduction

Experimental evidence is presented, demonstrating the feasibility of a surface-patterning strategy that allows stepwise electrochemical generation and subsequent in situ metallization of patterns of carboxylic acid functions on the outer surfaces of highly ordered OTS monolayers assembled on silicon or on a flexible polymeric substrate. The patterning process can be implemented serially with scanning probes, which is shown to allow nanoscale patterning, or in a parallel stamping configuration here demonstrated on micrometric length scales with granular metal film stamps sandwiched between two monolayer-coated substrates. The metal film, consisting of silver deposited by evaporation through a patterned contact mask on the surface of one of the organic monolayers, functions as both a cathode in the printing of the monolayer patterns and an anodic source of metal in their subsequent metallization. An ultrathin water layer adsorbed on the metal grains by capillary condensation from a humid atmosphere plays the double role of electrolyte and a source of oxidizing species in the pattern printing process. It is shown that control over both the direction of pattern printing and metal transfer to one of the two monolayer surfaces can be accomplished by simple switching of the polarity of the applied voltage bias. Thus, the patterned metal film functions as a consumable "floating" stamp capable of two-way (forward-backward) electrochemical transfer of both information and matter between the contacting monolayer surfaces involved in the process. This rather unusual electrochemical behavior, resembling the electrochemical switching in nanoionic devices based on the transport of ions in solid ionic-electronic conductors, is derived from the nanoscale thickness of the water layer acting as an electrolyte and the bipolar (cathodic-anodic) nature of the water-coated metal grains in the metal film. The floating stamp concept introduced in this report paves the way to a series of unprecedented capabilities in surface patterning, which are particularly relevant to nanofabrication by chemical means and the engineering of a new class of molecular nanoionic systems.     

Single-feature inking and stamping: a versatile approach to molecular patterning

A technique for micrometer-scale patterning of multiple functional biological molecules on surfaces is demonstrated. The technique is referred to as single-feature inking and stamping (SFINKS). It combines elements of dip-pen nanolithography and microcontact printing. "Inked" atomic force microscopy probes are used to ink individual features of an elastomer stamp. From a single stamp, we printed three different probe ssDNA with <10 mum resolution and showed that they specifically hybridize the complementary DNA labeled with different fluorophores. As a further demonstration of SFINKS' versatility, we patterned a silane onto a silicon wafer consisting of four subpatterns separated by >100 mum and composed of 2 mum lines. We discuss why patterns such as these are impractical with available techniques. Furthermore, we comment on the prospects for multiple stamping after a single inking.     

DNA biosensor based on the electrochemiluminescence of Ru(bpy)3(2+) with DNA-binding intercalators

This paper reports a novel detection method for DNA hybridization based on the electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) with a DNA-binding intercalator as a reductant of Ru(bpy)(3)(3+). Some ECL-inducible intercalators have been screened in this study using electrochemical methods combined with a chemiluminescent technique. The double-stranded DNA intercalated by doxorubicin, daunorubicin, or 4',6-diamidino-2-phenylindole (DAPI) shows a good ECL with Ru(bpy)(3)(2+) at +1.19 V (versus Ag/AgCl), while the non-intercalated single-stranded DNA does not. In order to stabilize the self-assembled DNA molecules during ECL reaction, we constructed the ECL DNA biosensor separating the ECL working electrode with an immobilized DNA probe. A gold electrode array on a plastic plate was assembled with a thru-hole array where oligonucleotide probes were immobilized in the side wall of thru-hole array. The fabricated ECL DNA biosensor was used to detect several pathogens using ECL technique. A good specificity of single point mutations for hepatitis disease was obtained by using the DAPI-intercalated Ru(bpy)(3)(2+) ECL.