DNA microarray synthesis by using PDMS molecular stamp (II) Oligonucleotide on-chip synthesis using PDMS stamp

Based on the standard phosphoramidites chemistry protocol, two oligonucleotides synthetic routes were studied by contact stamping reactants to a modified glass slide. Route A was a contact coupling reaction, in which a nucleoside monomer was transferred and coupled to reactive groups (OH) on a substrate by spreading the nucleoside activated with tetrazole on a polydimethylsiloxane (PDMS) stamp. Route B was a contact detritylation, in which one nucleoside was fixed on the desired synthesis regions where dimethoxytrityl (DMT) protecting groups on the 5’-hydroxyl of the support-bound nucleoside were removed by stamping trichloroacetic acid (TCA) distributed on features on a PDMS stamp. Experiments showed that the synthetic yield and the reaction speed of route A were higher than those of route B. It was shown that 20 mer oligonucleotide arrays immobilized on the glass slide were successfully synthesized using the PDMS stamps, and the coupling efficiency showed no difference between the PDMS stamping and the conventional synthesis methods.     

DNA microarray synthesis by using PDMS molecular stamp (Ⅱ)——Oligonucleotide on-chip synthesis using PDMS stamp

Based on the standard phosphoramidites chemistry protocol, two oligonucleotides synthetic routes were studied by contact stamping reactants to a modified glass slide. Route A was a contact coupling reaction, in which a nucleoside monomer was transferred and coupled to reactive groups (OH) on a substrate by spreading the nucleoside activated with tetrazole on a polydimethylsiloxane (PDMS) stamp. Route B was a contact detritylation, in which one nucleoside was fixed on the desired synthesis regions where dimethoxytrityl (DMT) protecting groups on the 5'-hydroxyl of the support-bound nucleoside were removed by stamping trichloroacetic acid (TCA) distributed on features on a PDMS stamp. Experiments showed that the synthetic yield and the reaction speed of route A were higher than those of route B. It was shown that 20 mer oligonucleo-tide arrays immobilized on the glass slide were successfully synthesized using the PDMS stamps, and the coupling efficiency showed no difference between the PDMS stamping and the co     

Transferring complementary target DNA from aqueous solutions onto solid surfaces by using affinity microcontact printing

In this paper, we report a method of transferring complementary target DNA from an aqueous solution onto a solid surface by using affinity microcontact printing. In this approach, the probe DNA is first immobilized on the surface of an aminated poly(dimethylsiloxane) (PDMS) stamp. After a complementary target DNA hybridizes with the probe DNA on the stamp surface, the PDMS stamp is printed on an aminated glass slide. By using fluorescent microscopy, we show that only complementary target DNA, but not noncomplementary DNA, can be captured onto the surface of the stamp and then transferred to the aminated glass slide. The transfer of DNA can be attributed to the stronger electrostatic attraction between DNA and amine groups compared to the hydrogen bonds between the hybridized DNA molecules. We also investigate several factors that may influence the transfer of DNA, such as the surface density of amine groups, hybridization conditions, and contamination from unreacted PDMS monomers.     

DNA微阵列原位合成中的氧化剂体系改进

DNA芯片的制备方法主要有离片合成法和在片合成法．离片合成是用点样法将DNA探针固定在基片上;在片合成是在基片表面直接原位合成寡核苷酸探针阵列．目前国外已有多种DNA芯片原位合成方法的报道,但其中仅Fordor等提出的光脱保护DNA固相原位合成技术已商品化,该方法制备成本高,     

Fine synthetic nucleoside chemistry based on nucleoside natural products synthesis

Synthetic nucleoside chemistry based on nucleoside natural products synthesis were described. First, a samarium diiodide (SmI 2)-promoted aldol reaction with the use of alpha-phenylthioketone as an enolate was developed. The characteristics of this reaction are that the enolate can be regioselectively generated and the aldol reaction proceeds under near neutral condition. This reaction is proved to be a powerful reaction for the synthesis of complex nucleoside natural products, and herbicidin B and fully protected tunicaminyluracil, which were undecose nucleoside natural products, were synthesized. Next, the synthetic methodology of the caprazamycins, which are promising antibacterial nucleoside natural products, was also developed by the strategy including beta-selective ribosylation without using a neighboring group participation. Our synthetic route provided a range of key analogues with partial structures to define the pharmacophore. Simplification of the caprazamycins was further pursued to develop diketopiperazine analogs.     

利用软模板和紫外光固化技术制备超疏水表面

研究了一种制备超疏水表面的新方法.该方法以复制了荷叶表面结构的聚二甲基硅氧烷(PDMS)弹性体为软模板.利用可紫外光交联预聚物在模板压印条件下固化成型,得到了具有微乳突结构的仿荷叶表面.制备的仿荷叶表面表现出了超疏水性能.通过对紫外光固化体系中的单体含量、交联剂含量、引发剂含量、以及紫外曝光时间等因素的研究,得到了使仿荷叶表面的疏水性优化的条件.     

Total synthesis and antifungal activity of a carbohydrate ring-expanded pyranosyl nucleoside analogue of nikkomycin B

In a study aimed at investigating an as yet unknown structure-activity relationship of the nikkomycin family of antifungal peptidyl nucleoside antibiotics, the present research reports the synthesis and antifungal evaluation of a carbohydrate ring-expanded pyranosyl nucleoside analogue of nikkomycin B. Employing a convergent synthetic route, independent synthesis of the N-terminal amino acid side chain and a stereoselective de novo construction of the desired pyranosyl nucleoside amino acid fragment was followed by peptidic coupling of the two components, leading to the first synthesis of a carbohydrate ring-enlarged pyranosyl nikkomycin B analogue. In vitro biological evaluation of the above analogue against a variety of human pathogenic fungi demonstrated significant antifungal activity against several fungal strains of clinical significance.     

Total synthesis of an antitumor antibiotic,Fostriecin (CI-920)

The total synthesis of an antitumor antibiotic, fostriecin (CI-920), via a highly convergent route is described. A characteristic feature of the present total synthesis is that the synthesis was achieved via a coupling procedure of three segments A, B, and C. The unsaturated lactone moiety of fostriecin, corresponding to segment A, was constructed from a known Horner-Emmons reagent, and the stereochemistry of the C-5 position was introduced by asymmetric reduction with (R)-BINAl-H. Segment B having a series of stereogenic centers was synthesized from (R)-malic acid and the stereogenic centers at the C-8 and C-9 positions were prepared by a combination of Wittig reaction and Sharpless asymmetric dihydroxylation reaction. The conjugated Z,Z,E-triene moiety of fostriecin, corresponding to segment C, was eventually constructed by Wittig reaction and Stille coupling reaction. The phosphate moiety, which is known to be essentially important for the antitumor activity, was introduced via two routes: (i) direct phosphorylation of the monohydroxyl derivative in which other hydroxyl groups are protected with silyl groups; (ii) cyclic phosphorylation and selective cleavage of the cyclic phosphate derivative. Although the former route is basically the same as those reported by other groups, the latter route is novel and more effective than the former one. The present total synthesis would serve as a versatile synthetic route to not only fostriecin, but also its various analogues including stereoisomers.     

Creating patterned carbon nanotube catalysts through the microcontact printing of block copolymer micellar thin films

We report a route for synthesizing patterned carbon nanotube (CNT) catalysts through the microcontact printing of iron-loaded poly(styrene-block-acrylic acid) (PS-b-PAA) micellar solutions onto silicon wafers coated with thin aluminum oxide (Al(2)O(3)) layers. The amphiphilic block copolymer, PS-b-PAA, forms spherical micelles in toluene that can form quasi-hexagonal arrays of spherical PAA domains within a PS matrix when deposited onto a substrate. In this report, we dip a poly(dimethylsiloxane) (PDMS) molded stamp into an iron-loaded micellar solution to create a thin film on the PDMS features. The PDMS stamp is then put in contact with a substrate, and uniaxial compressive stress is applied to transfer the micellar thin film from the PDMS stamp onto the substrate in a defined pattern. The polymer is then removed by oxygen plasma etching to leave a patterned iron oxide nanocluster array on the substrate. Using these catalysts, we achieve patterned vertical growth of multiwalled CNTs, where the CNTs maintain the fidelity of the patterned catalyst, forming high-aspect-ratio standing structures.     

Poly(dimethylsiloxane) contamination in microcontact printing and its influence on patterning oligonucleotides

It is well-established that, during microcontact printing (muCP) using poly(dimethylsiloxane) (PDMS)-based stamps, some unexpected siloxane fragments can be transferred from the stamp to the surface of the sample. This so-called contamination effect coexists with the delivery of the molecules constituting the ink and by this way influences the printing process. The real impact of this contamination for the muCP technique is still partially unknown. In this work, we investigate the kinetics of this contamination process through the surface characterization of both the sample and the stamp after imprinting. The way both the curing conditions of the PDMS material and the contact time influence the degree of contamination of the surface is investigated on silicon and glass substrates. We propose a cleaning process of the stamp during several hours which eliminates any trace of contamination during printing. We show that hydrophobicity recovery of PDMS surfaces after hydrophilic treatment using oxygen plasma is considerably slowed down when the PDMS material is cleaned using our procedure. Finally, by comparing cleaned and uncleaned PDMS stamps, we show the influence of contamination on the quality of muCP using fluorescent DNA molecules as an ink. Surprisingly, we observe that the amount of DNA molecules transferred during muCP is higher for the uncleaned stamp, highlighting the positive impact of the presence of low molecular weight siloxane fragments on the muCP process. This result is attributed to the better adsorption of oligonucleotides on the stamp surface in presence of these contaminating molecules.     

A short and efficient synthesis of the tRNA nucleosides PreQ0 and archaeosine

Modified nucleosides in tRNAs play an important role in the translational process. They fine tune the codon-anticodon interactions and they influence the folding and stabilisation of the tRNA structure. Herein, we present a novel synthetic route to the highly modified nucleosides PreQ(0) and archaeosine. The synthesis involves coupling of a protected 7-cyano-7-deazaguanosine nucleobase with a TBDMS and isopropylidene protected chloro-ribose unit yielding the PreQ(0) nucleoside after deprotection. This PreQ(0) nucleoside is then used as the starting material for the synthesis of archaeosine providing the first total synthetic access to this hypermodified RNA nucleoside.     

Microscale plasma-initiated patterning (muPIP)

A novel technique to create biomolecular micropatterns of varying complexity on several types of polymer substrates is presented. This method uses a patterned PDMS stamp to preferentially expose or protect areas of an underlying polymer substrate from oxygen plasma. Following plasma treatment, the substrate is immersed in a biomolecular ink, whereby molecules preferentially adsorb to either the plasma-exposed or plasma-protected substrate regions, depending on the particular substrate/ink combination. Using this method, polyethylene (PE), polystyrene (PS), poly(methyl methacrylate) (PMMA), poly(dimethylsiloxane) (PDMS), and poly(hydroxybutyrate/hydroxyvalerate) (PHBV) were micropatterned with different aqueous-based biomolecular inks (i.e., goat anti-rabbit immunoglobulin G, poly-l-lysine, and bovine serum albumin (BSA)). Water contact angle measurements performed on substrates after oxygen plasma exposure showed that the hydrophilicity of substrate areas exposed to plasma was significantly greater than that of areas protected from plasma by the PDMS stamp. In addition, scanning electron microscopy results demonstrated that substrate areas exposed to plasma were physically modified (e.g., roughened) compared to adjacent, protected areas. Areas in contact with a patterned PDMS stamp during plasma exposure were found to be physically unaffected by plasma treatment, and exhibited spatial features/dimensions consistent with the corresponding features of the patterned stamp. Last, protein patterns of BSA on the polymer substrates were stable and distinct after 4 weeks of incubation at 37 degrees C.     

Nucleosides XI. Synthesis and antiviral evaluation of 5'-alkylthio-5'-deoxy quinazolinone nucleoside derivatives as S-adenosyl-L-homocysteine analogs

4-amino-1-(beta-D-ribofuranosyl)quinazolin-2-one (3) was prepared by a direct glycosylation of 4-aminoquinazolin-2-one (7) using the Vorbruggen's silylation method and provided exclusively the beta-anomer. This quinazoline nucleoside and its 2',3'-O-isopropylidene derivative (9) did not undergo the coupling reaction with dialkyl disulfides in the presence of tri-n-butylphosphine unless their 4-amino groups were protected by N,N-dimethylaminomethylidene. This approach provides a viable alternative synthetic route to 5'-alkylthio-5'-deoxy nucleosides.     

Photopatternable silicon elastomers with enhanced mechanical properties for high-fidelity nanoresolution soft lithography

Soft lithography has been widely used in stamping and printing processes for microfabrication as a low cost alternative to photolithography. However, conventional poly(dimethyl)siloxane (PDMS) stamp materials have limitations, especially in the submicrometer range, due to their low physical toughness and requirements for thermocuring. A new version of functional stamp materials with adjustable physical toughness has been developed for advanced soft lithography. We thus demonstrate here its photopatternability and nanoresolution soft lithography, which have proven to be difficult using commercial stamp materials.     

Construction of microfluidic chips using polydimethylsiloxane for adhesive bonding

A thin layer of polydimethylsiloxane (PDMS) prepolymer, which is coated on a glass slide, is transferred onto the embossed area surfaces of a patterned substrate. This coated substrate is brought into contact with a flat plate, and the two structures are permanently bonded to form a sealed fluidic system by thermocuring (60 degrees C for 30 min) the prepolymer. The PDMS exists only at the contact area of the two surfaces with a negligible portion exposed to the microfluidic channel. This method is demonstrated by bonding microfluidic channels of two representative soft materials (PDMS substrate on a PDMS plate), and two representative hard materials (glass substrate on a glass plate). The effects of the adhesive layer on the electroosmotic flow (EOF) in glass channels are calculated and compared with the experimental results of a CE separation. For a channel with a size of approximately 10 to 500 microm, a approximately 200-500 nm thick adhesive layer creates a bond without voids or excess material and has little effect on the EOF rate. The major advantages of this bonding method are its generality and its ease of use.     

Light stamping lithography: microcontact printing without inks

We report a new patterning method, called light-stamping lithography (LSL), that uses UV-induced adhesion of poly(dimethylsiloxane) (PDMS). LSL is based on the direct transfer of the contact surface of the PDMS stamp to a substrate via a UV (254 nm)-induced surface bonding between the stamp and the substrate. This procedure can be adopted in automated printing machines that generate patterns with a wide range of feature sizes on diverse substrates. To demonstrate its usefulness, the LSL method was applied to prepare several PDMS patterns on a variety of substrates. The PDMS patterns were then used as templates for selective deposition of TiO2 thin film using atomic layer deposition as well as resists for selective wet etching.     

Surface energy modified chips for detection of conformational states and enzymatic activity in biomolecules

A novel patterning method for anchoring biomolecules and noncovalent assembled conjugated polyelectrolyte (CPE)/biomolecule complexes to a chip surface is presented. The surface energy of a hydrophilic substrate is modified using an elastomeric poly(dimethylsiloxane) (PDMS) stamp, containing a relief pattern. Modification takes place on the parts where the PDMS stamp is in conformal contact with the substrate and leaves low molecular weight PDMS residues on the surface resulting in a hydrophobic modification, and then biomolecules and CPE/biomolecule complexes are then adsorbed in a specific pattern. The method constitutes a discrimination system for different conformations in biomolecules using CPEs as reporters and the PDMS modified substrates as the discriminator. Detection of different conformations in two biomacromolecules, a synthetic peptide (JR2E) and a protein (calmodulin), reported by the CPE and resolved by fluorescence was demonstrated. Also, excellent enzyme activity in patterned CPE/horseradish peroxidase (HRP) enzyme was shown, demonstrating that this method can be used to pattern biomolecules with their activity retained. The method presented could be useful in various biochip applications, such as analyzing proteins and peptides in large-scale production, in making metabolic chips, and for making multi-microarrays.     

Modification of micro-channel filling flow by poly(dimethylsiloxane) surface functionalization with fluorine—Substituted aminonaphthols

Microfluidics based on the capillarity-induced filling of elastomeric channels by a suitable liquid or solution represents a useful route for realizing portable diagnostic devices designed without additional mechanical or electrical micropumps. In this study, an elastomeric mold made of poly(dimethylsiloxane) (PDMS), containing relief patterns placed in intimate contact with a silicon substrate, is utilized to create a continuous network of rectangular micro-channels for the motion of water fluid. The immobilization on activated PDMS surface of suitable functional molecules such as hydrophilic and hydrophobic fluorine-containing aminonaphthols, obtained through a straightforward and versatile synthetic procedure, allowed us to modulate PDMS surface properties depending on the structural characteristics of the employed derivative. In this context, the incorporation of fluorine groups is important for improving biocompatibility of the resulting device, providing surfaces that could be chemically and biologically inert as well as resistant to surface adhesion phenomena. The functionalization from liquid phase of PDMS replicas, involving a covalent derivatization via silanization reaction of the above mentioned compounds to an oxidized PDMS surface, resulted in a successful modification of microfluidic motion of water in rectangular capillaries, moreover contact angle values evidence also how wettability of PDMS films could be modulated, with the fluorinated aminonaphthols fuctionalized PDMS exhibiting higher contact angles.     

Hydrophobization of glass surface by adsorption of poly(dimethylsiloxane)

Silica or glass particles are introduced in a poly(dimethylsiloxane) (PDMS) matrix for various applications. A particular feature of these systems is that PDMS adsorbs on the surface of the dispersed particles, thus rendering them more hydrophobic with time. The mechanism of this process of in situ hydrophobization is still poorly understood. The major aims of the present study are (1) to quantify the rate of surface hydrophobization by PDMS and, on this basis, to discuss the mechanism of the process; (2) to compare the contact angles of surfaces that are hydrophobized by different procedures and are placed in contact with different fluid interfaces-PDMS-water, hexadecane-water, and air-water; and (3) to check how the type of surfactant affects the contact angles, viz., the effective hydrophobicity of the surface. We present experimental results for the kinetics of hydrophobization of glass surfaces, which are characterized by measuring the three-phase contact angle of glass-surfactant solution-PDMS. The data reveal two consecutive stages in the hydrophobization process: The first stage is relatively fast and the contact angle increases from 0 degrees to about 90 degrees within several minutes. This stage is explained with the physical adsorption of the PDMS chains, as a result of hydrogen-bond formation with the surface silanol groups. The second stage is much slower and hours or days are required at room temperature to reach the final contact angle (typically, 150-160 degrees). This stage is explained as grafting of the PDMS molecules on the surface by chemical reaction with the surface silanol groups. If the glass surface had been pretreated by hexamethyldisilazane (HMDS), so that CH(3) groups had blocked most of the surface silanol groups, the first stage in the hydrophobization process is almost missing-the contact angle slowly changes at room temperature from about 90 degrees up to 120 degrees. The experiments aimed to compare several hydrophobization procedures showed that PDMS ensures larger contact angle (more hydrophobic surface) than grafted alkyl chains. The contact angles at the PDMS-water and hexadecane-water interfaces were found to be very similar to each other, and much larger than that at the air-water interface. Interestingly, we found that the ionic surfactants practically do not affect the contact angle of PDMS-hydrophobized surface, whereas the nonionic surfactants reduce this angle. Similar trends are expected with silica surfaces, as well.     

Surface modification of elastomeric stamps for microcontact printing of polar inks

Chemical modification of the surface of a stamp used for microcontact printing (microCP) is interesting for controling the surface properties, such as the hydrophilicity. To print polar inks, plasma polymerization of allylamine (PPAA) was employed to render the surface of poly(dimethylsiloxane) (PDMS), polyolefin plastomers (POP), and Kraton elatomeric stamps hydrophilic for long periods of time. A thin PPAA film of about 5 nm was deposited on the stamps, which increased the hydrophilicity, and which remained stable for at least several months. These surface-modified stamps were used to transfer polar inks by microCP. The employed microCP schemes are as follows: (a) a second generation of dendritic ink having eight dialkyl sulfide end groups to fabricate patterns on gold substrates by positive microCP, (b) fluorescent guest molecules on beta-cyclodextrin (beta-CD) printboards on glass employing host-guest recognition, and (c) Lucifer Yellow ethylenediamine resulting in covalent patterning on an aldehyde-terminated glass surface. All experiments resulted in an excellent performance of all three PPAA-coated stamp materials to transfer the polar inks from the stamp surface to gold and glass substrates by microCP, even from aqueous solutions.