Quanguo He

Self-assembly monolayer of mercaptopropyltrimethoxysilane for electroless deposition of Ag

Mercaptopropyltrimethoxysilane (MPTS) was used to form self-assembly monolayers (SAMs) on glass slides, which was verified by using X-ray photoelectron spectroscopy (XPS) and auger electron spectroscopy (AES). Electroless plating of Ag was performed on the SAMs-modified glass slide. XPS study showed that Ag colloids formed in solution were successfully and hard anchored on SAMs through chemical bonds. Scanning electron microscopy (SEM) analysis illustrated that Ag film on the SAMs-modified glass showed more predominant in durability of temperature than that on conventionally modified glass.

In situ synthesis of oligonucleotide arrays by using soft lithography

This paper describes the in situ synthesis of oligonucleotide arrays on glass surfaces by using soft lithography. In this method, based on the standard phosphoramidite chemistry protocol, the coupling was achieved by the glass slide being printed with a set of polydimethylsiloxane (PDMS) microstamps, on which was spread nucleoside monomer and tetrazole mixed solution. The elastic characteristic of PDMS allowed it to make conformal contact with the glass slide in the printing coupling. With regard to the efficiency of the printing coupling, the hybridization microscope images of 20-mer oligonucleotide synthesized via the directly drip-dropped coupling and the contact coupling were compared; the fluorescence intensities of the two methods showed no significant differences. The coupling efficiency was also investigated via an end-labelled fluorescence nucleotide method and a stepwise yield of 97% was obtained. A high-quality, high spatial resolution and large-scale PDMS stamp, which was developed by integrating 168 different microstamps on one glass substrate for synthesizing oligonucleotide arrays. The stamp was modified to improve the surface wettability by plasma discharge treatments, so that microstamps could be used to fabricate oligonucleotide arrays. A motional printing head was developed to improve the contact effect of the glass slide with different microstamps. A higher boiling point solvent was used in the printing coupling to inhibit solvent volatilization and to maintain the consistency of reagents on different features of the microstamp. An effective method was used to eliminate residual reactive nucleosides on chips with small molecules containing a hydroxyl group. A specific oligonucleotide array of four probes both matched and mismatched with the target sequence was fabricated to identify the perfect match and mismatch sequences.

DNA microarray synthesis by using PDMS molecular stamp (II)

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.

Soft lithography for oligonucleotide arrays fabrication

A method for fabricating high-density oligonucleotide arrays directed by a set of molecular stamps is reported in this paper. In this method, based on the standard phosphoramidites chemistry protocol, the coupling was conducted under soft lithography. A 20-mer DNA microarray with 10,000 probes has been successfully fabricated, which shows the advantages of accurate, reliable operation and low-cost production of high-density DNA chips.

Mosaic DNA chip fabrication and its time-resolved fluorescence detection

Lab-on-a-Chip (LOC) and μ-TAS (micro-total analytical system) are based on miniaturized integrated platforms that have the potential to revolutionize chemical, biological, and biochemical synthesis and analysis. Here, we demonstrated a process of fabricating a mosaic DNA chip and a corresponding detection method by time-resolved fluorescence (TRF) labeling. We synthesized oligonucleotide sequences in situ on glass slides directly, and then sliced them up into small pieces and patched up the pieces with different sequences to generate a mosaic DNA chip. With multiple BCPDA (BCPDA, abbreviated from 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid) labeling method based on biotin-avidin amplification, we established a TRF detection format on the mosaic DNA chip. The detection method allows discriminatory signals for perfect match, one-base mismatch, two-base mismatch and three-base mismatch by TRF labeled hybridization, whereby Europium (III, Eu3+) was captured and released on the principle of complexation and dissociation interaction between BCPDA and Eu3+ solution when the BCPDA-tagged avidin and biotin-ended oligonucleotide sequence linked. The fluorescence spectra and related lifetimes were determined. Also, we compared the TRF detection mode with the conventional fluorescence one. These results showed the former is more reliable and stable than the latter, especially for the mosaic DNA chip. Likewise, by applying TRF probing (or labeling) to specific bio-systems, the discovery is of fundamental interest and has significant implications to time-resolved-fluorescence based detection on biosensor.

Hydrophilic surface formation on PDMS stamp by microwave plasma-induced grafting

In this paper, a method of microwave plasma-induced grafting to enhance PDMS surface hydrophilicity was developed and its durable hydrophilicity resulted from an oriented grafting of acrylonitrile (AN) was described. In the investigations, the microwave plasma treatment parameters and post-wet chemical grafting were optimized, and their effects on hydrophilicity were also investigated. Contact angle measurements were used to assess the hydrophilicity of the various modified PDMS surfaces. Moreover, attenuated total reflectance Fourier transform infrared (ATR-FT-IR) spectroscopic characterization was used to evaluate PDMS samples variation. It has been found that the cyano groups bonded onto PDMS showed the best hydrophilicity among the modified surfaces, which showed a better affinity to acetonitrile. Therefore the hydrophilic surface was formed on PDMS stamps and the results implied promising applications of DNA microarray.

Fabrication of microstamps used for oligonucleotide array synthesis

A polydimethylsiloxane (PDMS) stamp with well-defined features on its surface was fabricated through soft lithography. First, we intended to increase the molecular interaction between the PDMS materials and its supporting substrate through surface modification with self-assembly technique. A silane molecular monolayer has been produced on a glass plate by treatment with 5% (CH3)2SiCl2 in CH3Cl. Secondary, we optimized the thickness of the PDMS stamps between 6x10-4m and 1.0x10-3m, and the feature highness 1.5x10-5m, and thus the stamp shrinkage could be controlled within 0.0417% in linearity or within 0.286% in area, respectively. Third, we modified the surface of the master through chemical plating silver or aluminum to prevent the PDMS from sticking to the master surface. During curing process of the stamp fabrication, PDMS intends to shrink towards the silanized glass plate from the master surface, which helps to peel stamp from the master without damaging its features.

Planar distortion quantification to evaluate PDMS stamps affixed on glass support

This article describes a planar distortion quantification method for PDMS stamps used in soft lithography by introducing an angular parameter θ; the distortion θ is proportional to planar distortion in magnitude. We employ this method to evaluate PDMS stamps planar distortions supported on different treated glass with Micron XYZ Scope measurements. The average planar distortion of individual pattern (absolute distortion θ1) and their pattern-to-pattern distortion (relative distortion θ2) of PDMS stamps were determined by angular discrepancies (θ). The planar distortion quantification was evaluated among four different PDMS stamps affixation treatments, and the PDMS stamps supported on silane-modified glass showed strong binding and minimal planar distortion, its absolute angular distortion θ1 was 3.98x10-3 and relative angular distortion θ2 1.22x10-3. Such distortion quantification agreed with the results of linear and area shrinkages on the stamps surface patterns, the results showed high reliability and fidelity of PDMS stamps and similar elastomer micro-patterns supported on silane-modified glass by photo lithographic microfabrication method and their promising prospects for on-chip synthesis of DNA microarray and bio-devices fabrication in soft lithography. The distortion evaluations demonstrate a versatile method for quantifying and comparing planar distortions among patterns as well as screening elastomer stamps support in soft lithography.

Improvement of hybridization signals of colorimetric gene detection based on porous polypropylene membranes

Porous polypropylene (PP) membranes were modified by the plasma treatment in order to graft amino functional group (-NH2) onto the membrane surface. Oligonucleotides were in situ synthesized on the aminated polypropylene support. Gold nanoparticle labeled DNAs were bybridized to the synthesized oligonucleotide array. The membranes were exposed to the Silver Enhance Solution for singla amplification. The Hybridization signals of amino plasma-grafted polypropylene membranes were stronger than the commercial polyacrylamide modified polyproplylene membranes that load 0.07 μmol/cm2 free primary amino functions. Complementary and mismatched sequences were clearly distinguished. The diameter of nanogold particles and the concentration of thiol DNA modified gold nanoparticles were investigated to improve the hybridization signals. Bigger nanoparticle diameter, as well as higher concentration of thiol DNA modified gold nanoparticles lead to stronger hybridization signals.

Shrinkage of polyurethane molecular stamp fixed on epoxy resin modified glass substrate

The shrinkage of polyurethane stamps used for the in situ synthesis of DNA microarrays via molecular stamping method was studied with Micron XYZ Scope. It was found that the polyurethane stamp fixed on the epoxy resin modified glass strongly and showed minimum linear shrinkage. The linear shrinkage of the whole polyurethane stamp and that of each feature of polyurethane stamp were controlled within 0.0341% and 0.309%, respectively, which were due to the strong van der Waals forces and hydrogen bonds between polyurethane and epoxy resin. It was also confirmed by scanning electron microscope that the polyurethane stamp fixed on the epoxy resin modified glass replicated the patterns of motherboard with a high fidelity. All these underlay the synthesis of DNA microarray through molecular stamping method.