Porous polypropylene (PP) membranes were modified by the plasma treatment in order to graft amino functional group (-NH<sub>2</sub>) 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/cm<sup>2</sup> 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.
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, Eu<sup>3+</sup>) was captured and released on the principle of complexation and dissociation interaction between BCPDA and Eu<sup>3+</sup> 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.
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 θ<sub>1</sub>) and their pattern-to-pattern distortion (relative distortion θ<sub>2</sub>) 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 θ<sub>1</sub> was 3.98x10<sup>-3 </sup>and relative angular distortion θ<sub>2</sub> 1.22x10<sup>-3</sup>. 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.
The shrinkage of polyurethane stamps used for the <i>in situ</i> 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.
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% (CH<SUB>3</SUB>)<SUB>2</SUB>SiCl<SUB>2</SUB> in CH<SUB>3</SUB>Cl. Secondary, we optimized the thickness of the PDMS stamps between 6x10<SUP>-4</SUP>m and 1.0x10<SUP>-3</SUP>m, and the feature highness 1.5x10<SUP>-5</SUP>m, 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.
Polyurethane based on polypropylene glycol (PPG) and Toluene diisocyanate (TDI) using 3,3'-dichloride-4,4'- methylenedianiline (MOCA) as the crosslinker is presented for the first time to fabricate molecular stamps (PU stamps) for the synthesis of DNA microarray with contact procedure. The predictability of the process is achieved by utilizing commercially available starting materials. SEM analysis of the morphology of PU stamps and master showed that PU elastometer could replicate subtly the motherboard's patterns with high fidelity. It was proved from the contact angle measurement that PU stamps surface has good affinity with acetonitrile, which guarantee the well-distribution of DNA monomers on patterned stamps. Laser confocal fluorescence microscopy images of oligonucleotide arrays confirmed polyurethane is an excellent material for molecular stamps.
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.