Micropatterning of proteins on nanospheres

Currently micropatterning of proteins is mainly carried out on a planar substrate, which involves multi-step surface modifications directly on the substrate. Efficiency of chemical reactions is usually low, resulting in low signal-to-noise (S/N) ratio and poor repeatability of results. Here we presented a micropatterning method using polystyrene nanospheres with non-planar surface as a solid support for attaching proteins, which introduces many advantages. The patterning of proteins was carried out in two approaches: one was to dispense polystyrene nanospheres into an array of microwells and then attach proteins onto the nanospheres, and another was to coat polystyrene nanospheres with proteins first and then deposit the spheres into the microwells. For both approaches, a uniform pattern of proteins was generated. The amount of proteins attached via nanospheres was much higher than that on planar surface.     

Optimization of DNA-tagged liposomes for use in microtiter plate analyses

Dye-encapsulating unilamellar DNA oligonucleotide-tagged liposomes were prepared and characterized for use as signal-enhancing reagents in a microtiter plate sandwich-hybridization analyses of single-stranded RNA or DNA sequences. The liposomes were synthesized using the reversed-phase evaporation method and tagged with DNA oligonucleotides by adding cholesteryl-modified DNA reporter probes to the initial lipid mixture. Liposomes were prepared using probe coverages of 0.0013–0.103 mol% of the total lipid input, several hydrophobic and poly(ethylene glycol)-based spacers between the cholesteryl anchor and the probe, and liposome diameters ranging from 200 nm to 335 nm. Their signal enhancement functionality was compared by using them in microtiter plate sandwich-hybridization assays for the detection of single-stranded DNA sequences. In these assays, an optimal reporter probe concentration of 0.103 mol%, a liposome diameter of 274 nm, and a phospholipid concentration of 0.3 mM were found. The length between the cholesteryl anchor and the probe was optimal when a spacer composed of TEG+(CH₂O)₃ was used. Under optimal conditions, a detection limit of 0.5 nM for a truncated synthetic DNA sequence was found with a coefficient of variation of 4.4%. A 500-fold lower limit of detection using fluorescence was found using lysed dye-encapsulating liposomes versus a single fluorescein-labeled probe. Finally, when this method was applied to the detection of atxA RNA extracted from E.coli SG12036-pIu121 and amplified using NASBA, a minimum extracted concentration of RNA of 1.1×10⁻⁷ μg/μL was found.     

Micropatterning of Plasma Fluorinated Super-hydrophobic Surfaces

Photolithographic patterning of super-hydrophobic CF₄ plasma fluorinated polybutadiene films is shown to provide sufficient surface energy contrast to facilitate spatially organized deposition of metal salts and polystyrene microspheres from a nebulized mist or solution.     

铂纳米颗粒自组装膜的微图像化

从硝基重氮树脂(NDR)与包裹巯基乙酸的铂纳米颗粒(MA-PtNP)的静电自组装,制备了感光性的自组装多层超薄膜.经选择性紫外曝光和十二烷基硫酸钠(SDS)水溶液显影,光照部分的膜,因层与层之间的离子键转变为共价键,不再被SDS水溶液洗脱而留下来;未光照部分的膜,层与层之间仍是离子键,在显影时被除去,从而形成图像.用AFM和SEM对形成的图像进行了表征.     

Micropatterning neuronal cells on polyelectrolyte multilayers

This paper describes an approach to adhere retinal cells on micropatterned polyelectrolyte multilayer (PEM) lines adsorbed on poly(dimethylsiloxane) (PDMS) surfaces using microfluidic networks. PEMs were patterned on flat, oxidized PDMS surfaces by sequentially flowing polyions through a microchannel network that was placed in contact with the PDMS surface. Polyethyleneimine (PEI) and poly(allylamine hydrochloride) (PAH) were the polyions used as the top layer cellular adhesion material. The microfluidic network was lifted off after the patterning was completed and retinal cells were seeded on the PEM/PDMS surfaces. The traditional practice of using blocking agents to prevent the adhesion of cells on unpatterned areas was avoided by allowing the PDMS surface to return to its uncharged state after the patterning was completed. The adhesion of rat retinal cells on the patterned PEMs was observed 5 h after seeding. Cell viability and morphology on the patterned PEMs were assayed. These materials proved to be nontoxic to the cells used in this study regardless of the number of stacked PEM layers. Phalloidin staining of the cytoskeleton revealed no apparent morphological differences in retinal cells compared with those plated on polystyrene or the larger regions of PEI and PAH; however, cells were relatively more elongated when cultured on the PEM lines. Cell-to-cell communication between cells on adjacent PEM lines was observed as interconnecting tubes containing actin that were a few hundred nanometers in diameter and up to 55 microm in length. This approach provides a simple, fast, and inexpensive method of patterning cells onto micrometer-scale features.     

Micropatterning of cells using modulated magnetic fields

A new technique of cell micropatterning was presented. Mouse osteoblast cells (MC3T3-E1) were seeded on a substrate whose surface was exposed to a periodically modulated magnetic field (a line pattern with a 200- or 600-microm pitch) produced by a field modulator inserted into a homogeneous magnetic field of 1 T generated by an electromagnet. The cells were trapped consistent with the line profile of the modulated field. The trapping efficiency was enhanced by adding Mn(II)EDTA (paramagnetic) to the cultivation medium. The cells were subsequently incubated in the magnetic field. The same technique was applied to whole blood to pattern red blood cells.     

Micropatterning of polystyrene nanoparticles and its bioapplications

Micropatterning of biomolecules forms the basis of cell culture, biosensor and microarray technology. Currently, the most widely used techniques are photoresist lithography, soft lithography or using robots which all involve multi-step surface modification directly on a planar substrate. Here we report a method to pattern biomolecules through self-assembling polystyrene nanoparticles in arrayed microwells on a solid surface to form well-ordered patterning, followed by attaching biomolecules to the assembled nanoparticles. The formation of colloidal patterns depends on capillary force, surface wettability and physical confinement. This method can be used for micropatterning a variety of biomolecules such as protein and antibody.     

Optimization of DNA-tagged dye-encapsulating liposomes for lateral-flow assays based on sandwich hybridization

A novel protocol for the synthesis of dye-encapsulating liposomes tagged with DNA oligonucleotides at their outer surface was developed. These liposomes were optimized for use as signal enhancement agents in lateral-flow sandwich-hybridization assays for the detection of single-stranded RNA and DNA sequences. Liposomes were synthesized using the reverse-phase evaporation method and tagged with oligonucleotides by adding cholesteryl-modified DNA probes to the initial lipid mixture. This resulted in a greatly simplified protocol that provided excellent control of the probe coverage on the liposomes and cut the preparation time from 16 hours to just 6 hours. Liposomes were prepared using probe concentrations ranging from 0.00077 to 0.152 mol% of the total lipid, several hydrophobic and polyethylene glycol-based spacers between the cholesteryl anchor and the probe, and liposome diameters ranging from 208 nm to 365 nm. The liposomes were characterized by dynamic light scattering, visible spectroscopy, and fluorescence spectroscopy. Their signal enhancement functionality was compared by using them in lateral-flow optical biosensors for the detection of single-stranded DNA sequences. In these assays, an optimal reporter probe concentration of 0.013 mol%, liposome diameter of 315 nm, and liposome optical density of 0.4–0.6 at 532 nm were found. The spacer length between the cholesteryl anchor and the probe showed no significant effect on the signals in the lateral-flow assays. The results presented here provide important data for the general use of liposomes as labels in analytical assays, with specific emphasis on nucleic acid detection via lateral flow assays.     

Superhydrophobic-Superhydrophilic Micropatterning: Towards Genome-on-a-Chip Cell Microarrays

High-density cell microarrays based on superhydrophilic microspots separated by superhydrophobic barriers have been realized. The microspots absorb water solutions, while the barriers prevent cross-contamination, thus allowing the spots to be used as reservoirs for transfection mixtures and preventing cell proliferation and cell migration between the microspots. The picture shows four cell types after two days of culturing on the microarray.     

Micropatterning of living cells on a heterogeneously wetted surface

We report a simple approach to fabricate heterogeneously wetted surfaces which can be used to pattern living cells or biomolecules. An array of pedestals composed of SU-8 was fabricated on a glass surface which was then derivatized with a hydrophobic silane. Upon addition of aqueous solutions to the array, air was trapped within the hydrophobic cavities between the pedestals. The trapped air formed a "virtual wall" blocking access to these cavities. Cells cultured on the array were forced to grow only on the tops of the pedestals, i.e., the surfaces wetted by aqueous media. The virtual walls were stable during manipulation of the array and over long time periods (months).     

Micropatterning of lanthanum-based oxide thin film on self-assembled monolayers

We studied the direct micropatterning of a lanthanum-based thin film on a template of self-assembled monolayers in an aqueous solution at 80 degrees C. The template composed of silanol and octadecyl areas was prepared by UV-modified octadecyltrichlorosilane SAMs through a photomask. The amorphous La(2)O(CO(3))(2) x H(2)O thin films were selectively deposited in the silanol regions. Crystallized La(2)O(3) was obtained after heating at 800 degrees C in air.     

Micropatterning Electrospun Scaffolds to Create Intrinsic Vascular Networks

Sufficient vascularization is critical to sustaining viable tissue‐engineered (TE) constructs after implantation. Despite significant progress, current approaches lack suturability, porosity, and biodegradability, which hinders rapid perfusion and remodeling in vivo. Consequently, TE vascular networks capable of direct anastomosis to host vasculature and immediate perfusion upon implantation still remain elusive. Here, a hybrid fabrication method is presented for micropatterning fibrous scaffolds that are suturable, porous, and biodegradable. Fused deposition modeling offers an inexpensive and automated approach to creating sacrificial templates with vascular‐like branching. By electrospinning around these poly(vinyl alcohol) templates and dissolving them in water, microvascular patterns were transferred to fibrous scaffolds. Results indicated that these scaffolds have sufficient suture retention strength to permit direct anastomosis in future studies. Vascularization of these scaffolds is demonstrated by in vitro endothelialization and perfusion.     

聚合物微球的自组装与微图形材料

苯乙烯;丙烯酸;共聚物;聚合物微球的自组装与微图形材料     

Micropatterning of Sol-Gel Derived Thin Films Using Hydrophobic-Hydrophilic Patterned Surface

Formation process of convexly shaped oxide micropatterns using hydrophobic-hydrophilic patterned surface has been examined, and this technique was applied to several oxide thin films such as SnO₂, ZrO₂, TiO₂ and Al₂O₃. Hydrophobic-hydrophilic patterned surfaces were prepared on glass substrates by selective UV irradiation through a photomask on double-layered films of a very thin TiO₂ gel film as the underlayer and a hydrolyzed fluoroalkyltrimethoxysilane layer as the top layer. Precursor solutions were then spin-coated on the hydrophobic-hydrophilic patterns, and the coated substrates were dried at room temperature. The micropatterns of oxides were very difficult to be formed on the hydrophobic-hydrophilic patterned surfaces from metal-alkoxides as a precursor solution, but convexly shaped micropatterns were formed on the hydrophilic regions of the pattern when metal chlorides or oxychlorides were used as starting materials. This patterning technique potentially has a wide variety of applications such as fabrication of micro-optical components and finely patterned transparent electrodes.     

Cell Adhesion Micropatterning by Plasma Treatment of Alginate Coated Surfaces

Air plasma treatment, coupled to a masking technique, was used to promote micropatterned cell adhesion onto a cell-adhesion-resistant alginate coated surface. L-929 mouse fibroblasts were successfully confined into 50 m diameter cell-adhesive areas patterned inside a cell-resistant layer. The plasma treatment performed, albeit very mild, destroys the molecular architecture of the hydrophilic polysaccharide coating, leading to an enhancement of protein adsorption and hence of cell-adhesion. Both the cell-adhesion-resistant and the cell-adhesive regions are hydrophilic, yet they show a completely different behavior towards cells. Thus, they are a very interesting subject in the study of interfacial interactions in aqueous media, and, in particular, on the mechanisms of bio-adhesion at hydrophilic surfaces.     

Micropatterning of ZnO nanoarrays by forced hydrolysis of anhydrous zinc acetate

Micropatterns of ZnO nanoarrays were simply and successfully fabricated in an aqueous solution without any high-temperature treatment and/or expensive catalyst. In situ forced hydrolysis of patterned anhydrous zinc acetate, derived by ultraviolet irradiation with a photomask, resulted in heterogeneous nucleation and growth to form ZnO nanoarrays. Micropatterns of ZnO nanoarrays were characterized by FE-SEM and XRD. ZnO nanoarrays were well site-selectively deposited on anhydrous zinc acetate coated regions at 88 degrees C. HR-TEM clarified the formation mechanism in which anhydrous zinc acetate showed a tendency of forced hydrolyzation to ZnO nanocrystals at the initial stage in the reaction solution.     

Micropatterning topology on soft substrates affects myoblast proliferation and differentiation

Micropatterning techniques and substrate engineering are becoming useful tools to investigate several aspects of cell-cell interaction biology. In this work, we rationally study how different micropatterning geometries can affect myoblast behavior in the early stage of in vitro myogenesis. Soft hydrogels with physiological elastic modulus (E = 15 kPa) were micropatterned in parallel lanes (100, 300, and 500 μm width) resulting in different local and global myoblast densities. Proliferation and differentiation into multinucleated myotubes were evaluated for murine and human myoblasts. Wider lanes showed a decrease in murine myoblast proliferation: (69 ± 8)% in 100 μm wide lanes compared to (39 ± 7)% in 500 μm lanes. Conversely, fusion index increased in wider lanes: from (46 ± 7)% to (66 ± 7)% for murine myoblasts, and from (15 ± 3)% to (36 ± 2)% for human primary myoblasts, using a patterning width of 100 and 500 μm, respectively. These results are consistent with both computational modeling data and conditioned medium experiments, which demonstrated that wider lanes favor the accumulation of endogenous secreted factors. Interestingly, human primary myoblast proliferation is not affected by patterning width, which may be because the high serum content of their culture medium overrides the effect of secreted factors. These data highlight the role of micropatterning in shaping the cellular niche through secreted factor accumulation, and are of paramount importance in rationally understanding myogenesis in vitro for the correct design of in vitro skeletal muscle models.     

Micropatterning of functional conductive polymers with multiple surface chemistries in register

A versatile procedure is presented for fast and efficient micropatterning of multiple types of covalently bound surface chemistry in perfect register on and between conductive polymer microcircuits. The micropatterning principle is applied to several types of native and functionalized PEDOT (poly(3,4-ethylenedioxythiophene)) thin films. The method is based on contacting PEDOT-type thin films with a micropatterned agarose stamp containing an oxidant (aqueous hypochlorite) and applying a nonionic detergent. Where contacted, PEDOT not only loses its conductance but is entirely removed, thereby locally revealing the underlying substrate. Surface analysis showed that the substrate surface chemistry was fully exposed and not affected by the treatment. Click chemistry could thus be applied to selectively modify re-exposed alkyne and azide functional groups of functionalized polystyrene substrates. The versatility of the method is illustrated by micropatterning cell-binding RGD-functionalized PEDOT on low cell-binding PMOXA (poly(2-methyl-2-oxazoline)) to produce cell-capturing microelectrodes on a cell nonadhesive background in a few simple steps. The method should be applicable to a wide range of native and chemically functionalized conjugated polymer systems.     

Micropatterning of bioactive glass nanoparticles on chitosan membranes for spatial controlled biomineralization

Bioactive glass nanoparticles (BG-NPs) capable of inducing apatite precipitation upon immersion in simulated body fluid (SBF) were patterned on free-standing chitosan membranes by microcontact printing using a poly(dimethylsiloxane) (PDMS) stamp inked in a BG-NPs pad. Formation of the patterns was characterized by scanning electron microscopy (SEM). Mineralization of the bioactive glass patterns was induced in vitro by soaking the samples in SBF over different time points up to 7 days. The confined apatite deposition in the patterned regions with diameters of 50 μm was confirmed by Fourier-transformed infrared spectroscopy (FTIR), energy-dispersive X-ray (EDX) analysis, and SEM. In vitro tests confirmed the preferential attachment and proliferation of L929 cells to the areas printed with BG-NPs of the membranes. This approach permits one to spatially control the properties of biomaterials at the microlevel and could be potentially used in guided tissue regeneration for skin, vascular, articular, and bone tissue engineering and in cellular cocultures or to develop substrates able to confine cells in regions with controlled geometry at the cell's length scale.     

自组装单层膜表面制备BiFeO3图案化薄膜的研究

采用短波紫外光(UV)对十八烷基三氯硅烷(OTS)自组装单分子层(SAMs)进行刻蚀,利用化学液相法在OTS-SAMs模板表面制备出铁酸铋(BiFeO₃)图案化薄膜。通过接触角、X射线衍射(XRD)、扫描电镜(SEM)、X射线能谱(EDS)等测试手段对OTS膜和BiFeO₃薄膜进行表征。结果表明：以OTS为模板利用化学液相法成功制备出边缘轮廓清晰、条纹宽度为10~20 μm的BiFeO₃图案化薄膜。