Microcontact printing has become a versatile soft lithography technique used to produce molecular micro- and nano-patterns consisting of a large range of different biomolecules. Despite intensive ...research over the last decade and numerous applications in the fields of biosensors, microarrays and biomedical applications, the large-scale implementation of microcontact printing is still an issue. It is hindered by the stamp-inking step that is critical to ensure a reproducible and uniform transfer of inked molecules over large areas. This is particularly important when addressing application such as cell microarray manufacturing, which are currently used for a wide range of analytical and pharmaceutical applications. In this paper, we present a large-scale and multiplexed microcontact printing process of extracellular matrix proteins for the fabrication of cell microarrays. We have developed a microfluidic inking approach combined with a magnetic clamping technology that can be adapted to most standard substrates used in biology. We have demonstrated a significant improvement of homogeneity of printed protein patterns on surfaces larger than 1 cm2 through the control of both the flow rate and the wetting mechanism of the stamp surface during microfluidic inking. Thanks to the reproducibility and integration capabilities provided by microfluidics, we have achieved the printing of three different adhesion proteins in one-step transfer. Selective cell adhesion and cell shape adaptation on the produced patterns were observed, showing the suitability of this approach for producing on-demand large-scale cell microarrays.
The main goal of this paper is to probe mechanical properties of living and dead bacteria via atomic force microscopy (AFM) indentation experimentations. Nevertheless, the prerequisite for bioAFM ...study is the adhesion of the biological sample on a surface. Although AFM has now been used in microbiology for 20 years, the immobilization of micro-organisms is still challenging. Immobilizing a single cell, without the need for chemical fixation has therefore constituted our second purpose. Highly ordered arrays of single living bacteria were generated over the millimeter scale by selective adsorption of bacteria onto micrometric chemical patterns. The chemically engineered template surfaces were prepared with a microcontact printing process, and different functionalizations of the patterns by incubation were investigated. Thanks to this original immobilization strategy, the Young moduli of the same cell were measured using force spectroscopy before and after heating (45 degrees C, 20 min). The cells with a damaged membrane (after heating) present a Young modulus twice as high as that of healthy bacteria.
BAP31 is one of the most abundant endoplasmic reticulum (ER) membrane proteins. It is a chaperone protein involved in several pathways, including ER-associated degradation, export of ER proteins to ...the Golgi apparatus, and programmed cell death. BAP31 is encoded by BCAP31, located in human Xq28 and highly expressed in neurons. We identified loss-of-function mutations in BCAP31 in seven individuals from three families. These persons suffered from motor and intellectual disabilities, dystonia, sensorineural deafness, and white-matter changes, which together define an X-linked syndrome. In the primary fibroblasts of affected individuals, we found that BCAP31 deficiency altered ER morphology and caused a disorganization of the Golgi apparatus in a significant proportion of cells. Contrary to what has been described with transient-RNA-interference experiments, we demonstrate that constitutive BCAP31 deficiency does not activate the unfolded protein response or cell-death effectors. Rather, our data demonstrate that the lack of BAP31 disturbs ER metabolism and impacts the Golgi apparatus, highlighting an important role for BAP31 in ER-to-Golgi crosstalk. These findings provide a molecular basis for a Mendelian syndrome and link intracellular protein trafficking to severe congenital brain dysfunction and deafness.
Highly ordered arrays of single living bacteria were obtained by selective adsorption of bacteria onto chemical patterns with micrometric resolution. The chemically engineered template surfaces were ...prepared with the combination of microcontact printing process and a simple incubation technique. This methodology can be used for fundamental studies of bacterium's inner mechanisms and sub-cellular organization as well as for interfacing living bacteria with artificial microsystems.
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•Epoxy resist & polyurethane can be used to replicate nanostructured silicon masters.•Replicated line gratings (500nm width /1μm pitch) were obtained by nanoimprint.•These materials ...do not require an anti-adhesive treatment for PDMS stamp unmolding.•Low cost polyurethane is a good candidate for soft lithography industrial processes.
Soft lithography at the nanoscale requires a nanostructured silicon master mold generated by ebeam lithography and reactive ion etching. Such a fabrication is both expensive and time consuming for an expected industrial use of polydimethylsiloxane (PDMS) stamps in soft lithography. Our work focuses on a easy – low cost – technology to duplicate silicon master molds with nanoscale structures. Hence, master silicon molds patterned with nanometer scale lines have been replicated into epoxy resist and polyurethane by a process that implicated the following steps. First, the PDMS stamp made from a silicon master mold was used as template to copy this original mold into epoxy resist or polyurethane layer by nanoimprint. After characterization by Atomic Force Microscopy (AFM), these replicated molds were used to generate a second generation of PDMS stamps, which were ultimately employed to transfer labeled DNA probes on epoxysilane slides by soft lithography. Fluorescent microscopy reveals that molecular nanoscale patterns produced by these second generation PDMS stamps were exquisitely identical to those from the original PDMS stamp. As a conclusion, the epoxy resist or polyurethane are very attractive and cost-effective substrate to reproduce at large scale PDMS stamps initially made on a nanostructured silicon master mold.
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•A capillary assembly module was implemented onto a microcontact printing automate.•The assembly and nanocontact printing of biomolecules is fully automated.•Large-scale arrays of ...elongated DNA molecules and F-actin were achieved.•Single molecule deposition is obtained in a repeatable manner.•Local in situ F-actin polymerization during capillary assembly is observed.
Up to date, a large amount of research studies have been carried out to manipulate and arrange biomolecules at the single molecule scale using capillary forces. However, many of these techniques remain in the fundamental research field, their industrial transfer being restricted by poor repeatability and user-dependent processes.
We present an automated process for the controlled and large scale deposition of single biomolecules, relying on the use of directed capillary assembly and nano-contact printing processes. The adjustment and control of physical parameters allow for single molecule deposition, and the use of an automate operating arm ensures high reproducibility and freedom in the assembly architectures to create.
This methodology was used to assemble DNA molecules and actin filaments (or F-actin), evidencing two distinct assembly mechanisms. In the first one we use capillary forces to trap and elongate preformed 1D structures such as DNA molecules. In the second case, fluid flows created upon evaporation and local pinning of the meniscus favor the F-actin polymerization through continuous supply of monomers. In this way, we introduce a new concept of using capillary assembly as a construction tool to assemble 1D nano-structures. Additionally, by sequentially aligning and printing multiple single molecule assemblies, large-scale multi-layer architectures of single molecules were also obtained.
The large-scale capabilities and reliability of our fabrication process render sophisticated single molecule biophysical measurements possible with systematic analysis over a large population for statistical relevance.
•A new concept is presented: magnetic field assisted microcontact printing.•An automated system results from this concept: the INNOSTAMP40.•The genericity of this concept is proven by using standard ...stamp and macrostamp.•The homogeneity of the deposit is studied.•A developed model permits to find some trends in order to calibrate the INNOSTAMP40.
Microcontact printing (μ-CP) is a well-known and easy-to-use technology which is well established worldwide. This technology opens new opportunities for various applications. Though there is a wide range of applications, there is no standardized or calibrated system to easily reproduce microcontact printing results or to transfer scientific research to industrial applications. One of the critical points affecting the quality of μCP is being able to control the force applied on the stamp during the printing step. Up until this point, existing technologies have been based on a mechanical force. However, the drawback to this system is that stamp geometry has to be adapted to the mechanical system.
In this work, we propose a new concept of magnetic field assisted microcontact printing. We report theoretical and experimental studies of the homogeneity of the force applied and the resulting deposit. Theoretical models allow the prediction of a trend between the thickness of the magnetic stamp, the iron powder concentration and the pressure applied. The versatility of this concept is proven thanks to the development of an automated prototype, the INNOSTAMP40.
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•We obtain homogeneous DNA transfer in μ-contact printing by dynamic inking process.•The speed of the triple line is found critical in the quality of dynamic inking process.•DNA ...inking time in μ-contact printing (μCP) is not key to inking quality.•DNA consumption is reduced 10-folds.•This method paves the way to μCP as an industrial solution for biochips fabrication.
Microcontact printing (μCP) is used as a patterning technique to produce simple, rapid and cost-effective DNA microarrays. The accuracy of the final transferred pattern drastically depends on the inking step. The usual way to ink a PDMS stamp by droplet deposition of labeled biomolecules using a pipette, results in irregular transfer of the biomolecules on the chip surface and leads to poor and irreproducible fluorescent signals. These drawbacks are likely due to irregular ‘coating’ of the biomolecules on the PDMS stamp. In this work, a novel approach for inking PDMS with DNA molecules is presented. It is based on the continuous displacement of the meniscus formed by the inking solution over the surface of the stamp. When compared with the conventional technique, this dynamic PDMS inking method proved to be very reproducible for producing regular prints/spots on a functionalized glass slide, and this method could be easily extrapolated at an industrial scale.
We have developed a novel method for multiplexing the Micro-Contact Printing (μCP) technique which targets the deposition of 100–1000 different molecules in one step. For this purpose we have ...fabricated so-called macrostamps containing millimetric pads arranged in a periodic array compatible with titration plates. Through a very simple procedure of molding, each of this pad includes topographical micro and nanoscale features suitable for μCP on its surface. Then by inking the macrostamp in a single step through a titration plate, it becomes possible to print different inks in one step. The validations of this process are presented with the latest results showing that without any trace of contamination up to 800 different biological inks can be printed in one step. This method could be efficiently exploited for generating at low cost new slides for DNA Micro-arrays.
Microarrays are established research tools for genotyping, expression profiling, or molecular diagnostics in which DNA molecules are precisely addressed to the surface of a solid support. This study ...assesses the fabrication of low-density oligonucleotide arrays using an automated microcontact printing device, the InnoStamp 40(®). This automate allows a multiplexed deposition of oligoprobes on a functionalized surface by the use of a MacroStamp(TM) bearing 64 individual pillars each mounted with 50 circular micropatterns (spots) of 160 µm diameter at 320 µm pitch. Reliability and reuse of the MacroStamp(TM) were shown to be fast and robust by a simple washing step in 96% ethanol. The low-density microarrays printed on either epoxysilane or dendrimer-functionalized slides (DendriSlides) showed excellent hybridization response with complementary sequences at unusual low probe and target concentrations, since the actual probe density immobilized by this technology was at least 10-fold lower than with the conventional mechanical spotting. In addition, we found a comparable hybridization response in terms of fluorescence intensity between spotted and printed oligoarrays with a 1 nM complementary target by using a 50-fold lower probe concentration to produce the oligoarrays by the microcontact printing method. Taken together, our results lend support to the potential development of this multiplexed microcontact printing technology employing soft lithography as an alternative, cost-competitive tool for fabrication of low-density DNA microarrays.
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