The present invention relates to a kit-of-parts for attaching a preferably biological object and/or a non-biological object on a substrate according to claim 1, a method of producing a kit-of-parts according to claim 20, the use of a substrate for attaching a preferably biological object and/or a non-biological object according to claim 22, the use of a first solution for attaching a preferably biological object and/or a non-biological object on a functionalized substrate according to claim 28, a method of attaching a preferably biological object and/or a non-biological object on a substrate according to claim 34, the use of a kit-of-parts in a method of attaching a preferably biological object and/or non-biological object according to claim 43, and the substrate comprising a preferably biological object and/or non biological object being attached thereon according to claim 44, respectively.
Attachment of biological matter to abiotic surface is of general interest in many industries. It is a natural, yet often an undesired phenomenon, for example in the case of biofilms which can lead to microbial influenced corrosion, infestation of medical devices, clogging (e.g. pipes, bioreactors, or wastewater plants), and so forth. Likewise, the attachment of biological matter can also be intended, for example in medical implants, biosensors, fuel cells, imaging, cell cultures, bioreactors, and numerous other research and industry applications.
One very recent application for the attachment of biological matter is the measurement of nanomotion as disclosed in EP 2 766 722 B1. Namely, EP 2 766 722 B1 discloses a rapid antibiotic susceptibility test (AST) that is based on atomic force microscopy. The AST is called nanomotion AST because it uses movements of AFM cantilevers that are caused by microorganisms and other biological material attached to these cantilevers. The nanomotion AST provides the same results within a few minutes to hours regardless of strain identity. It comprises a device and a cantilever mounted in the device. Once an effective toxin, for example an antibiotic, is added, cells attached to the cantilevers die and the movements of the cantilevers' cease. Obviously, this technology requires robust cell attachment.
A major issue with nanomotion ASTs is cell attachment to cantilevers. It is not unusual that only one out of five attempts to attach cells to cantilevers is successful. Other bioactivity tests require cell attachment as well. For example, cells can be attached on a surface to detect their movements using microscopy. In other cases, cellular vital signs are not necessarily of interest, for example in the case of cell attachment aided by polymers on Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) trays. However, the use of such polymers as additives leads to cell aggregation and cell death which is often not desired, for example when vital signs are to be recorded. Some negative consequences of cell aggregation are that a clean distinction of single cells is impossible, that cell aggregates change their metabolism and that biofilms form, or that cellular signals become unevenly distributed. In summary, the applications for bio-immobilization are plentiful and there is a need for rapid and robust cell attachment to abiotic surfaces, avoiding cell death or aggregation.
It is therefore an object of the present invention to enable an improved attachment of a biological object on a substrate. In particular, it is an object to enable an attachment of a biological object on a substrate in a rapid and robust manner.
This object is achieved with a kit-of-parts according to claim 1, with a method of producing a kit-of-parts according to claim 20, with the use of a substrate for attaching an object according to claim 22, with the use of a first solution for attaching an object on a functionalized substrate according to claim 28, with a method of attaching an object on a substrate according to claim 34, the use of a kit-of-parts in a method of attaching an object according to claim 43, and with a substrate comprising an object being attached thereon according to claim 44, respectively.
In particular, a kit-of-parts for attaching a biological object on a substrate is provided, which comprises (i) at least a first solution comprising at least one first compound, wherein the first compound is at least one of a gelling agent, a gellable agent, and a thickening agent, and (ii) at least a first substrate comprising a surface. The first solution is suitable for forming at least a first dispersion of at least one biological object in the first solution when at least one biological object is added to the first solution. The first dispersion is suitable for attaching the biological object on the surface of the substrate when the first dispersion is added to the surface of the substrate.
The kit-of-parts preferably further comprises instructions for the attachment of the object, wherein the instructions comprise the step of preparing the first dispersion by dispersing the at least one object in the first solution. Optionally, the instructions may further comprise the step of adding the first dispersion to the optionally functionalized surface of the substrate so as to attach the object on the optionally functionalized surface of the substrate.
To this end it is conceivable that at least a first part of the surface of the substrate is functionalized, see further below. In this case, the first dispersion and the functionalized part of the substrate are preferably suitable for attaching the biological object on the functionalized surface of the substrate when the first dispersion is added to the functionalized surface of the substrate.
Since the first solution is used for forming a dispersion comprising the potentially living objects to be attached it is preferred that the first solution comprises a pH-value that is physiological. In particular, it is preferred that the pH-value of the first solution is in the range of about 6 to 8, particularly preferably about 7. It is furthermore preferred that a temperature of the first solution is and/or remains below a temperature being lethal for the objects to be attached. As such, it is conceivable that the instructions of the kit-of-parts furthermore comprise the step of i) preparing the first dispersion at a temperature being at or below about 37° C. and/or in the range of between about 15° C. to 40° C., more preferably in the range of about 20° C. to 25° C. and/or ii) adding the first dispersion to the optionally functionalized surface of the substrate at a temperature being at or below about 37° C. and/or in the range of about 15° C. to 40° C., more preferably in the range of about 20° C. to 25° C. The instructions may further comprise the step of heating the first solution to a temperature of at least 40° C. or more, for example of at least 60° C. or more such as 90° C. or more so as to dissolve the first compound. Consequently, the instructions may further comprise the step of cooling the heated first solution to a temperature of 37° C. or less before the one or more objects are added to the first solution.
Likewise, a method of producing a kit-of-parts for attaching a biological object on a substrate is provided, the method comprising the steps of (i) providing at least a first solution comprising at least one first compound, wherein the first compound is at least one of a gelling agent, a gellable agent, and a thickening agent, and (ii) providing at least a first substrate comprising a surface. The first solution is suitable for forming at least a first dispersion of at least one biological object in the first solution when at least one biological object is added to the first solution. The first dispersion is suitable for attaching the biological object on the surface of the substrate when the first dispersion is added to the surface of the substrate.
Also, in this case it is conceivable that at least a first part of the surface of the substrate is functionalized, wherein the first dispersion and the functionalized part of the substrate are preferably suitable for attaching the biological object on the functionalized surface of the substrate when the first dispersion is added to the functionalized surface of the substrate.
Furthermore, the present invention also relates to the use of substrate for attaching at least one biological object dispersed in at least a first solution, wherein the first solution comprises at least one first compound, wherein the first compound is at least one of a gelling agent, a gellable agent, and a thickening agent, and wherein at least a first part of the surface of the substrate is preferably functionalized.
In addition, the present invention also relates to the use of a first solution for attaching at least one biological object dispersed in the first solution on a surface of a substrate, wherein the first solution comprises at least one first compound, wherein the first compound is at least one of a gelling agent, a gellable agent, and a thickening agent, and wherein at least a first part of the surface of the substrate is preferably functionalized.
Moreover, a method of attaching a biological object on a substrate is provided, which method comprises the steps of (i) preparing at least a first dispersion of at least one biological object in a first solution, and (ii) adding the first dispersion to the surface of the substrate, whereby the biological object is attached on the surface of the substrate. The first solution comprises at least one first compound, wherein the first compound is at least one of a gelling agent, a gellable agent, and a thickening agent.
Furthermore, the present invention relates to the use of a kit-of-parts as described above and further below in a method of attaching an object on a surface of a substrate as described above and further below.
The present invention also relates to a substrate comprising at least one object being attached thereon, wherein the substrate comprises at least one surface and at least one first layer being arranged on at least a part of the surface and being formed from at least a first dispersion as obtained in the method of attaching an object on a surface of a substrate as described above and further below.
For the sake of completeness, it is mentioned again that it is conceivable that at least a first part of the surface of the substrate is functionalized, wherein the first dispersion and the functionalized part of the substrate are preferably suitable for attaching the biological object on the functionalized surface of the substrate when the first dispersion is added to the functionalized surface of the substrate.
The inventors have surprisingly found out that the use of a first solution comprising at least one of a gelling agent, gellable agent, and a thickening agent possibly in combination with a functionalized surface of the substrate results in an improved attachment of the biological object as compared to the attachment methods known in the state of the art. In particular, the invention enables an attachment of the biological objects in high numbers. Furthermore, the formation of an agglomerations of the attached objects is reduced or even prevented. In other words, a more homogeneous attachment is achieved. At the same time, it also enables a rapid and facile attachment that can be carried out using standard laboratory equipment. Besides, and as opposed to substrates known in the art which have to be used within a few hours, the first solution comprising the at least one first compound and the preferably functionalized substrate can be stored over several weeks. To this end it is therefore preferred to provide the first solution comprising the first compound in a storage means such as a container or the like.
The object preferably is a biological object and/or a non-biological object. That is, it is conceivable to attach one or more biological objects, or one or more non-biological objects, or a mixture of one or more biological objects as well as one or more non-biological objects. The biological object preferably is at least one of a cell, a virus such as a phage, and a matter of biological origin such as peptides, proteins, polysaccharides, vesicles, protein-RNA co-polymers, protein-DNA co-polymers, capsules, spores, and so forth. The matter of biological origin is preferably particulate. The cell can correspond to pathogenic or non-pathogenic prokaryotic cells, eukaryotic cells, aggregates thereof, or tissue. Pathogenicity may occur in humans, animals, plants, or fungi. The cells can have different genotypic and phenotypic traits and do not need to be of the same identity. However, it is important to note that further biological objects that have not been explicitly mentioned here can be likewise attached. However, it is also conceivable that the object is a non-biological object such as a protein, lipid, nucleic acid such as DNA, a nanotube or nano bead made of elementary carbon metal oxides such as titanium oxide, a nanodevice, a glucide, a hydrocarbon, an aliphatic or aromatic polymer such as a phenolic polymer, and the like.
In the following, further aspects of the kit-of-parts, the method of producing a kit-of-parts, the use of the substrate for attaching the biological object and/or the non-biological object, the use of the first solution for attaching the biological object and/or the non-biological object on the substrate, the method of attaching the biological object and/or the non-biological object on the substrate, the use of a kit-of-parts in a method of attaching an object on a surface of a substrate, as well as the substrate comprising at least one object being attached thereon are discussed. For convenience, no explicit distinction is made between the kits-of-parts, the methods, the uses, and the substrate. Instead, any explanations provided below likewise apply to all of them.
As already mentioned, it is conceivable that at least a first part of the surface of the substrate is functionalized, and wherein the first dispersion and the functionalized surface of the substrate are suitable for attaching the object on the functionalized surface of the substrate when the first dispersion is added to the functionalized surface of the substrate. To this end, pre-treated substrates having a functionalized surface can be provided. However, it is likewise conceivable to provide untreated, i.e. non-functionalized substrates together with means for functionalizing said substrates. In the former case the kit-of-parts could further comprise the pre-treated substrates. In the latter case the kit-of-parts could further comprise the untreated substrates as well as the means for functionalizing said untreated substrates. In the latter case it is conceivable that the instructions furthermore comprise the step of adding the second solution comprising the second compound to the surface of the substrate so as to functionalize the surface of the substrate. This step of adding the second solution to the substrate is particularly preferably performed prior to the step of adding the first dispersion to the substrate.
It should be noted that only one part of the surface of the substrate can be functionalized, or that two or more parts of the surface of the substrate can be functionalized. Said two or more functionalized parts could be arranged immediately adjacent to one another or at a distance from one another. However, it is likewise conceivable that the entire surface of the substrate is functionalized. Explanations provided herein with reference to a partial functionalization of the surface of the substrate likewise apply to an entire functionalization of the surface of the substrate.
That is to say, it is conceivable that at least one of i) at least a first part of the surface of the substrate is preferably physically and/or chemically functionalized and ii) the kit-of-parts further comprises at least one second compound being suitable for chemically functionalizing at least a first part of the surface of the substrate.
Hence, the functionalization of the surface of the substrate can correspond to a chemical functionalization that is achieved by applying at least one second compound to the surface of the substrate, wherein the second compound interacts with the surface of the substrate. Said interaction between the second compound and the surface of the substrate results in the functionalization of the surface of the substrate. In other words, the surface of the substrate is functionalized by its interaction with the second compound. To this end it is therefore conceivable to provide at least a second compound in the kits-of-parts. This is particularly preferred when the second compound is directly applicable onto the surface of the substrate. However, the second compound can also be provided in a solution. Hence, the kit-of-parts could further comprise at least a second solution comprising the at least one second compound. Said second solution is preferably provided in a suitable storage means such as a container or the like. Hence, during the method of attaching the biological object and/or the non-biological object on the surface of the substrate it is conceivable to prepare at least one second solution and to add that second solution to the substrate so as to functionalize the surface of the substrate in a first step, and to then add the first dispersion comprising the first solution and the biological objects and/or the non-biological objects to the functionalized surface of the substrate in a second step. However, it is likewise conceivable to provide the kit-of-parts with a pretreated substrate, wherein its surface has been functionalized as just described before the functionalized substrate is arranged in the kits-of-parts. In this case a user can simply prepare the first dispersion and can readily add said first dispersion to the functionalized substrate.
Additionally, or alternatively, the functionalization of the surface of the substrate can correspond to a physical functionalization that is achieved by generating a surface structure in the surface of the substrate and/or by generating at least one layer on the surface of the substrate.
The generation of a surface structure in the surface of the substrate is preferably performed by surface modification methods that are well-known in the art. Said surface modification methods can correspond to chemical or physical surface modification methods and include, without limitation: etching with bases (e.g. KOH), etching with acids (e.g. HF), ion milling, deep reactive ion etching (DRIE), focused ion beam (FIB), scanning electron microscopy (SEM), as high energy electrons can modify surfaces, ion implantation/doping, electroplating, epitaxy methods such as liquid phase epitaxy (LPE), molecular beam epitaxy (MBE), VPE—vapour phase epitaxy (VPE), metal-organic vapor phase epitaxy (MOVPE), sputtering methods such as thin film sputtering methods, DC-sputtering, RF-sputtering, magnetron sputtering, as well as mechanical modifications such as scratching or laser-based modifications.
The surface structure can be seen as a surface relief that is generated in the surface of the substrate. That is, it is preferably constituted by several elevations and recesses. The dimensions of the surface structure, i.e. a height or depth of said elevations and recesses, preferably are microscopic dimensions. In other words, the dimensions of the surface structure preferably essentially correspond to the size of the object that shall be attached.
The one or more layers that are generated on the surface of the substrate are preferably a monolayer, particularly preferably an atomic monolayer. A preferred thickness of the one or more layers lies in the range of several hundred nanometers.
The at least one layer can comprise at least one metal compound and/or at least one oxide compound and/or at least one silicon compound and/or at least one nitride compound and/or at least one sulphide compound. The metal compound preferably comprises or consists of a noble metal such as gold, platinum, and palladium and combinations thereof. The oxide compound is preferably selected from titanium oxide, iron oxide, nickel oxide, aluminium oxide, silicone dioxide, cupric oxide, cuprous oxide and combinations thereof, such as an iron-nickel-oxide. The nitride compound preferably corresponds to silicon nitride. The sulphide compound is preferably selected from molybdenum sulphide, iron sulphide, nickel sulphide, iron-nickel sulphide, manganese sulphide, copper sulphide, titanium sulphide, uranium sulphide, cobalt sulphide, aluminium sulphide, chromium sulphide, yttrium sulphide and combinations thereof.
The second compound preferably is at least one of a polymer or a copolymer thereof, a polymerizable agent, a cross-linking agent, and a compound comprising at least one functional group. The polymer or the copolymer thereof and/or the polymerizable agent can be at least one of a polysaccharide compound, a polyaminosaccharide compound, a polyaminoacid compound, a polydopamine compound, a glycoproteine compound, a nucleic acid compound, an epoxy resin compound, a polysilane compound, a polysiloxane compound, a polyphosphate compound, a boron nitride polymer compound, a fluoropolymer compound, a polyallylamine compound, a polysulphide compound, a polyphenol compound, and a silicon-based polymer. The polyaminosaccharide compound preferably is chitosan. Additionally or alternatively the polyaminoacid compound preferably is polylysine, particularly preferably poly-D-lysine. Additionally or alternatively the glycoprotein compound preferably is laminin. Additionally or alternatively the nucleic acid compound preferably is desoxy ribonucleic acid. Additionally or alternatively the epoxy resin compound preferably is at least one of a bisphenol polymer compound and polyacetylene compound. Additionally or alternatively the polyphenol compound preferably is a polyphenolic protein, preferably a polyphenolic protein secreted by Mytilus sp., such as the polyphenolic protein secreted by Mytilus edulis. 110-140 kDa that is commercially available as Cell-Tak™. Additionally or alternatively, the second compound can be a recombinant Mytilus protein, preferably a recombinant Mytilus protein being produced by bacteria such as MAPTRix™, 23 kDa. Additionally or alternatively, the silicon-based polymer preferably corresponds to a polymeric organosilicon compound, preferably polydimethylsiloxane (PDMS). If polydimethylsiloxane is used as the second compound it is particularly preferred to additionally provide one or more curing agents that are configured to cure the said second compound. Additionally or alternatively the polyallylamine compound preferably comprises primary and/or secondary and/or tertiary polymers and preferably corresponds to a copolymer of polyallylamine and polystyrene. A conceivable copolymer of polyallylamine and polystyrene corresponds to the commercially available compound Bacpro® II. The cross-linking agent can be at least one of a homobifunctional cross-linking agent, a heterobifunctional cross-linking agent, and a photoreactive cross-linking agent, preferably an aldehyde-comprising cross-linking agent, particularly preferably glutaraldehyde. A homobifunctional cross-linking agent is understood as an agent comprising identical reactive groups at either ends. A heterobifunctional cross-linking agents is understood as an agent that possesses two different reactive groups. A photoreactive cross-linking agent is understood as a heterobifunctional cross-linking agent that become reactive upon exposure to radiation. The functional group can be at least one of an organic group, an inorganic group, and an organometallic group, preferably an organosilicon compound or an organosulfur compound, particularly preferably (3-aminopropyl)triethoxysilane (APTES) or 4-aminothiophenol (4-ATP).
The second solution preferably comprises at least one of a protic solvent, an aprotic solvent, a nonpolar solvent, a polar solvent, an organic compound, an inorganic compound, a liquid gas, and a melt. For example, the second solution could comprise acetone, ethanol, ethylene glycol, toluene, or naphthalene. It is preferred that the second solution is an aqueous solution, particularly preferably an aqueous solution that further comprises at least one of a polar water-soluble solvent such as an alcohol, a dissolved salt such as sodium chloride, and an acid such as acetic acid or hydrochloric acid. Depending on the chemical or physical properties of these compounds, it is preferred to apply the second solution to the substrate at an elevated temperature and/or pressure, see below.
The first compound can be at least one of a polymer and a polymerizable agent. Preferably the first compound is at least one of a polysaccharide, an amide-based polymer, a silicon-based polymer, and an ionomer. The polysaccharide is preferably selected from agarose, agar, alginate, dextran. Additionally or alternatively the amide-based polymer preferably corresponds to polyacrylamide. Additionally or alternatively the silicon-based polymer preferably corresponds to a polymeric organosilicon compound, preferably polydimethylsiloxane. Additionally or alternatively, the ionomer preferably corresponds to an inorganic polymer, preferably to a fluorinated polymer. The ionomer particularly preferably corresponds to the commercially available compound known as Nafion®.
In other words, the first compound may be chosen among saccharides, disachharides, oligosacchraides, or polysachharides and their respective mixtures. Suitable monosaccharides are in particular glucose, fructose and galactose. Suitable disaccharides are lactose, sucrose and maltose. Suitable polysaccharides are agarose, galactan, agaropectin, alginate, and mixtures thereof. An example of such a mixture of polysaccharides is agar. The first compound may further be chosen among synthetic polymers such as polyacrylamide, polyalkylene glycols, polysiloxanes or fluorpolymers. Suitable polyalkylene glycols may be chosen among polyethylene glycols, polypropylene glycols or copolymers thereof. Suitable polysiloxanes may be chosen among polydimethylsiloxane. Suitable fluoropolymers may be chosen among polymers or copolymers of tetrafluoroethylene such as polytetrafluoroethylene (PTFE) or sulphonated tetrafluoroethylene (Nafion®).
The second compound may be chosen among aldehydes, dialdehydes or polyaldehydes and their respective mixtures. Suitable dialdehydes are aliphatic dialdehydes or aromatic dialdehydes. An example of an aliphatic dialdehyde is glutaraldehyde. The second compound can be further chosen among polyelectrolytes such as poly(sodium-p-styrene sulfonate), poly(allylamine hydrochloride) or copolymers thereof, polynucleotides, polypeptides, polysaccharides such as a polyaminosaccharide for example polyglucosamin, also known as chitosan, polypeptides such as poly-alpha-lysine or poly-D-lysine, proteins such as collagens, glycoproteins such as laminins or mussel adhesive proteins, enzymes, or aminosilanes such as APTES (3-aminopropyl)-triethoxysilane, APDEMS (3-aminopropyl)-diethoxy-methylsilane, APDMES (3-aminopropyl)-dimethyl-ethoxysilane, or APTMS (3-aminopropyl)-trimethoxysilane. The second compound may be further chosen among polystyrenes or polyallylamines and mixtures thereof. Examples of polystyrenes are polymers of sodium-styrene sulfonate, such as poly(sodium-p-styrene sulfonate. A suitable mixture of polystyrenes and polyallylamines is Poly(sodium-p-styrene sulfonate)/poly(allylamino hydrochloride), also known as PSS/PAH, i.e. a polyelectrolyte. The PAH-PSS co-polymer is a so-called layer-by-layer polymer, wherein one layer is formed by PAH (poly(allylamino hydrochloride)) and the other layer is formed by PSS (Poly(sodium-p-styrene sulfonate)). PAH is charged positively and PSS is charged negatively, which makes the PAH-PSS co-polymer advantageous for attaching positively or negatively charged objects. The layer being proximate to the object to be attached is chosen in accordance with the charge of the object to be attached. An example of a mussel adhesive protein is the Mussel Adhesion Protein extracellular matrix (MAPTrix™). The Mussel Adhesion Protein extracellular matrix (MAPTrix™) as used herein has been commercially bought as the MAPTrix™ Adhesive Kit from the supplier Sigma-Aldrich, which indicates Kollodis Biosciences as producer. It corresponds to a Tyrosinase-pretreated powder with a molecular weight of about 23 kDa. The MAPTrix™ Adhesive Kit is a formulation of polyphenolic mussel adhesive proteins recombinantly produced in Kollodi's proprietary E. coli expression system. The recombinant mussel adhesive protein is a hybrid of Mytilus edulis fp-1 and fp-5 or a hybrid of Mytilus edulis fp-1, fp-3 and fp-5. The second compound may be further chosen among thiols such as aromatic thiols. An example of an aromatic thiol is tiophenol. A suitable tiophenol is aminothiophenol such as 2-aminothiopnelol, 3-aminothiopnelol, or 4-aminothiopnelol. As mentioned earlier, the object preferably is a biological object. Suitable biological objects are cells. The cells may be prokaryotic cells and/or eukaryotic cells. Examples of prokaryotic cells are bacteria such as enterobacteria and mycobacteria. Examples of eukaryotic cells are mammalian cells and yeast. An example of enterobacteria is Escherichia coli. An example of mycobacteria is Mycobacterium smegmatis. An example of mammalian cells are Vero cells. An example of yeast is Candida albicans.
Preferred first compounds used for forming the first dispersion and preferred second compounds to which the first dispersion is added in order to attach the object to the substrate are the following.
For example, it is preferred to use agarose as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use agar as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use alginate as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use Nafion® as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and poly-D-lysine as the second compound.
It is furthermore preferred to use agarose as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and laminin as the second compound.
It is also preferred to use agar as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and laminin as the second compound.
It is also preferred to use alginate as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and laminin as the second compound.
It is also preferred to use Nafion® as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and laminin as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, laminin as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and laminin as the second compound.
It is furthermore preferred to use agarose as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and chitosan as the second compound.
It is also preferred to use agar as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and chitosan as the second compound.
It is also preferred to use alginate as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and chitosan as the second compound.
It is also preferred to use Nafion® as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and chitosan as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, chitosan as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and chitosan as the second compound.
It is furthermore preferred to use agarose as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use agar as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use alginate as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use Nafion® as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and glutaraldehyde as the second compound.
It is furthermore preferred to use agarose as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use agar as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use alginate as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use Nafion® as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is furthermore preferred to use agarose as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use agar as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use alginate as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use Nafion® as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is furthermore preferred to use agarose as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use agar as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use alginate as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use Nafion® as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is furthermore preferred to use agarose as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use agar as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use alginate as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use Nafion® as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, an Enterobacterium such as Escherichia coli as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is furthermore preferred to use agarose as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use agar as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use alginate as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use Nafion® as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and poly-D-lysine as the second compound.
It is furthermore preferred to use agarose as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and laminin as the second compound.
It is also preferred to use agar as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and laminin as the second compound.
It is also preferred to use alginate as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and laminin as the second compound.
It is also preferred to use Nafion® as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and laminin as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, laminin as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and laminin as the second compound.
It is furthermore preferred to use agarose as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and chitosan as the second compound.
It is also preferred to use agar as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and chitosan as the second compound.
It is also preferred to use alginate as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and chitosan as the second compound.
It is also preferred to use Nafion® as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and chitosan as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, chitosan as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and chitosan as the second compound.
It is furthermore preferred to use agarose as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use agar as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use alginate as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use Nafion® as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and glutaraldehyde as the second compound.
It is furthermore preferred to use agarose as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use agar as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use alginate as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use Nafion® as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is furthermore preferred to use agarose as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use agar as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use alginate as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use Nafion® as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is furthermore preferred to use agarose as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use agar as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use alginate as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use Nafion® as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is furthermore preferred to use agarose as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use agar as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use alginate as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use Nafion® as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, a Mycobacteria such as Mycobacterium smegmatis as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is furthermore preferred to use agarose as the first compound, Mammalian cells such as Vero cells as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use agar as the first compound, Mammalian cells such as Vero cells as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use alginate as the first compound, Mammalian cells such as Vero cells as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use Nafion® as the first compound, Mammalian cells such as Vero cells as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and poly-D-lysine as the second compound.
It is furthermore preferred to use agarose as the first compound, Mammalian cells such as Vero cells as the object to be attached, and laminin as the second compound.
It is also preferred to use agar as the first compound, Mammalian cells such as Vero cells as the object to be attached, and laminin as the second compound.
It is also preferred to use alginate as the first compound, Mammalian cells such as Vero cells as the object to be attached, and laminin as the second compound.
It is also preferred to use Nafion® as the first compound, Mammalian cells such as Vero cells as the object to be attached, and laminin as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, Mammalian cells such as Vero cells as the object to be attached, laminin as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and laminin as the second compound.
It is furthermore preferred to use agarose as the first compound, Mammalian cells such as Vero cells as the object to be attached, and chitosan as the second compound.
It is also preferred to use agar as the first compound, Mammalian cells such as Vero cells as the object to be attached, and chitosan as the second compound.
It is also preferred to use alginate as the first compound, Mammalian cells such as Vero cells as the object to be attached, and chitosan as the second compound.
It is also preferred to use Nafion® as the first compound, Mammalian cells such as Vero cells as the object to be attached, and chitosan as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, Mammalian cells such as Vero cells as the object to be attached, chitosan as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and chitosan as the second compound.
It is furthermore preferred to use agarose as the first compound, Mammalian cells such as Vero cells as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use agar as the first compound, Mammalian cells such as Vero cells as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use alginate as the first compound, Mammalian cells such as Vero cells as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use Nafion® as the first compound, Mammalian cells such as Vero cells as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and glutaraldehyde as the second compound.
It is furthermore preferred to use agarose as the first compound, Mammalian cells such as Vero cells as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use agar as the first compound, Mammalian cells such as Vero cells as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use alginate as the first compound, Mammalian cells such as Vero cells as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use Nafion® as the first compound, Mammalian cells such as Vero cells as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is furthermore preferred to use agarose as the first compound, Mammalian cells such as Vero cells as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use agar as the first compound, Mammalian cells such as Vero cells as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use alginate as the first compound, Mammalian cells such as Vero cells as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use Nafion® as the first compound, Mammalian cells such as Vero cells as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is furthermore preferred to use agarose as the first compound, Mammalian cells such as Vero cells as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use agar as the first compound, Mammalian cells such as Vero cells as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use alginate as the first compound, Mammalian cells such as Vero cells as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use Nafion® as the first compound, Mammalian cells such as Vero cells as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is furthermore preferred to use agarose as the first compound, Mammalian cells such as Vero cells as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use agar as the first compound, Mammalian cells such as Vero cells as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use alginate as the first compound, Mammalian cells such as Vero cells as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use Nafion® as the first compound, Mammalian cells such as Vero cells as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, Mammalian cells such as Vero cells as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is furthermore preferred to use agarose as the first compound, yeast such as Candida albicans as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use agar as the first compound, yeast such as Candida albicans as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use alginate as the first compound, yeast such as Candida albicans as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use Nafion® as the first compound, yeast such as Candida albicans as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, yeast such as Candida albicans as the object to be attached, and poly-D-lysine as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, yeast such as Candida albicans as the object to be attached, and poly-D-lysine as the second compound.
It is furthermore preferred to use agarose as the first compound, yeast such as Candida albicans as the object to be attached, and laminin as the second compound.
It is also preferred to use agar as the first compound, yeast such as Candida albicans as the object to be attached, and laminin as the second compound.
It is also preferred to use alginate as the first compound, yeast such as Candida albicans as the object to be attached, and laminin as the second compound.
It is also preferred to use Nafion® as the first compound, yeast such as Candida albicans as the object to be attached, and laminin as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, yeast such as Candida albicans as the object to be attached, laminin as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, yeast such as Candida albicans as the object to be attached, and laminin as the second compound.
It is furthermore preferred to use agarose as the first compound, yeast such as Candida albicans as the object to be attached, and chitosan as the second compound.
It is also preferred to use agar as the first compound, yeast such as Candida albicans as the object to be attached, and chitosan as the second compound.
It is also preferred to use alginate as the first compound, yeast such as Candida albicans as the object to be attached, and chitosan as the second compound.
It is also preferred to use Nafion® as the first compound, yeast such as Candida albicans as the object to be attached, and chitosan as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, yeast such as Candida albicans as the object to be attached, chitosan as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, yeast such as Candida albicans as the object to be attached, and chitosan as the second compound.
It is furthermore preferred to use agarose as the first compound, yeast such as Candida albicans as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use agar as the first compound, yeast such as Candida albicans as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use alginate as the first compound, yeast such as Candida albicans as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use Nafion® as the first compound, yeast such as Candida albicans as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, yeast such as Candida albicans as the object to be attached, and glutaraldehyde as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, yeast such as Candida albicans as the object to be attached, and glutaraldehyde as the second compound.
It is furthermore preferred to use agarose as the first compound, yeast such as Candida albicans as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use agar as the first compound, yeast such as Candida albicans as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use alginate as the first compound, yeast such as Candida albicans as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use Nafion® as the first compound, yeast such as Candida albicans as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, yeast such as Candida albicans as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, yeast such as Candida albicans as the object to be attached, and (3-Aminopropyl)triethoxysilane (APTES) as the second compound.
It is furthermore preferred to use agarose as the first compound, yeast such as Candida albicans as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use agar as the first compound, yeast such as Candida albicans as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use alginate as the first compound, yeast such as Candida albicans as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use Nafion® as the first compound, yeast such as Candida albicans as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, yeast such as Candida albicans as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, yeast such as Candida albicans as the object to be attached, and poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer (PSS/PAH) as the second compound.
It is furthermore preferred to use agarose as the first compound, yeast such as Candida albicans as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use agar as the first compound, yeast such as Candida albicans as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use alginate as the first compound, yeast such as Candida albicans as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use Nafion® as the first compound, yeast such as Candida albicans as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, yeast such as Candida albicans as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, yeast such as Candida albicans as the object to be attached, and Mussel Adhesive recombinant protein (MAPTrix™) as the second compound.
It is furthermore preferred to use agarose as the first compound, yeast such as Candida albicans as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use agar as the first compound, yeast such as Candida albicans as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use alginate as the first compound, yeast such as Candida albicans as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use Nafion® as the first compound, yeast such as Candida albicans as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use polydimethylsiloxane (PDMS) as the first compound, yeast such as Candida albicans as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
It is also preferred to use polyethylene glycol (PEG) as the first compound, yeast such as Candida albicans as the object to be attached, and 4-Aminothiophenol (4-ATP) as the second compound.
The first solution preferably is an aqueous solution such as water, preferably an aqueous solution that may further comprise a buffer and/or a preferably dissolved salt such as sodium chloride. The buffer can be any buffer known in the art, such as a phosphate buffered saline (PBS) or another buffered solution such as Tris buffer, etc. The first solution may further comprise a growth medium.
The first solution preferably has a pH-value in the range of about 5 to 9, more preferably about 6 to 8, particularly preferably about 7. Furthermore, a rinsing solution can be provided. Said rinsing solution is preferably used to rinse the substrate after the biological object and/or non-biological object has been attached to the substrate in order to remove any non-attached objects from the substrate. The rinsing solution preferably corresponds to an aqueous solution such as water, phosphate buffered saline (PBS) or other buffered solutions such as Tris buffer, etc.
The growth medium is preferably used during the culturing of the biological objects that shall be attached to the substrate. When doing so the growth medium is added to a solution comprising the biological objects. Subsequently, said solution is washed, preferably by using a suitable buffer being known in the art. Thereafter, the at least one first compound is added to said solution, whereby the above-mentioned first dispersion is formed. Said first dispersion then comprises the biological objects, the at least one first compound, and the buffer. Then, said first dispersion is added to the substrate in order to attach the biological objects to the substrate. In a last step, the substrate comprising the attached biological objects is rinsed by the rinsing solution.
If a first solution comprising a growth medium is used, a growth medium known in the art is preferred. This could be, for example, at least one of pancreatic digest of casein, peptic digest of animal tissue, acid hydrolysate of casein, yeast extract, beef extract, starch such as corn starch, tryptone, peptone, dextrose and agar.
The first compound of the first solution preferably has a concentration in the range of between 0.0001% by weight to 10% by weight with respect to a total volume of the first solution, preferably between 0.001% by weight to 5% by weight with respect to the total volume of the first solution, particularly preferably between 0.02% by weight to 1% by weight with respect to the total volume of the first solution. Additionally or alternatively, the first compound of the first solution preferably has a concentration in the range of between 0.0001% by volume to 10% by volume with respect to a total volume of the first solution, preferably between 0.001% by volume to 5% by volume with respect to the total volume of the first solution, particularly preferably between 0.02% by volume to 1% by volume with respect to the total volume of the first solution. Additionally or alternatively the first compound of the first solution is preferably added to the first solution at a temperature between −20° C. to 120° C., preferably between 0° C. to 100° C., particularly preferably between 10° C. and 40° C. Here, the expression “volume with respect to the total volume” means “volume of the pure first compound per total volume of the first solution”.
The second compound of the second solution preferably has a concentration in the range of between 0.0001% by weight to 50% by weight with respect to a total volume of the second solution, preferably between 0.001% by weight to 5% by weight with respect to the total volume of the second solution, particularly preferably between 0.01% by weight to 2% by weight with respect to the total volume of the second solution. The second compound of the second solution preferably has a concentration in the range of between 0.0001% by volume to 50% by volume with respect to a total volume of the second solution, preferably between 0.001% by volume to 5% by volume with respect to the total volume of the second solution, particularly preferably between 0.01% by volume to 2% by volume with respect to the total volume of the second solution. Additionally or alternatively the second compound of the second solution is preferably added to the second solution at a temperature between −100° C. to 500° C., preferably between 0° C. and 100° C., particularly preferably between 10° C. and 40° C. Here, the expression “volume with respect to the total volume” means “volume of the pure second compound per total volume of the second solution”.
As indicated earlier, the second compound can be added to the substrate directly, i.e. in the absence of a second solution, or it can be provided together with a second solution. A preferred second solution is water. In the former case it is conceivable to apply the second compound to the surface of the substrate in the form of an aerosol, for example. In the latter case, it is conceivable to add the second compound to the second solution under standard pressure. However, depending on the chemical or physical characteristics of the second compound and the second solution, respectively, it might be preferred to add the second compound to the second solution under pressure and/or at high temperatures. For example, second compounds that are difficult to dissolve such as chitosan or APTES can be dissolved in the second solution, for example in water, at a pressure of between about 200 bar and at a temperature of about 370° C. Furthermore, more hydrophobic substances such as 4-ATP or APTES dissolve better in molten solids such as naphthalene or ethylene glycol. In order to melt these solids, temperatures above their melting points have to be applied. In the present example, said temperature should be above 197° C. in the case of ethylene glycol and above 218° C. in the case of naphthalene. Additionally or alternatively the second solution can comprise or consist of water or one or more solvents or mixtures thereof. The organic solvents are known in the art and can correspond to, e.g. ethanol or acetone and the like. An organic solvent is preferred in the event that the second compound is a hydrophobic compound.
As mentioned initially, it is conceivable to functionalize two or more parts of the surface of the substrate. In particular, at least a second part of the surface of the substrate can be functionalized, wherein a further first dispersion of a biological object and/or of a non-biological object in a further first solution is added to the functionalized second part of the surface, wherein the further first solution comprises at least one further first compound that differs from the first compound of the first solution that is added to the functionalized first part of the surface. Additionally or alternatively the functionalization of said second part of the surface of the substrate can differ from the functionalization of the first part of the surface of the substrate.
That is to say, it is conceivable to provide two or more first dispersions comprising two or more first compounds that differ from one another. To this end said two or more first compounds can differ in their chemical compositions. However, it is likewise conceivable that said two or more first compounds are chemically identical but differ in their concentration, for example. Additionally or alternatively it is conceivable to functionalize two or more parts of the surface of the substrate differently. For example, a first part could be chemically functionalized whereas a second part could be physically functionalized. However, it is likewise conceivable that both parts are chemically functionalized, wherein different second compounds are used for the surface treatment, or that both parts are physically functionalized, wherein the dimensions of their surface structures differ from one another. This procedure enables one to determine optimal compounds or conditions for the attachment of the biological objects and/or the non-biological objects in a rapid manner.
The substrate can be a flexible support, preferably a cantilever, a fibre such as a hollow fibre or a glass fibre, a membrane, a wire, a sponge, a flexible electrode, an integrated circuit, and a tuning fork. However, it is likewise conceivable that the substrate is a rigid support such as a glass cover slide, a ceramic tile, a rigid electrode, and a culture dish, for example. Additionally or alternatively the substrate can comprise a silicone-compound such as silicone dioxide or elementary silicone, plastic, ceramic, ceramic-metallic blend, a metal, a metal oxide or sulphide, and carbon such as graphite or diamond. The substrate preferably corresponds to a cantilever comprising or consisting of glass or quartz or to a silicone-chip.
Additionally or alternatively, at least part of the surface of the substrate can be coated with a coating prior to the functionalization of the surface of the substrate. The coating preferably comprising or consisting of at least one of a noble metal such as gold, a metal oxide such as titanium dioxide, a transition metal such as palladium, and a non-metal compound such as a nitride compound. For example, it is conceivable that part of the surface of the substrate, in particular a tip of the cantilever, is coated with a coating. A metal coating, for example, renders a flexibility of the substrate “tunable” as said coating increases the flexibility. A metallic surface, for example, has a better reactivity with reactive residues such as thiols. Metallic surfaces render the substrate conductive, making it an electrode, i.e. a sensor for other reactions such as the determination of a pH-value or the detection of redox compounds such hydrogen gas, quinones and so forth. Metal oxides or sulphides also confer further chemical properties to the substrate. Titanium dioxide, for example, can act as a catalyst in combination with ultraviolet radiation to kill microbes. Metal sulphides such as molybdenum sulphide can act as catalyst for redox reactions. Furthermore, oxides provide reactive surfaces that improve an attachment of the biological object and/or the non-biological object.
In any case it is preferred that after attachment of the object to the substrate the substrate comprises a layered structure. In particular, it is preferred that at least a first layer is arranged on at least a part of the surface of the structure, wherein said first layer is formed from at least a first dispersion as described above with reference to the method of attaching an object on a surface of the substrate. That is, the first layer is preferably formed from the first dispersion comprising the object, the first solution and the first compound. In fact, by adding the first dispersion to the substrate and subsequently incubating the substrate, a first layer can be formed. Said first layer can be directly arranged on the surface of the substrate. However, it is likewise conceivable that said first layer is indirectly arranged on the surface of the substrate. In fact, in this latter case it is conceivable that the surface of the substrate is functionalized, and wherein the first layer is arranged on the functionalized surface. For example, the functionalization of the substrate could be provided by means of a second layer being arranged on the surface of the substrate, and wherein the first layer in turn is arranged on said second layer. The second layer could be provided by the second solution comprising the second compound as described above, and wherein said second solution is allowed to solidify after its addition to the surface. For example, a solidification of the second solution could be achieved by allowing the second solution to dry.
A thickness of the first layer preferably is in the nanometer range to micrometer range or larger. For example, the first layer could have a thickness of 100 nanometer or more, for example of 1000 nanometer or more. Other thicknesses are however likewise conceivable and depend on the particular first compound, the particular object (the size of the object) the amount of first compound, etc. being used.
A thickness of the second layer preferably is in the nanometer range or larger. For example, the second layer could have a thickness of 10 nanometer or more, for example of 100 nanometer or more. Here, too, it should be noted that other thicknesses are however likewise conceivable and depend on the particular second compound, the amount of the second compound, etc. being used.
An overall thickness encompassing the first layer, the second layer and the objects attached thereby is preferably in the micrometer range or larger.
In the following, preferred examples for the first solution and the second solution and their application during the attachment of a biological object on a substrate are given.
For example, the biological objects can correspond to Escherichia coli ATCC 25922 strain or to Klebsiella pneumoniae ATCC 27736 strain that are grown on Columbia medium (contains sheep blood) agar plates at 37° C.
The Columbia agar ingredients are the following:
The pH-value is 7.3±0.2.
Once per week the transferred strain is renewed to reduce the risk of mutations. That is, a frozen −80° C. culture is defrosted and plated on Columbia agar. Cells for attachment tests are taken from these plates, starting with the first plate-to-plate transfer. To harvest the cells, a fair amount of material is scraped off the agar surface using an inoculation loop and used to inoculate 3 ml of lysogeny broth (LB), see below. This step stimulates the activity of the cells and can be omitted if this is not necessary. After 20 minutes of incubation at 37° C., the cells are precipitated by centrifuging at 5,000 rpm and re-suspended in 1 ml PBS at pH 7.4. In other cases, longer stimulation periods are necessary. The cell material is then washed in PBS by centrifuging 4 times at 5,000 rounds per minute and re-suspending the cells again. After the fourth centrifuging, the cells are re-suspended in 200 microlitre PBS.
A different number of centrifuging steps may be used, depending the cell material and the medium. The optical density (OD, wavelength of 600 nm) of the washed suspension is between 1.0 and 1.3. This OD600 corresponds to a McFarland standard between 8 and 15. To measure the corresponding McFarland turbidity, the cell suspension has to be diluted 1/10 in 0.85% NaCl. The OD may also be lower or higher or determined at a different wavelength. If a higher cell concentration is needed, the cell suspension is precipitated again and re-suspended in a lower volume of the buffer, for example in ⅕th. Starting from the final dilution, cell forming units (CFU) are estimated using Mueller-Hinton agar plates as shown below. While in this example cells of E. coli are used, other cells, tissues, organisms, or viruses may be used as well. For example, yeasts such as Saccharomyces cerevisiae may be used. Cells of S. cerevisiae are grown on yeast extract peptone dextrose agar (YPD, see below) overnight. The cells are then stimulated for 2 h in the YPD medium and harvested in the same fashion as described above with reference to the E. coli ATCC 25922 strain.
The LB medium ingredients are the following:
The pH-value is 7.0±0.2.
The Mueller-Hinton agar ingredients are the following:
The pH-value is 7.3±0.1.
The yeast extract peptone dextrose agar (YPD) medium ingredients are the following:
The pH-value is 6.5±0.2.
The first compound and its usage in the above examples 1 to 7 can be replaced by a first compound according to one of the below examples 8 to 11.
In one example polydimethylsiloxane (PDMS) was used as silicone defoamer.
In another example, Nafion™ was not diluted prior to the addition of the cell suspension. Instead, Nafion™ was diluted to a dispersion having a final concentration of 0.25% (w/v) Nafion with the cell suspension. In this case, water served as the solvent instead of 2-propanol or ethanol.
The biological object suspension is removed and the result is verified using a microscope. If desired, steps (7) and (8) are repeated.
Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,
In fact, the substrate 1′ depicted in
As has already been mentioned in the introduction, the attachment of biological objects is of great interest in the field of nanomotion AST, for example. In doing so the biological objects can be attached to a flexible support such as a cantilever, and wherein the movements of the cantilever that are caused by the attached biological objects are measured.
As has already been discussed in great detail above, the inventors have found out that the use of a first solution comprising at least one of a gelling agent, gellable agent, and a thickening agent in combination with a functionalized surface 3, 3′ of the substrate 1, 1′ results in an improved attachment of the biological object as compared to the attachment methods known in the state of the art.
Namely,
The same findings are found with respect to
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Regarding the attachment of other objects such as DNA the following is noted. DNA is negatively charged just as Gram-neg and Gram-pos bacteria. DNA backbone is constituted of phosphate groups which are negatively charged. In Gram-positive bacteria the reason of this negative charge is the presence of teichoic acids linked to either the peptidoglycan or to the underlying plasma membrane. These teichoic acids are negatively charged because of presence of phosphate in their structure. The Gram-negative bacteria have an outer covering of phospholipids and lipopolysaccharides. The lipopolysaccharides impart a strongly negative charge to surface of Gram-negative bacterial cells. The addition of a first compound according to the invention, i.e. the addition of a gelling agent and/or gellable agend and/or thickening agent, will help with the DNA distribution and the attachment on the substrate. Besides, it is noted that polysaccharides such as agar and agarose are commonly used in laboratories working with DNA already in these days, wherein agar and agarose are used to create hydrogels allowing DNA extraction and verification, for example.
Number | Date | Country | Kind |
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PCT/EP2019/086974 | Dec 2019 | EP | regional |
Filing Document | Filing Date | Country | Kind |
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PCT/EP2020/087821 | 12/23/2020 | WO |