Patent Application
20110046013
The invention relates to a lithographic method for generating micro- or nano-patterns of biomolecules by controlling locations and amounts of a multiplicity of different biomolecules. More particularly, the invention is directed to a method to obtain predetermined patterned surfaces for bioanalysis, diagnostics, drug delivery, gene delivery, and/or cell differentiation.
The construction of microarrays and use of such microarrays in complex analysis has been employed for almost 20 years. Various techniques for providing such arrays are known in the art, with varying levels of efficiency, compactness, and sensitivity. Such microarrays or nanoarrays are typically used to analyze compositions containing proteins, nucleic acids, small molecules, and the like. There is an extensive literature on preparing the micro or nanoarrays, and the use of lithographic techniques has been exploited. For example, dip pen approaches, nanografting, and electron beam lithography have been used. All of these are serial point-to-point techniques which are limited by their low speed. Nanocontact printing and nanoimprint lithography have been used to generate biomolecular nanopatterns, but it is difficult to generate heterogeneous patterns with a multiplicity of different biomolecules using these techniques. Other techniques, such as bottom-up self-assembled particles, copolymers and the use of oligonucleotides to bind the relevant biomolecules, have also been used.
The present invention offers an efficient, scalable technique to generate complex patterns of biomolecules which technique controls locations and amounts of a multiplicity of different biomolecules.
The present invention is a high-speed, high-throughput, low-cost technique to generate microscale or nanoscale patterns composed of multiple different biomolecules. Distinct biomolecules are optionally conjugated with micro- or nano-particles to form bioconjugates, and the particles or biomolecules can be selectively assembled onto a template surface to form micro- or nano-scale patterns with controllable biomolecular types, densities, and amounts on each pattern. The biomolecular micro- or nano-patterns can be printed from the resulting template to a biocompatible matrix or product film for various biological and medical applications.
Thus, in one aspect, the invention is directed to a method to provide a template which comprises a support having a multiplicity of portions arranged in a master pattern with a multiplicity of different biomolecules associated with the different portions of the support.
The different biomolecules are provided in the form of biomolecules coupled to nanoparticles or microparticles to form bioconjugates. The bioconjugates are coupled to the various portions in the master pattern by virtue of an attractive force applied to these portions and to which the bioconjugates or biomolecules are responsive. The attractive force may be an electrical force, a magnetic force, a hydrophilic interaction, hydrophobic interaction, molecular interaction, or combinations thereof.
The bioconjugates may be responsive by virtue of the nature of the particles, but the inherent properties of the biomolecules may themselves provide the appropriate response. For example, if the attractive force is an electrical force, a negatively charged molecule, such as DNA may provide the response to a positive gradient applied to the relevant portion. In any case, magnetic and electrically charged nanoparticles and microparticles are well known in the art as will further be described below.
There are a number of ways that the process itself may be performed. For suitable dimensions of the master pattern, microfluidics may provide a means whereby suspensions containing the various bioconjugates can be individually directed to individual portions simultaneously. Alternatively, the bioconjugates may be supplied sequentially by providing a first suspension containing a first bioconjugate which is then subjected to an attractive force applied to a first portion of the micropattern. Any bioconjugates or biomolecules that are not entrapped are then washed away, and a second suspension containing a second bioconjugate is supplied and an attractive force applied to a second portion of the micropattern, and so on.
These methods apply regardless of the choice of electrical, magnetic, hydrophilic or hydrophobic interactions, or molecular interactions. In the embodiments wherein a magnetic field is used as the attractive force, for preparing templates that will contain coated portions with diameters of the order of millimeters, as opposed to microns or nanometers, the dimensions of the coated portions may be determined without pre-patterning simply by applying magnetic fields only to certain portions of the support. Again, either simultaneous application through appropriate individual channels to individual portions or sequential application to the various portions can be employed. In the embodiments wherein a hydrophilic or hydrophobic interaction is used as the attractive force, the wetting property of the surface is alternated between hydrophilic and hydrophobic. Electrowetting is a method to change the surface wetting property by an applied electric field. In the embodiments wherein the molecular attraction is used as the force, templates contain portions containing molecules that interact or bind specifically to supplied bioconjugates. These may be members of specific binding pairs such as antibodies or fragments thereof and antigens, complementary oligonucleotides, ligand receptor pairs and the like.
In another aspect, the invention is directed to transferring a pattern of biomolecules contained on a template as described above (comprising a support composed of multiplicity of different biomolecule-conjugates or biomolecules entrapped at an upper surface of each of a multiplicity of portions of the support) to a matrix, which method comprises casting said matrix onto the upper surface of said biomolecule conjugate-populated support or template, effecting transfer into the matrix, and entrapping the patterned biomolecule conjugates or biomolecules into a product film formed by said matrix and then removing said film.
The matrix can be crosslinkable polymer or hydrogel, which is cast in an un-crosslinked form onto the upper surface of the biomolecule conjugate-populated support. Bonding interaction between the bioconjugates or biomolecules and matrix then occurs. The forces between the matrix and the particles are stronger than the forces between the particles and the portions on the template. The bioconjugates or biomolecules are entrapped by crosslinking (chemical or physical) the polymer or hydrogel by UV exposure, heat, physical association or chemical reactions to form the product film. When the film is separated from the support, the bioconjugates or biomolecules in the pattern on the template are retained in the product film. The template containing the master pattern can then be reused in the process outlined above.
In one embodiment, transferring a pattern of biomolecules contained on a template to a matrix can be achieved by applying an electric field. Portions of the template are made of conductive materials. After the template is prepared, it is covered by a matrix, such as a chemically crosslinked, or physically associated polymer or hydrogel, for example, an agarose gel. The matrix can also be an uncrosslinked or unassociated polymer or hydrogel, and after being cast onto the template, the matrix is crosslinked or associated. A conductive plate is then added as a counter electrode to generate parallel electric fields inside the matrix. The matrix is sandwiched between the portions and the counter electrode, creating a gel electrophoresis apparatus. The bioconjugates migrate into the matrix and also migrate inside the matrix under the influence of the electric field.
In another embodiment, transferring a pattern of biomolecules contained on a template to a matrix can be achieved by pressing a solid plate onto the top surface of the template. After the bioconjugates attached to the solid plate by chemical crosslinking or physical association, the plate is removed from the template and the bioconjugates or biomolecules in the pattern are transferred to the said solid plate.
In another aspect, the invention is directed to the products of the processes set forth above and to the combination of these two processes.
Both the product film and the template may be used for analysis of samples by detecting binding of components of these samples to the various biomolecules displayed according to the master pattern using techniques well known in the art. For example, if the biomolecules are a variety of proteins and the product film is used to analyze a fluid for human antibodies to these proteins, binding of any human antibodies from, the sample could be detected at the various locations by use of antihuman antibodies provided with labels.
Both the bioconjugates or biomolecules patterned on the portions of the substrate and the bioconjugates or biomolecules patterned in the matrix (after transfer) can be used for bioanalysis, diagnostics, drug delivery, gene delivery, cell differentiation and morphogenesis, tissue regeneration, and the like.
a-1e show one example of a schematic for particle printing lithography.
a-4f show an alternate scheme for preparing a product film with a pattern of biomolecule conjugates.
a-5d show atomic force microscopy (AFM) images of three different types of nanoparticles assembled sequentially onto three 70 nm-wide Pt nanoelectrodes as the boundaries of the characters “1”, “2”, and “3”. The nanoelectrodes are shown as lines, and the nanoparticles are shown as bright dots in the AFM images. The assembled nanoparticles are then transferred from the nanoelectrodes to a polymer film.
a-6b show a fluorescence microscopy image (6(a)) and corresponding scheme (6(b)) for the three different types of nanoparticles labeled with red, green, and blue fluorophores that had been sequentially assembled on nanoelectrodes on a stamp; transferred onto a polymer film; and incubated with biotinylated BSA and then streptavidin labeled with orange fluorophore.
a-7h show the results of the magnetic electric lithography (MEL) process illustrated in Example 2 below.
a and 8b show treating the entrapped biomolecule conjugates in the product film with labeled probes to demonstrate retention of activity.
a-9d show the results of patterning on a nanometer scale using gold electrodes providing labeled polymeric film product alongside a scheme of the process.
a-10c show the process (
a and 11b show the densities of microparticles that can be obtained as immobilized on a master pattern by applying different voltages and durations.
The methods of the invention can conveniently be designated particle printing lithography (PPL). PPL is a micro- and nano-scale lithographic technique for biomolecular patterning.
Particles coupled to a desired biomolecule, i.e., biomolecule conjugates, contained in a suspension are supplied to the upper surface of the support or “stamp” and a suitable force, e.g., electrical or magnetic, is applied from the portions of the master pattern.
In the embodiment shown in
The template in
In one embodiment, the assembly of the particles onto the master patterns on the template can be controlled by applying electric fields on portions of conductivity arranged in a master pattern as diagrammed in
In another embodiment, magnetic forces are applied. The assembly of the particles onto the master patterns can also be achieved by applying a magnetic field, just as the electrical force can be focused successively on each portion to be coated with each different biomolecule, so too can the magnetic field. The magnetic field on the portions can be selectively activated and deactivated by the local external magnetic field generated by a system such as a magnetic writing head used in the magnetic memory system. The ferromagnetic or paramagnetic particles in suspension on the upper surface of the support can be attracted and immobilized to the portions by the activated magnetic field. The magnetic (ferromagnetic or paramagnetic) particles can also be electrically charged by coating electrically charged molecules on the surfaces of the particles, so a repulsive force between the particles will avoid the formation of the clusters of the magnetic particles. After a single layer of the close-packed desired biomolecules conjugated with magnetic particles is assembled on the selected portions, the electric repulsive force between the charged magnetic particles can avoid assembly of the other magnetic particles on the top of the assembled magnetic particles to avoid mixture of the magnetic particles with different biomolecules. The different biomolecule conjugates may be assembled to the different portions on the said support sequentially by activating the magnetic field on the different corresponding portions.
The magnetic field can also be generated by electrically conductive coils embedded in the support underneath the portion. The magnetic field and its gradient are generated near the coil by passing electric current through the coil. The magnetic particles in the suspension that on the upper surface of the support are thus attracted and immobilized to the portions by the magnetic field or gradient of the magnetic field.
In another embodiment, hydrophilic forces are applied. The support is made to be hydrophobic. The surface wetting property of each portion can be patterned by applying an electric field. When the aqueous liquid containing bioconjugates or biomolecules is applied to the support, the aqueous liquid will go to the hydrophilic portion.
This can be employed in reverse by supplying a hydrophobic liquid containing the desired bioconjugates in an oil or other hydrophobic fluid.
In another embodiment, molecular interactions are applied as attractive forces. Each portion is composed of one type of molecule or molecular combination. These capture molecules can specifically bind to their target complementary molecules in the supplied bioconjugates or biomolecules. The supplied bioconjugates are sorted according to the molecules on each portion. This embodiment employs specific binding pairs such as antigen/antibody, receptor/ligand, or complementary oligonucleotides.
The molecular interactions can be combined with electric or magnetic forces, or hydrophilic or hydrophobic forces. The electric magnetic force or hydrophilic or hydrophobic interaction attracts the bioconjugates to the vicinity of portions containing capture molecules—members of the bonding pairs; then the bioconjugates containing target complementary molecules can be captured by the capture molecules through molecular interactions and immobilized on the portions.
As with the use of electrical forces, the patterning process can be implemented by introducing a first suspension containing a first desired biomolecule conjugate to the upper surface of the support to assemble the biomolecules to a first portion, and then repeated with second, third, etc., suspensions to form patterns of different biomolecules on the different portions on the support. The same implementation can be used when using magnetic force, hydropilic/hydrophobic force, or molecular interaction as attractive force.
As stated above, in either case, by employing appropriate microfluidic techniques, individual suspensions of different bioconjugates may be supplied simultaneously to individual portions of the master pattern and the appropriate force applied to the various portions of the master pattern. The multiple portions of the master pattern can, for example, be controlled by electric circuits embedded in the support. The sizes of the biomolecular patterns can be assembled on the template in parallel over an area range between 100 nm2 to 100 cm2.
This is illustrated in
A slight modification of the process described above is found in
Transferring bioconjugates to matrix can also be achieved by applying electric fields in a process similar to electrophoresis. A 3D pattern inside the matrix can be obtained using this method. The portions of the template from which transfer is to occur are made with conductive materials. After the bioconjugates are immobilized on the portions, the template is contacted with the matrix which is chemically crosslinked, or physically associated polymer or hydrogel, for example, an agarose gel. The matrix can also be an uncrosslinked or unassociated polymer or hydrogel and subsequently crosslinked or associated. A conductive plate is then added as a counter electrode to generate parallel electric fields in the matrix between the template and the plate. The magnitude and duration of the electric field applied will depend on the pore size of the polymer or the hydrogel, the size of the bioconjugates, and the destination position of the bioconjugates inside the matrix. This system works as a conventional gel electrophoresis apparatus. After transferring, the matrix is removed from the support which can then be reused.
Transferring bioconjugates to matrix can also be achieved by pressing a solid plate onto the template. The bioconjugates or biomolecules then chemically react or physically associated with the solid plate. For example, thiol containing biomolecules react with Au plate to form S—Au bond. The solid plate can then be removed from the template with the bioconjugates or biomolecules attached to it. After transferring, the support can be reused.
As further described below, if the bioconjugates comprise particles or polymers coupled to biomolecules through a cleavable linker, the template can be subjected to a cleavage agent or condition and only the biomolecule portion of the conjugate transferred to the film.
The sizes and shapes of the biomolecule-conjugate patterns are determined by the sizes and shapes of master patterns of the portions that permit effectiveness of the attractive force, i.e., the pattern of relevant portions contained on the support, and by the sizes of the particles.
The pattern of the biomolecules themselves also depends on the size and shape of the particles to which they are conjugated and the number of biomolecules contained on each particulate support.
The distribution of the densities of the distinct biomolecules assembled on each master pattern is determined by controlling the assembly conditions such as the concentrations and types of the distinct particles in the solution, and the duration and magnitude of the assembly forces.
As used herein, the term “substrate” or “support” refers to the material that forms the supporting platform corresponding, for example, to 14 in
“Master pattern” refers to the pattern of portions responsive to the attractive force displayed on or within an upper surface of the support or substrate, in which one or more attractive force can be applied to attract and immobilize the bioconjugates from the suspension.
Portions on the support to which an electrical force, an electrostatic force, an electrophoretic force, an electromagnetic force can be applied are made from conductive materials including but not limited to Au, Pt, Ag, Ti, Si, TiO2, or semiconductors including but not limited to Si, Ge, GaAs and GaN. Portions on the support to which a magnetic force can be applied are themselves magnetic and composed of, for example, Fe3O4, Co, Ni, Dy, Gd or NIB. Portions on the support in which hydrophilic interactions occur are coated with hydrophilic materials such as polyethylene glycol (PEG) or hydrophilic proteins. The portions can also be made hydrophilic by patterning certain nanopatterns on it. Portions that provide for hydrophobic interaction are coated with hydrophobic materials such as waxes or Teflon®. Portions on the support where molecular interaction occurs are made by immobilizing the capture molecules on the portions by chemical or physical bonding.
The support itself, where the force is electrical, may be constructed of, for example, SiO2 and Si3N4 or other insulators. Use of magnetic force requires a support made by non-magnetic material, such as insulators (SiO2, Si3N4), metals (Au, Pt), semiconductors (Si, GaN), or polymers (PMMA, PDMS). Use of hydrophilic interaction requires a support made with hydrophobic material such as PDMS, Teflon®, or coated with hydrophobic molecules. This pattern may also be used to take advantage of hydrophobic interactions by use of hydrophobic solvents for the bioconjugate.
The sizes in the portions of the master pattern range between 1 nm to 1 cm. The master patterns on the template surface can be made from lithographic techniques including but not limited to optical lithography, X-ray lithography, e-beam lithography, imprint lithography, ion-beam lithography, dip-pen lithography, contact printing lithography, nanografting lithography, etc, or self-assembly techniques.
“Biomolecule conjugate” or “conjugated biomolecule” or “bioconjugate” refers to a molecule of interest coupled to an appropriate microparticle or nanoparticle. The “biomolecule” should be understood to include not only molecules that occur in nature, but also any organic molecule of interest. The term “bio” molecule is used for convenience as in most (but not all) applications, the molecules concerned will be those that either occur in nature or are relevant to interacting with biological systems.
The biomolecules assembled onto the particles include DNA, RNA, proteins, antibodies, antigens, drug molecules, and any other organic molecules of interest.
The micro- or nanoparticles may be composed of a variety of materials and may range in size from 1 nm-100 μm. For most biological applications, the particles will be in the range of 10-600 nm. A variety of particles is well known in the art, including magnetic particles, metal particles, oxide particles, complex particles with core/shell structure, biomolecular particles, virus, cells, liposomes, perfluorocarbon particles, etc. Microparticles include microspheres, microbeads and any particles measured on a micro scale regardless of shape. Nanoparticles include nanospheres, nanocrystals, nanorodes, nanburgers, and the like, i.e., any particles measured on a nanoscale regardless of shape, and quantum dots.
Biomolecules can be conjugated with ferromagnetic or paramagnetic particles made by magnetic materials (e.g., Fe3O4, Co, Ni, Dy, Gd, NIB), wherein when the size of the magnetic particles are reduced below a critical value, the ferromagnetic particles become paramagnetic particles. The magnetic (ferromagnetic or paramagnetic) particles can also be electrically charged by coating electrically charged molecules on the surfaces of the particles, therefore the electrical repulsive force between the particles will avoid the formation of the clusters of the magnetic particles due to the attractive magnetic force between the particles.
The micro- or nanoparticles for use in the invention may be coupled only to a single biomolecule, to a multiplicity of identical biomolecules, or to a multiplicity of different biomolecules. In some cases the desired property to effect attraction to the conductive portion may be provided by the biomolecule itself or by coupling the biomolecule with a charged substance other than the particle. For example, DNA is negatively charged at neutral pH.
The micro- or nanoparticles of the invention may be coupled to a biomolecule through a linker. The linker is typically bifunctional having a functional group for coupling directly to the substrate and a functional group for coupling to the biomolecule. Means for coupling biomolecules to particles through such linkers are well known and linkers are commercially available, for example, from Pierce Chemical Company. The linkers may include a cleavage site so that if desired, the biomolecules may be released, for example, during the process of transferring the pattern of the template onto a film for further use. Thus, the bioconjugates may themselves be transferred, or the biomolecules may be cleaved and transferred absent the particles. Cleavage sites include those susceptible to light, those susceptible to pH value, and those susceptible to enzyme cleavage. Thus, the linker might be a peptide containing a cleavage site for trypsin or a polysaccharide containing a cleavage site for a specific polysaccharide hydrolysis enzyme. Other cleavage sites independent of enzymes, pH value, or light may include those specifically cleavable chemically, such as disulfide bonds which are cleavable with reducing agents. A variety of such sites is available for use in the invention.
It should be noted that in some embodiments biomolecular may be used without the need to couple them to particles. In the general description herein, “bioconjugate” includes both biomolecules alone and biomolecules coupled to particles. This simplification is to avoid excess verbiage; as that it is not necessary always to say “bioconjugate or biomolecule.”
“Template” refers to the completed arrangement of biomolecule conjugates on the upper surface of the support according to the master pattern thereon, such as shown in
“Matrix” refers to an un-crosslinked polymer or hydrogel composition used to obtain the transferred pattern from the template to produce ultimately the “product film” which reflects the master pattern populated with biomolecule conjugates and which then may be used in various assays. In general, “product film” refers to this useable result of the invention method.
The crosslinkable matrix can be polymers or hydrogels made from poly(ethylene glycol) (PEG), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), hyaluronic acid (HA), gelatin, polysaccharides such as agarose, etc. The polymers or hydrogels are cast onto the template, and cross-linked, for example, by UV exposure, thermal curing, physical association, or chemical reaction. After the crosslinking, the polymers or hydrogels entrap the patterned biomolecule conjugates. When the cross-linked polymer or hydrogel product films are peeled off from the template, the patterned biomolecule conjugates are removed from the template to the product film. The master patterns on the support can thus be reused repeatedly.
The matrix can be organic or inorganic molecules that can bond with biomolecule conjugates in the template, such as glasses, amorphous materials, insulators, metals or semiconductors.
In an illustrative example, similar to the sequence in
During the assembly and immobilization step, the assembly speed and density of conjugates can be controlled. The first of conjugate DNA/particle conjugate, is delivered to the master pattern at the upper surface of the support by a fluidic channel. A magnetic field is generated to bring the conjugates to the vicinity of master pattern. This short step increases the local concentration of the conjugates. The magnetic field is then removed, and an electrical potential (usually +1.0V˜2.1 V vs. counter electrode if using two-electrode system, or +0.7V˜2.1 V vs. reference electrode if using three-electrode system) is applied on a first conductive portion of the master pattern for 40-600 s to immobilize the conjugate. The larger the electrical potential and the longer the time duration, the higher the density of the conjugate immobilized on master patterns. Excess conjugate is removed by washing. The second DNA particle conjugate is then delivered to the master pattern area, the magnetic concentration is repeated, and these conjugates immobilized to a second portion of the master pattern. Hybrid portions can also be made if both types of conjugates are immobilized to the same portion. Excess conjugate is removed by washing. The aforementioned steps are repeated until all the portions contain immobilized pre-designed conjugates.
Once the assembly process is finished, all the biomolecule-conjugates can be transferred to a polymer or hydrogel, such as the hydrogel formed by PEG-DA (polyethylene glycol diacrylate) and PBS buffer containing, e.g., 1% photoinitiator. The hydrogel concentration can be controlled by varying the percentage of the two components. The hydrogel solution is cast onto the master pattern with the immobilized biomolecule-conjugates. By irradiation with UV, the polymer is crosslinked within 10 s. The crosslinked film is peeled off from the master pattern, leaving the clean pattern for the next use.
As noted above, the complete biomolecule conjugates may be transferred to the film or, if desired, the biomolecules may be cleaved from the particles in only the biomolecules transferred.
Some of the advantages of the present invention are that heterogeneous biomolecular nanopatterns can be generated with high speed, high throughput, high resolution and low cost, and can employ all kinds of biomolecules, such as proteins, nucleic acids, small molecules. The process is substantially independent of the nature of the biomolecules. Therefore, no special protocol is needed for each different set of biomolecules and can employ a large variety of materials, depending on the desired applications.
The biomolecular patterns may contain distinct biomolecules, ranging between 2 to 107 different kinds of biomolecules, and can generate biomolecular patterns with a high speed. The assembly process can be completed within 0.1 s to 10 hours.
The following examples are offered to illustrate but not to limit the invention.
Metal nanoelectrodes with a width of ≈70 nm were fabricated by e-beam lithography on a 100 nm-thick SiO2 layer on a 1 inch×1 inch Si stamp. The nanoelectrodes composed of 20 nm-thick Pt (top) and 5 nm-thick Ti (bottom) layers were deposited by e-beam evaporation. The electric connections with the nanoelectrodes were established by microscale Pt/Ti metal wires fabricated by optical lithography on the same substrate. The metal connection wires were covered with a 300 nm thick SiO2 insulating layer by plasma-enhanced chemical vapor deposition (PECVD). To prevent nonspecific binding of nanoparticles to the SiO2 surface on the stamp, PEG silane was grafted onto the SiO2 surface by incubating the stamp in a silane solution consisted of 0.5% methoxy(polyethyleneoxy) propyltrimethoxysilane (SIM6492.7, Gelest, Inc.) and 0.05% triethylamine in anhydrous toluene in a desiccator for 1 h. To remove excess PEG silane on the stamp surface, the stamp was rinsed with toluene for 2 min, and sonicated in a toluene bath for 1 min. The stamp was rinsed consecutively with isopropanol and DI water for 30 s, and finally dried by blowing with nitrogen.
Five different types of negatively charged polystyrene nanoparticles were used:
Type I: 40 nm nanoparticles coated with streptavidin and labeled by green fluorophores with excitation/fluorescence peak wavelengths at 505 nm/515 nm (F8780, Invitrogen);
Type II: 40 nm nanoparticles coated with biotin and labeled by green fluorophores with excitation/fluorescence peak wavelengths at 505 nm/515 nm (F8766, Invitrogen);
Type III: 20 nm nanoparticles labeled by red fluorophores with excitation/fluorescence peak wavelengths at 580 nm/605 nm (F8786, Invitrogen), and coated with DNA sequence 5′-AAAAAAAAAAAAGGGGGGGGGGGG-3′;
Type IV: 54 nm nanoparticles labeled by red fluorophores with excitation/fluorescence peak wavelengths at 660 nm/690 nm (FS02F-7102, Bangs Laboratories, Inc.);
Type V: 60 nm nanoparticles labeled by blue fluorophores with excitation/fluorescence peak wavelengths at 360 nm/420 nm (FS02F-2598, Bangs Laboratories, Inc.).
In this example, the metal electrodes (portions) were arranged on the stamp as shown in
The nanoparticle/biomolecule patterns were then printed from the stamp to a polyethylene glycol diacrylate (PEG-DA) polymer film by immersing the stamp surface in the PEG-DA solution (n=400, Polysciences, Inc.) covered by a glass substrate. This solution consisted of 80% PEG-DA, 17% phosphate buffered saline, pH 7.4, 0.01 M, 2% bis (2,4,6-trimethyl-benzoyl)-phenylphosphineoxide (Irgacure™ 819, Ciba) as a photoinitiator and 1% Tween 20 as surfactant. The PEG-DA solution was cured by UV light with a UV intensity of 11.0 mW cm−2 for 4 s. The fluorescence images were taken using a fluorescence microscope (Nikon Eclipse E400) with different filter sets. The nanoscale structures were observed by an AFM (Digit Instruments Dimension 3100) and an SEM (JEOL S-4300).
After the polymer solution was cured, the nanoparticles were transferred to the solidified PEG-DA file by peeling off the polymer film from the stamp. The AFM image of the transferred image is shown in
The streptavidin coupled to the particles that form numeral 2 was still active. The PEG-DA film was soaked in 30 μg/ml biotinylated BSA (more than 8 mol biotin per mol BSA) (29130, Pierce) in a 0.01 M PBS solution for 30 min. After rinsing, the film was incubated in a PBS solution consisting of 10 μg/ml orange Alexa Fluor®-546 coupled to streptavidin (with excitation/fluorescence peak wavelengths at 546 nm/573 nm), 0.1% BSA and 0.02% Tween 20 for 30 min.
In
Thus, the results confirm that streptavidin remains active.
Water-soluble iron oxide (Fe3O4) magnetic nanoparticles (MNPs) with an average diameter of approximately 10 nm were synthesized and capped with positively charged 2-pyrrolidinone (Li, Z., et al., Chem. Mater. (2004) 16:1391-1393), and a negatively charged poly(styrene sulfonate) (PSS) layer was then self-assembled onto the MNPs (Schneider, G., Nano Lett.) 2004) 4:1833-1839). Streptavidin was physically adsorbed to the PSS-coated MNPs (Norde, W., et al., J. Colloid Interface Sci. (1979) 71:350-366), and three different biotinylated DNA oligonucleotides (S1, S2, and S3, Table 1) were conjugated with the streptavidin on the MNPs.
[a]The maximum excitation wavelengths.
[b]The maximum emission wavelengths.
The cross-linkable polymer solution for MNP transfer contains 66 wt % poly(ethylene glycol) diacrylate (PEG-DA, n=400), 3.0 wt % 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone as photoinitiator, 30 wt % phosphate-buffered saline (PBS), and 1 wt % surfactant (Tween 20). The PEG polymer solution was cross-linked by UV exposure with an intensity of 11.0 mW cm−2 for 30 seconds to form PEG hydrogel. After the MNPs were transferred to the PEG polymer substrate, complementary DNA strands (F1, F2, and F3, Table 1) were hybridized with the DNA (100 nM) on the MNPs in a buffer solution (1 M NaCl, 10 mM 2-amino-2-hydroxymethyl-propane-1,3-diol (tris) HCl, 1 mM ethylenediamine tetraacetic acid (EDTA), and 0.01% (w/v) sodium dodecyl sulfate (SDS), pH 7.4) at room temperature (25° C.) for 1 h. The biotin was conjugated with the streptavidin on the MNPs in a solution of 10 μg ml−1 biotin in PBS solution for 1 hour.
Method and Results
Gold nanowire electrodes were used to form the boundaries of the letters M, E and L on a SiO2 surface (
After the MNPs were transferred to the PEG polymer substrate as described above, the AFM image of the template surface (
The template surface morphology was faithfully embossed onto the polymer product film (
The bioactivity and specificity of the biomolecular nanopatterns on the polymer substrate was demonstrated by exposing the polymer substrate to a mixed solution of three different fluorophore-labeled DNA oligonucleotides (F1, F2, and F3 in Table 1) that are complementary to the aforementioned S1, S2, and S3 DNA strands coated on the MNPs. A superimposed fluorescence image of the complementary DNA nanopatterns after DNA hybridization is shown in
To demonstrate the high resolution of magnetic electric lithography (MEL), a template consisting of two parallel 8 nm wide Au nanoelectrodes was fabricated on a SiO2 surface. Streptavidin-coated single MNPs with a diameter of approximately 10 nm were immobilized and aligned in a row along the Au nanoelectrodes. The MNPs were coated with streptavidin labeled with green fluorophores (Alexa Fluor®-488); therefore, a green fluorescent line can be observed along the nanoelectrodes by the fluorescence microscope, but the double nanoelectrodes cannot be distinguished owing to the limitation of the microscope resolution. After the MNPs were transferred to a PEG polymer substrate, the streptavidin units on the MNPs were treated with biotin labeled with a red fluorophore (Atto-590). A red fluorescent line can then be observed along the nanoelectrodes. The experimental results indicate that MEL can generate biomolecular nanopatterns with a resolution down to approximately 10 nm. The resolution is defined by the sizes of the MNPs and of the nanoelectrodes on the template. This is illustrated in
To explore biomolecular nanopatterns by a parallel MEL process over a large area, a template was fabricated with an array of nanoholes through a SiO2 layer over an area of approximately 0.5 cm2, and microscale Au electrodes buried underneath the SiO2 layer were exposed to the template surface through the nanoholes (
During the electrophoretic deposition of nanoparticles onto nanoelectrodes on a stamp surface, the density of the nanoparticles assembled on the surfaces of the nanoelectrodes can be modified by adjusting the magnitude and duration of the potential applied on the electrodes. For example, the streptavidin-coated polystyrene nanoparticles were assembled on Pt nanoelectrodes. In
Bioconjugates are prepared by adding 3.6 μl of a plasmid for expression of EGFP (1 mg/ml), 13 μl linear PEI (1 mg/ml) to 65 μl DI water, and mixing by sonication. The hydrodynamic diameters of the conjugates are around 30 nm measured by Zeta nanosizer. The surface potential of the conjugates is around +30 mV.
The bioconjugates (EGFP plasmid/PEI polyplexes) were assembled and immobilized on an Au electrode. Uncured PDMS (RTV-615 A:B=10:1, General Electric) was cast on the chip. Once cured, polyplexes were transferred to PDMA and the PDMS film was peeled off. The PDMS was coated with fibronectin and cells are grown on the surface of PDMS. After 48 hours, the cells grew only on the polyplexes showing bright green fluorescence, from the expressed GFP proteins.
This application claims benefit of U.S. application Ser. No. 61/227,372 filed 21 Jul. 2009. The contents of this application are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61227372 | Jul 2009 | US |
