Patent Application
20190030530
The invention is related to the area of micro- and nanocontact printing. In particular, it is related to micro- and nanocontact printing with aminosilanes: patterning surfaces of microfluidic devices for multi-plexed bioassays.
Since the early 1980's, microfluidic systems have advanced significantly to satisfy the growing demand for the miniaturization of bioassay devices with applications ranging from disease diagnostics (W. Su, X. Gao, L. Jiang, and, J. Qin, Journal of Chromatography A, 2015, 1377, 13-26; D. G. Rackus, M. H. Shamsi and A. R. Wheeler, Chemical Society Reviews, 2015, 44, 5320-5340.; M. Karle, S. K. Vashist, R. Zengerle and F. von Stetten, Analytica Chimica Acta, 2016, 929, 1-22.; V. C. Rucker, K. L. Havenstrite, B. A. Simmons, S. M. Sickafoose, A. E. Herr and R. Shediac, Langmuir, 2005, 21, 7621- 7625.) to cell behavior studies (G. Du, Q. Fang and J. M. den Toonder, Analytica Chimica Acta, 2016, 903, 36-50.; A. Karimi, D. Karig, A. Kumar and A. Ardekani, Lab on a Chip, 2015, 15, 23-42.; N. D. Gallant, J. L. Charest, W. P. King and A. J. Garcia, Journal of nanoscience and nanotechnology, 2007, 7, 803-807.; S. Takayama, J. C. McDonald, E. Ostuni, M. N. Liang, P. J. Kenis, R. F. Ismagilov and G. M. Whitesides, Proceedings of the National Academy of Sciences, 1999, 96, 5545-5548.), with the goal to provide cheaper, simpler and more reliable means for simultaneous analysis of multiple biosensing reactions (P. Angenendt, J. Glokler, Z. Konthur, H. Lehrach and D. J. Cahill, Analytical chemistry, 2003, 75, 4368-4372.; S. Choi, M. Goryll, L. Y. M. Sin, P. K. Wong and J. Chae, Microfluidics and Nanofluidics, 2011, 10, 231-247.; B. S. Munge, T. Stracensky, K. Gamez, D. DiBiase and J. F. Rusling, Electroanalysis, 2016.; C. K. Tang, A. Vaze, M. Shen and J. F. Rusling, ACS sensors, 2016, 1, 1036-1043.). Out of the available platforms, surface-based microfluidic bioassay devices are rising to the forefront, owing to the enhanced sensitivity and ease of detection provided by these systems, largely attributed by the precise control of reaction sites by surface patterning (M. Zimmermann, E. Delamarche, M. Wolf and P. Hunziker, Biomedical microdevices, 2005, 7, 99-110.). Notably, the performance of these technologies is highly influenced by the quality of biomolecule surface patterning, ie., the surface density, orientation, and biofunctionality of patterned biomolecules. Additionally, the ease of operation, handling, and integration of the surface patterns into the devices is another important factor that influences the overall impact of these systems.
Existing techniques to pattern biomolecules on surfaces at the micro- and nanoscales, include physical patterning approaches such as photolithography (E. E. Hui and S. N. Bhatia, Langmuir, 2007, 23, 4103-4107.), adsorption of biomolecules confined to microfluidic networks (J. L. Garcia-Cordero and S. J. Maerkl, Chemical Communications, 2013, 49, 1264-1266.), and colloidal lithography (M. A. Ray, N. Shewmon, S. Bhawalkar, L. Jia, Y. Yang, and E. S. Daniels, Langmuir, 2009, 25, 7265-7270.). These techniques are either plagued by high costs, low throughput, or limited control over the geometry and functional properties of the achieved patterns. Particularly, nanopatterning of biomolecules has been laborious and integration of these patterned substrates into microfluidic devices has been a challenge. A recent report proposed a self-assembly-based colloidal lithography technique to generate nanopatterns which were then sealed into PDMS microchannels to immobilize proteins onto the nanopatterns via non-covalent coupling (A. S. Andersen, W. Zheng, D. S. Sutherland and X. Jiang, Lab on a Chip, 2015, 15, 4524-4532.). Although the proposed method enables the successful generation of multiple protein nanopatterns within a microfluidic channel, the major drawbacks are the requirement of complex fabrication techniques to create the nanopatterned substrates, the repeated fabrication of new surfaces prior to each use, and the non-covalent coupling of proteins onto the nanopatterns inducing potential desorption when subjected to flow.
One of the simpler and preferred methods of patterning micro- and nanoscale features is microcontact printing (μCP), where chemical or biological molecules are transferred in designated patterns from an elastomeric poly(dimethylsiloxane) (PDMS) stamp onto a substrate with higher surface energy (M. Mrksich and G. M. Whitesides, Trends in biotechnology, 1995, 13, 228-235.; L. Filipponi, P. Livingston, O. Kaspar, V. Tokarova and D. V. Nicolau, Biomedical microdevices, 2016, 18, 1-7.; R. Castagna, A. Bertucci, E. A. Prasetyanto, M. Monticelli, D. V. Conca, M. Massetti, P. P. Sharma, F. Damin, M. Chiari, L. De Cola et al., Langmuir, 2016, 32, 3308-3313.). Although these microcontact printed biomolecules have been successfully incorporated into microfluidic devices (R. S. Kane, S. Takayama, E. Ostuni, D. E. Ingber and G. M. Whitesides, Biomaterials, 1999, 20, 2363-2376.; E. B. Chakra, B. Hannes, J. Vieillard, C. D. Mansfield, R. Mazurczyk, A. Bouchard, J. Potempa, S. Krawczyk and M. Cabrera, Sensors and Actuators B: Chemical, 2009, 140, 278-286.), several challenges remain. First, as patterned biomolecules are physically adsorbed onto the surfaces driven by hydrogen-bonding and van der Waals forces (W. Norde, Colloids and Surfaces B: Biointerfaces, 2008, 61, 1-9.), they are unable to withstand high shear stresses introduced by the flow present in microfluidic channels. As a result, it gives rise to gradual desorption and degradation of patterned biomolecules that lead to reduced device performance and poor shelf life. Secondly, since partial dehydration of biomolecules is a prerequisite to the μCP technique, the probability of protein denaturation and impaired biological activity is high.
Additionally, the lack of control over the orientation of the printed proteins has been lamented and could be responsible for the suboptimal interactions in bioassays due to the inaccessibility of the binding sites. Lastly, patterning a substrate with multiple biomolecules proves to be difficult and time-consuming, as each individual stamp can only be utilized to pattern a single ink at a time.
To address these challenges, pre-patterned substrates have been used to covalently link the biomolecules from solution to pattern sensitive biomolecules. For example, Teerapanich, et al. recently achieved real-time monitoring of protein-binding kinetics by creating patterned gold films to stably and covalently immobilize antibodies within nanofluidic channels (P. Teerapanich, M. Pugniere, C. Henriquet, Y.-L. Lin, C.-F. Chou and T. Leichle, Biosensors and Bioelectronics, 2016). Others have reported a simpler and more stable surface patterning technique that employs covalent coupling of proteins and nucleotides to silane treated substrates (Y. Wu, T. Buranda, R. L. Metzenberg, L. A. Sklar and G. P. Lopez, Bioconjugate Chemistry, 2006, 17, 359-365.; H. H. Weetall, Applied Biochemistry and Biotechnology, 1993, 41, 157-188). Recently, Lin et al. demonstrated a novel method of covalently patterning multiple proteins to a (3-glycidyloxypropyl) trimethoxysilane modified substrate enclosed within nanochannels by using a robotic microarray spotter (Y.-L. Lin, Y.-J. Huang, P. Teerapanich, T. Leichle and C.-F. Chou, Biomicrofluidics, 2016, 10, 034114.). However, alignment of the channels with the patterns is difficult to achieve since the proteins are deposited onto the substrates prior to the bonding of the device. Additionally, as the proteins are dried briefly before alignment, viability for long-term studies is a concern due to their potential degradation.
Alternatively, (3-aminopropyl)triethoxysilane (APTES), an amine-NH2 terminated silane can be used to form covalent siloxane bonds with silica substrates under pertinent conditions (N. Aissaoui, L. Bergaoui, J. Landoulsi, J.-F. Lambert and S. Boujday, Langmuir, 2011, 28, 656-665.). Anhydrous organic solvents like toluene have been widely used to achieve homogeneity of the formed monolayers and to ensure covalent binding of APTES with the glass substrates (R. M. Pasternack, S. Rivillon Amy and Y. J. Chabal, Langmuir, 2008, 24, 12963-12971.). These terminal amine groups then serve to covalently couple biomolecules with the help of appropriate linkers (S. K. Vashist, E. Lam, S. Hrapovic, K. B. Male and J. H. Luong, Chemical reviews, 2014, 114, 11083-11130.). Several studies have demonstrated the potential of μCP to create patterns of APTES monolayers within microfluidic channels that are then covalently coupled with biomolecules from solution (T. F. Didar, A. M. Foudeh and M. Tabrizian, Analytical chemistry, 2011, 84, 1012-1018.; G. Arslan, M. Ozmen, I. Hatay, I. H. Gubbuk and M. Ersoz, Turkish Journal of Chemistry, 2008, 32, 313-321.). Although this method provides simplicity and potential for achieving multiplexing in microfluidic devices, the resolution of obtained features is not only limited by the microfluidic channel dimensions, but also by the printing process since existing μCP methods rely on the use of organic solvents that can potentially swell the PDMS substrate and increase the dimensions of the patterned features (J. N. Lee, C. Park and G. M. Whitesides, Analytical Chemistry, 2003, 75, 6544-6554). Although the degree of PDMS swelling does not significantly affect micron-size features in the stamps, it proves to be a limiting factor while attempting to achieve nanoscale APTES patterns. Notably, similar to thiols, silanes being small molecules, can diffuse into the PDMS stamp upon long incubation times (T. E. Balmer, H. Schmid, R. Stutz, E. Delamarche, B. Michel, N. D. Spencer and H. Wolf, Langmuir, 2005, 21, 622-632.). As a result, during the printing step (on the order of minutes), silane molecules tend to diffuse out of the stamp along with the solvent molecules, reducing the resolution of the patterned features (Y. Xia and G. M. Whitesides, Annual review of materials science, 52. 1998, 28, 153-184.).
NPL 3: M. Karle, S. K. Vashist, R. Zengerle and F. von Stetten, Analytica chimica acta, 2016, 929, 1-22.
NPL 5: G. Du, Q. Fang and J. M. den Toonder, Analytica chimica acta, 2016, 903, 36-50.
NPL 7: N. D. Gallant, J. L. Charest, W. P. King and A. J. Garcia, Journal of nanoscience and nanotechnology, 2007, 7, 803-807.
NPL 9: P. Angenendt, J. Glokler, Z. Konthur, H. Lehrach and D. J. Cahill, Analytical chemistry, 2003, 75, 4368-4372.
NPL 12: C. K. Tang, A. Vaze, M. Shen and J. F. Rusling, ACS sensors, 2016, 1, 1036-1043.
NPL 13: M. Zimmermann, E. Delamarche, M. Wolf and P. Hunziker, Biomedical microdevices, 2005, 7, 99-110.
NPL 18: M. Mrksich and G. M. Whitesides, Trends in biotechnology, 1995, 13, 228-235.
NPL 19: L. Filipponi, P. Livingston, O. Kaspar, V. Tokarova and D. V. Nicolau, Biomedical microdevices, 2016, 18, 1-7.
NPL 30: S. K. Vashist, E. Lam, S. Hrapovic, K. B. Male and J. H. Luong, Chemical reviews, 2014, 114, 11083-11130.
NPL 31: T. F. Didar, A. M. Foudeh and M. Tabrizian, Analytical chemistry, 2011, 84, 1012-1018.
NPL 35: Y. Xia and G. M. Whitesides, Annual review of materials science, 1998, 28, 153-184.
As the functionality of surface-based microfluidic bioassay devices is determined by the efficiency and accuracy of surface patterning of biomolecules, there is an increasing demand for new technologies to create surface patterns at micro- and nanoscales. It is an object of the present invention to achieve rapid surface patterning of biomolecules within microfluidic devices with high reproducibility.
In this work, we present a new means of creating micro- and nano-patterns of aminosilanes within microfluidic devices via an aqueous based microcontact printing technique. To minimize the diffusion of molecules into the PDMS stamp, we use water as the inking solvent and enforce short incubation and contact times during the printing process to preserve the pre-defined resolution of patterned features 36. These patterns then serve as the building block to couple multiple biomolecules in solution onto a single surface for subsequent bioassays. To validate the functionality of the coupled biomolecules, we carry out an aptamer based immunoassay to detect Interleukin 6 (IL6) and an antibody based immunoassay for the detection of human C-reactive protein (hCRP). We probe the stability of APTES patterns and demonstrate the possibility of fabricating pre-stored and ready-to-use bioassay devices with a shelf life of at least 3 months. Finally, we verify the multiplexing capability on a single patterned surface by delivering different biomolecules to different regions of the patterned array with the help of microfluidic networks and liquid dispensing technologies.
The present inventions are as follows.
The present invention of a simple aqueous based microcontact printing (μCP) method can create stable micro- and nanopatterns of (3-aminopropyl)triethoxysilane (APTES) on glass substrates of microfluidic devices with feature sizes ranging from a few hundred microns to 200 nm. By combining our surface patterning technique with sensing technologies, highly sensitive bioassay systems at nanoscale can be developed in the near future.
The invention is related to the area of micro- and nanocontact printing. In particular, it is related to micro- and nanocontact printing with aminosilanes: patterning surfaces of microfluidic devices for multiplexed bioassays.
In this work, we present a new means of creating micro- and nano-patterns of aminosilanes within microfluidic devices via an aqueous based microcontact printing technique. To minimize the diffusion of molecules into the PDMS stamp, we use water as the inking solvent and enforce short incubation and contact times during the printing process to preserve the pre-defined resolution of patterned features (H. Li, J. Zhang, X. Zhou, G. Lu, Z. Yin, G. Li, T. Wu, F. Boey, S. S. Venkatraman and H. Zhang, Langmuir, 2009, 26, 5603-5609.). These patterns then serve as the building block to couple multiple biomolecules in solution onto a single surface for subsequent bioassays. To validate the functionality of the coupled biomolecules, we carry out an aptamer based immunoassay to detect Interleukin 6 (IL6) and an antibody based immunoassay for the detection of human C-reactive protein (hCRP). We probe the stability of APTES patterns and demonstrate the possibility of fabricating pre-stored and ready-to-use bioassay devices with a shelf life of at least 3 months. Finally, we verify the multiplexing capability on a single patterned surface by delivering different biomolecules to different regions of the patterned array with the help of microfluidic networks and liquid dispensing technologies.
Before the present invention is described in detail, it is to be understood that this invention is not limited to the particular methodology, devices, solutions, arrays, kits, substrates or apparatuses described, as such methodology, devices, solutions, arrays, kits, substrates or apparatuses can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
Unless defined otherwise or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described.
All publications mentioned herein are hereby incorporated by reference for the purpose of disclosing and describing the particular materials and methodologies for which the reference was cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Definitions
The term “biomolecule” is used herein to refer to any chemical or biochemical structure present in living things which includes, but is not limited to, nucleotides, peptides, antibodies, carbohydrates and lipids.
The term “array” is used herein to refer to proteins, peptides, antibodies, nucleic acids, carbohydrates and lipids microarrays. Specific proteins, peptides, antibodies, nucleic acids, carbohydrates and lipids can be immobilized on solid surfaces to form arrays.
The term “binding” is used herein to refer to an attractive interaction between two molecules which results in a stable association in which the molecules are in close proximity to each other. Molecular binding can be classified into the following types: non-covalent, reversible covalent and irreversible covalent. Molecules that can participate in molecular binding include proteins, peptides, antibodies, nucleic acids, carbohydrates and lipids. Polypeptides that form stable complexes with other molecules are often referred to as receptors while their binding partners are called ligands. Polynucleotides can also form stable complex with themselves or others, for example, DNA-protein complex, DNA-DNA complex, DNA-RNA complex.
The term “peptides” is used herein to refer to proteins, fragments of proteins, and peptides, whether isolated from natural sources, produced by recombinant techniques, or chemically synthesized.
The term “nucleic acids” is used herein to refer to a polymeric form of nucleotides of any length, and may comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide.
The term “probe” is used herein to refer to a structure comprising nucleic acids, as defined above that contains a nucleic acid sequence that can bind to a corresponding target. The nucleic acids regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs. “Probe” is also used herein to refer to a structure comprising proteins, peptides, antibodies, carbohydrates and lipids that can bind to a corresponding target.
The term “nano features” is used herein to refer to predefined depositions of a given material of biomolecule or silane where the size of the depositions is inferior to 1000 nm and where the structures are predefined such as nanodots, nanoposts, and nanoislands.
“Silane” herein refers to silane compounds such as, not limited to, 3-aminopropyltriethoxysilane(APTES), triethoxysilypropyl succinic anhydride (TESPSA), (3-Glycidyloxypropyl)trimethoxysilane (GPTMS), octadecyltrichlorosilane (OTS), trichloro(1H, 1H, 2H 2H-perfluorooctyl) silane, trichlorosilanes, methyltrimethoxysilane, methyltriethoxysilane, phenyltrimethoxysilane, phenyltriethoxysilane, dimethyldimethoxysilane, dimethyldiethoxysilane, diphenyldimethoxysilane, diphenyldiethoxysilane, diphenyldiethoxysilane, tetramethoxysilane, tetraethoxysilane and the like. 3-aminopropyltriethoxysilane (APTES) and silane terminated with amine, carboxylate or thiol groups are preferable.
“Multiplexing” or “a multiplexed bioassay” herein refers to an assay or other analytical method in which the presence of multiple target molecules can be assayed simultaneously by using more than one capture probe conjugate, each of which has at least one different detection characteristic, e.g., fluorescence characteristic (for example excitation wavelength, emission wavelength, emission intensity, FWHM (full width at half maximum peak height), or fluorescence lifetime).
It is understood that aspects and embodiments of the invention described herein include “consisting” and/or “consisting essentially of aspects and embodiments.
Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.
In a high-throughput manner, microarray technologies enable the evaluation of up to tens of thousands of molecular interactions simultaneously. Microarrays have made significant impact on biology, medicine, drug discovery. DNA microarray-based assays have been widely used, including the applications for gene expression analysis, genotyping for mutations, single nucleotide polymorphisms (SNPs), and short tandem repeats (STRs). And polypeptide and chemical microarrays have emerged as two important tools in the field of proteomics. Chemical microarray, a form of combinatorial libraries, can also be used for lead identification, as well as optimization of these leads. In this era of bioterrorism, the development of a microarray capable of detecting a multitude of biological or chemical agents in the environment will be of great interest to the law enforcement agencies.
According to some embodiments of the present invention, assay methods, a substrate, an array, a kit for analysis of molecular interactions are provided. The inventive technology improves specificity and sensitivity of microarray- based assays while reducing the cost of performing genetic assays.
Methods
1. Patterning and Fabrication Procedures of the Present Invention
1-1. Soft Lithography
Stamps and microfluidic devices can be designed with AutoCAD (AutoDesk, USA). Stamp designs can comprise, but not limited to, (i) 100 μm wide stripes with 100 μm spacing (schematic in
1-2. Microcontact Printing (μCP) in Microfluidic Devices
As illustrated in the schematic in
1-3. Nanopatterned Lift-Off Stamps
Nanopatterned (nano feature is pattered from 10 nm to 1000 nm, preferably 20 nm to 800 nm, more preferably 50 nm to 500 nm) PDMS replicas are fabricated using the protocol previously described by Ricoult, et al. (S. G. Ricoult, M. Pla-Roca, R. Safavieh, G. M. Lopez-Ayon, P. Grutter, T. E. Kennedy and D. Juncker, Small, 2013, 9, 3308-3313). Briefly, nanopatterns consisting of a square array (200 nm in length, 200 nm in width, with 2 μm in spacing) are first created using Clewin Pro 4.0 (Wieweb software, Hengelo, Netherlands). A 4-inch silicon wafer is coated with PMMA resist and the dot arrays are patterned by electron beam lithography (VB6 UHR EWF, Vistec), followed by 100 nm reactive ion etching (System100 ICP380, Plasmalab) into the Si. After cleaning, the wafer is coated with an anti-adhesive layer by exposing it to perfluorooctyltriethoxysilane (Sigma-Aldrich, Oakville, ON, Canada) in vapor phase in a desiccator. An inverse polymer copy of the Si wafer is obtained after curing PDMS on the patterned wafer as described in the previous section to generate nanopillars. The lift-off stamp consisting of nanoholes with an inverse copy of the PDMS master (
1-4. Lift-Off Nanocontact Printing
A flat PDMS stamp is inked for 1-3 min with the 1% silane such as APTESaq solution as mentioned above (
2. APTESaq-Biomolecule Grafting Within Patterned Devices
Following APTESaq patterning and device assembly, the unpatterned regions within the device were blocked for 30 min by flowing a solution of 2 wt % PEG-silaneaq through the device. The concentration of the PEG-silaneaq can be 1% to 5%. Fluorescently labelled Immunoglobulins or protein of interest at 10 μg/ml were covalently grafted on the APTESaq patterned surface by employing EDC-NHS chemistry at a 10-fold molar excess of EDC (2 μM) and NHS (5 μM) to protein (see
3. Imaging and Analysis
NOA63 lift-off stamps were imaged using Quanta 250 FEG scanning electron microscope (FEI, Japan) at 5 kV with a spot size of 3.5 using an ETD Detector to detect secondary electrons. Micro- and nanopatterns of fluorescently labeled protein were imaged on a Ti-E Eclipse inverted fluorescent microscope (Nikon, Japan) and an LSM 780 Confocal microscope (Zeiss, Japan). All images were captured with fixed exposure times within each experiment, which varied from 1 to 10 s for all the images shown in this work. Mean fluorescence intensity measurements were obtained by performing image analysis in ImageJ (NIH, USA). Images were processed post quantification to increase the contrast through linear modifications in ImageJ.
Arrays
A microarray is a multiplex technology widely used in molecular biology and medicine. Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins, photolithography using pre-made masks, photolithography using dynamic micromirror devices, inkjet printing, microcontact printing, or electrochemistry on microelectrode arrays. In standard microarrays, the probe molecules are attached via surface engineering to a solid surface of supporting materials, which include glass, silicon, plastic, hydrogels, agaroses, nitrocellulose and nylon.
The systems described herein may comprise two or more probes that detect the same target biomolecules. For example, in some embodiments where the system is a microarray, the probes may be present in multiple (such as any of 2, 3, 4, 5, 6, 7, or more) copies on the microarray. In some embodiments, the system comprises different probes that detect the same target biomolecules. For example, these probes may bind to different (overlapping or non-overlapping) regions of the target biomolecules.
Any probes that are capable of determining the levels of target biomolecules can be used. In some embodiments, the probe may be an oligonucleotide (nucleic acids), peptides, antibodies, carbohydrates or lipids. It is understood that, for detection of target biomolecules, certain sequence or structure variations are acceptable. In some embodiments, the probe comprises a portion for detecting the target biomolecules and another portion. Such other portion may be used, for example, for attaching the biomolecules to a substrate. In some embodiments, the other portion comprises a non-specific sequence or nonspecific structure for increasing the distance between the complementary structure portion and the surface of the substrate.
The present invention provides an array of biomolecules comprising a substrate and a probe molecule, wherein the surface of the substrate has patterned nano features of silane. The unpatterned surface of the substrate is preferably blocked with PEG-silane. In addition, the diameter of the nano features is patterned from 10 nm to 1000 nm, preferably 20 nm to 800 nm, more preferably 50 nm to 500 nm. The probe molecule is selected from the group consisting of proteins, peptides, antibodies, nucleic acids, carbohydrates and lipids. The probe molecule is conjugated onto nano features of silane on the substrate. The method section can be referred for details of the Arrays of the present invention.
The assays based on the arrays of the present invention may be implemented in a multiplex format. Multiplex methods are provided employing 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000 or more different capture probes which can be used simultaneously to assay for amplification products from corresponding different target polynucleotides. Methods amenable to multiplexing, such as those taught herein, allow acquisition of greater amounts of information from smaller specimens. The need for smaller specimens increases the ability of an investigator to obtain samples from a larger number of individuals in a population to validate a new assay or simply to acquire data, as less invasive techniques are needed.
Kits
The present invention provides a kit comprising a substrate and a probe molecule, wherein the surface of the substrate has patterned nano features of silane. The unpatterned surface of the substrate is preferably blocked with PEG-silane. In addition, from 10 nm to 1000 nm, preferably 20 nm to 800 nm, more preferably 50 nm to 500 nm. The probe molecule is selected from the group consisting of proteins, peptides, antibodies, nucleic acids, carbohydrates and lipids. The method section can be referred for details of the Kits of the present invention.
Substrates
The present invention provides a substrate for an array of biomolecules, wherein the surface of the substrate has patterned nano features of silane for conjugating a probe molecule. The substrates can be made of glass, silicon, or plastic, preferably glass or plastic, more preferably glass. The unpatterned surface of the substrate is preferably blocked with PEG-silane. In addition, from 10 nm to 1000 nm, preferably 20 nm to 800 nm, more preferably 50 nm to 500 nm. The probe molecule is selected from the group consisting of proteins, peptides, antibodies, nucleic acids, carbohydrates and lipids. The method section can be referred for details of the Substrates of the present invention.
APTESaq Micropatterns for Grafting of Biomolecules Within Microfluidic Devices
To facilitate the patterning of biomolecules within microfluidic devices regardless of the molecular charge, a microcontact printing process was developed to print aqueous APTES (APTESaq) in closed microfluidic devices (schematic in
In previously described methods (G. Arslan, M. Ozmen, I. Hatay, I. H. Gubbuk and M. Ersoz, Turkish Journal of Chemistry, 2008, 32, 313-321.), when the silanes were inked with toluene, there is a high probability of PDMS swelling and subsequent change in feature sizes.
Additionally, silane-toluene reservoirs are created within the stamp, which diffuse out of the stamp when printed onto glass surfaces for long contact times, 35 ultimately leading to loss of resolution. With our protocol, by using water as the inking solvent, we not only limit the probability of silane reservoir formation and swelling of the stamp, but also reduce leakage upon contact by using combined inking and printing times on the order of a few minutes. Thus, the desired patterning dimension is maintained during and after the printing process. These results depict the compatibility of this technique with glass-based microfluidic devices to covalently pattern not only proteins but also effectively couple other biomolecules such as DNA aptamers, carbohydrates, and lipids onto their substrates for subsequent bioassay applications.
Aminosilane Nanopatterns for Grafting of Biomolecules Within Microfluidic Devices
In addition to successfully creating micropatterns of APTES to covalently pattern biomolecules, we further demonstrate a simple lift-off nanocontact printing method for creating nanopatterns of APTESaq within microfluidic channels to subsequently graft biomolecules covalently. Following a previous protocol (S. G. Ricoult, M. Pla-Roca, R. Safavieh, G. M. Lopez-Ayon, P. Grutter, T. E. Kennedy and D. Juncker, Small, 2013, 9, 3308-3313), disposable epoxy lift-off stamps (
A flat PDMS stamp was then inked with the APTESaq solution, rinsed and dried before being pressed against the plasma activated lift-off stamp for 5 s (
Several prior studies have reported the potential of nanocontact printing to satisfy the growing demand of creating biomolecule nanopatterns to achieve single molecule detection (B. R. Takulapalli, M. E. Morrison, J. Gu and P. Zhang, Nanotechnology, 2011, 22, 285302.; H.-W. Li, B. V. Muir, G. Fichet and W. T. Huck, Langmuir, 2003, 19, 1963-1965.; J. Gu, X. Xiao, B. R. Takulapalli, M. E. Morrison, P. Zhang and F. Zenhausern, Journal of Vacuum Science & Technology B, 2008, 26, 1860-1865.). However, few reports have incorporated these patterns into microdevices for subsequent microfluidic bioassays (A. S. Andersen, W. Zheng, D. S. Sutherland and X. Jiang, Lab on a Chip, 2015, 15, 4524-4532.). Additionally, the reliance of the patterning processes on physisorption proves to be a drawback as the biomolecules are susceptible to detachment from the surface due to the presence of high shear stress introduced by the flow. Our new protocol here addresses all these challenges: by employing lift-off nanocontact printing of APTESaq on glass substrates, covalently tethered nanopatterns of proteins with a resolution of 200 nm can be easily integrated into microfluidic devices. Additionally, these protein nanopatterns are created with the same efficiency as the previously described direct nanocontact printing approach by Ricoult, et al. (S. G. Ricoult, M. Pla-Roca, R. Safavieh, G. M. Lopez-Ayon, P. Grutter, T. E. Kennedy and D. Juncker, Small, 2013, 9, 3308-3313.), see more details in
Aptamer-Based and Antibody-Based Immunoassays
Interleukin-6 (IL6) (J. S. Yudkin, M. Kumari, S. E. Humphries and V. Mohamed-Ali, Atherosclerosis, 2000, 148, 209-214.; A. G. Vos, N. S. Idris, R. E. Barth, K. Klipstein-Grobusch and D. E. Grobbee, PloS one, 2016, 11, e0147484.) and human C-reactive protein (hCRP) (I. Kushner, Science, 2002, 297, 520-521.; P. M. Ridker, Circulation, 2003, 107, 363-369.) are the most important biomarkers of neurological, cardiovascular and other pathophysiological conditions that arise from tissue inflammation or infection. Quantitative detection of these biomarkers has immensely helped in early diagnosis and treatment of these diseases. In order to accurately diagnose these diseases, sensitive assays and biosensing technologies are required to reliably detect minute quantities of these biomarkers (S. K. Vashist, A. Venkatesh, E. M. Schneider, C. Beaudoin, P. B. Luppa and J. H. Luong, Biotechnology advances, 2016, 34, 272-290.; A. Qureshi, Y. Gurbuz and J. H. Niazi, Sensors and Actuators B: Chemical, 2012, 171, 62-76.). Therefore, to test the sensitivity and biofunctionality of our APTESaq-micropatterned microfluidic devices, sandwich-based immunoassays were carried out to qualitatively and quantitatively detect IL6 and hCRP with the help of either aptamers or antibodies respectively.
For the aptamer-based immunoassay, —NH2-terminated aptamers specific to IL6 were grafted onto the APTESaq micropatterns via BS3 chemistry after blocking. Subsequently, 470 nM of IL6 was detected with the help of a complimentary biotinylated detection aptamer and streptavidin dye (see
To further characterize the sensitivity of these patterned devices, we focused on the antibody-based sandwich immunoassay. Varying concentrations from 2 nM to 217 nM of hCRP mixed in PBS were flowed through microchannels patterned with capture antibodies against hCRP grafted onto APTESaq via BS3 chemistry. A range of concentrations of hCRP from 4-200 nM was successfully detected via the detection antibody pair and qualitatively analyzed by fluorescence microscopy (more details are shown in Figure S4a-e in the SI document).
To estimate the detection of hCRP quantitatively, the normalized fluorescence intensity was calculated for each condition by measuring the ratio of mean pixel intensity of the patterned region (red) to that of the unpatterned region (black), averaged over 3 images each with 9 patterned squares in each image. A blank reaction was carried out by flowing the detection antibody pair over the grafted capture antibody to account for the nonspecific adsorption. The histogram in
LoB=Meanblank+1.645(SDblank), (1)
LoD=LoB+1.645(SDlcs) (2)
where LoB, SD, LoD and lcs are the limit of blank, standard of deviation, limit of detection and lowest concentration sample respectively.
The successful detection of clinically significant levels of IL6 and hCRP validates the biofunctionality of these patterned devices. The sensitivity of these devices can be significantly improved in the future with more specific aptamer or antibody combinations coupled with label-free detection systems.
Stability of Aminosilane Patterns
To assess the stability of the APTESaq patterns, microfluidic device substrates were pre-patterned with APTESaq by microcontact printing perpendicular to the microfluidic channels, in stripes of 100 μm separated by 100 μm in spacing. Thirty patterned and sealed devices were stored in plastic containers after blocking with PEG-silaneaq for up to 3 months at room temperature (25° C.) or at 4° C. in the absence of vacuum. Three devices per testing condition were characterized to determine the efficiency of grafting of fluorescently labeled IgGs (Immunoglobulins) on the APTESaq patterns.
Square fluorescent bands shown in
It is worth noting that the initially microcontact printed APTESaq patterns already have a small level of inhomogeneity as seen in
By printing aminosilanes that are insensitive to enzymes and subsequently capturing the biomolecules at the time of the bioassay, we highlight the following advantages: 1) the patterned substrates can be stored on the order of months before carrying out the bioassay, 2) biomolecules are less likely to be affected by denaturation associated with external stresses since they are delivered in solution, and 3) interaction sites can be accurately engineered by precisely designing the silanes and biomolecules thereby providing control over the orientation of the biomolecules. Additional experiments are being carried out to further probe and improve the chemical viability of APTESaq patterns on substrates upon storage, which will be reported in the future.
Multiplexing on Aminosilane Patterned Substrates
To overcome the one stamp-one ink characteristic of microcontact printing, we use APTESaq patterns to capture and covalently graft different locally delivered biomolecules. To visually demonstrate the capability of patterning multiple biomolecules onto a single surface, two different solutions of EDC-NHS activated Alexa-fluor 488 and 546-labelled fluorescent antibodies were delivered onto the patterned substrate by two modes of liquid delivery. First, an array of squares (50 by 50 μm) of APTESaq was patterned on a plasma activated glass slide by microcontact printing and blocked with 2% PEG-silaneaq. Liquid dispensing robots (Musashi Engineering, Japan) were then used to deliver microliter volumes of droplets containing the two protein solutions (
Alternatively, microfluidic devices (
One of the major obstacles in achieving multipatterning by microcontact printing has been the necessity of fabrication of complex stamps that either contained microfluidic circuits or gradient generators on the stamp to create patterned concentration gradients on substrates. In comparison, the aminosilane printing approach coupled with microfluidics introduced in this work, facilitates the creation of large and stable arrays composed of multiple biomolecules presented via covalent bonds in a single device. By making use of the localized delivery available in microfluidic devices or liquid dispensing platforms, multi-protein patterns could easily be achieved within a single array. Additionally, with the advent of nanofluidic devices and liquid dispensing robots delivering picolitre droplets, densely packed nanoarrays can undoubtedly be achieved in the near future.
To create biomolecular patterns within microfluidic channels, we introduced a micro- and nanocontact printing method to pattern amino terminated silanes on a desired planar surface, with feature sizes ranging from a few hundred microns down to 200 nm. This protocol provides several key advantages. First, owing to its compatibility with PDMS, water can be used as the inking solvent to pattern APTES onto glass substrates. Next, the microfluidic channels deliver a blocking solution, to (i) limit the diffusion of volatile silanes as well as (ii) inhibit biofouling. Micro- and nanopatterns can be grafted with different biomolecules such as proteins and DNA in controlled orientations for subsequent immunoassay applications within these devices. Additionally, the APTESaq patterns maintain their ability to covalently graft biomolecules to the surface for at least 3 months after printing with no significant difference between storage conditions at room temperature or at 4° C., thereby demonstrating their storage potentials. By grafting biomolecules onto pre-patterned substrates prior to use, it greatly preserves the functionalities of the grafted biomolecules with minimized risks of biodegradation, accompanied by simplified operation protocols.
By demonstrating successful DNA-based immunoassays and antibody-based immunoassays carried out on microcontact printed aminosilane patterns, we demonstrated the biofunctionality of these prints areas thereby describing the overall potential of this technology in the field of bioassay applications. To demonstrate the multiplexing potentials of this technology, localized delivery available in microfluidic devices or liquid dispensing platforms were used to achieve multi-protein patterning within a single array.
Applications for patterned surfaces are broad, but their translation from the lab to commercial products has been hindered by limited abilities to integrate the patterns into microfluidic devices with control. With this simple patterning technique, it could help in accelerating the translation of these patterned substrates from the lab to commercial products for the development of integrated bioassays suitable for commercialization in the near future.
Reagents and Materials
(3-Aminopropyl) triethoxysilane (APTES) and 2-methoxy (polyethyleneoxy) 6-9 propyl tricholoro silane (PEG-silane) were purchased from Nacalai, Japan. 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), BS3 crosslinker, phosphate buffered saline (PBS), HEPES, Glycine, and Streptavidin DyLight™ 550 Conjugated, were purchased from Thermo Fischer Scientific, Japan. Biotin-SP-conjugated AffiniPure goat anti-mouse antibody was purchased from Jackson ImmunoResearch labs, USA. Biotinylated aptamers specific to Interleukin6 (IL6) were obtained from BasePair Biotechnologies, USA. Recombinant human IL6 (PHC0066) was purchased from Life Technologies. Mouse anti-C Reactive Protein antibody [C5] ab8279 (Abcam, Japan) and recombinant human C-reactive protein (hCRP) were obtained from Oriental Yeast Co., Ltd., Japan. Alexa Fluor 488 conjugated chicken anti-goat, Alexa Fluor 546 conjugated rabbit anti-mouse and goat anti-chicken Immunoglobulins (IgGs) were purchased from Abcam, Japan.
The present invention of a simple aqueous based microcontact printing (μCP) method can create stable micro- and nanopatterns of (3-aminopropyl)triethoxysilane (APTES) on glass substrates of microfluidic devices with feature sizes ranging from a few hundred microns to 200 nm (for the first time). By combining our surface patterning technique with sensing technologies, highly sensitive bioassay systems at nanoscale can be developed in the near future.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2017/003621 | 2/1/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62290067 | Feb 2016 | US |
