Patent Application
20120141767
1. Field of the Invention
The present invention is generally related to a biochip, and more particularly to a biochip with a three-dimensional structure and a method for forming the same.
2. Description of the Prior Art
At present, the biochip detection technology becomes increasingly important in biotechnology. The biochip detection technology can simultaneously detect various pathogens on a single chip and break the detection limitation achieved by traditional technologies. A microarrayed biochip is generally prepared by aligning a large quantity of bio-probes (DNA's or proteins) on a chip substrate and is used for analyzing or testing samples by the hybridization of DNA-DNA or specific binding between proteins. According to the detection objectives, there are two major categories for microarrayed biochips: DNA chip and protein chip. DNA chips use nucleotide molecules as the probes to detect their nucleotide fragments. DNA chips can also be categorized into complimentary DNA (cDNA) chips and oligonucleotide chips, according to the length of the probes spotted on chips. cDNA chips are often used in the research of gene expressions; while oligonucleotide chips can also be used in diagnosis of pathogen and genotyping in addition to gene expression analysis.
For DNA chips, probes are immobilized on substrates and used to detect specific DNA fragments by the characteristic hybridization with complimentary DNA's. DNA chips can be applied on disease detection and shorten the time for developing new medicines. DNA chip is also a powerful tool for analyzing DNA's by appropriate dye labeling in visible emission lights. By different emission wavelengths, individual target DNA can be distinguished and analyzed.
The application of biochip is vary wide, including gene expression profiling, toxicology analysis, gene sequencing, SNP identification, forensics, immunoassays, protein chip, combat biowarfare, drug screening, hard drives and microprocessors.
The improvement of detection sensitivity by modifying the substrate surfaces of traditional biochips is currently still being sought to obtain amplified signals to facilitate further analysis. Thus, a novel biochip preparation method is proposed to achieve the high-sensitivity performance.
In accordance with the present invention, a biochip with a three-dimensional mesoporous layer and a method for forming the same are provided.
The three-dimensional mesoporous material is a network polymer with nano-scaled pores, such as aerogel material. Its porosity can be as high as 95%. Due to its high porosity, it possesses a variety of characteristics: high specific surface area, low density, low heat conductivity, low sound spreading speed, low dielectric constant, and so forth. Therefore, it can be applied in various fields, such as heat insulation, catalyst, adsorbent, electrodes, electronics, detectors, etc.
The first objective of the present invention is to synthesize materials on the top of a flat substrate to form a three-dimensional mesoporous layer using the sol-gel technique.
The second objective of the present invention is to utilize the large three-dimensional inner specific surface area to recognize labeled DNAs, proteins, peptides, saccharides, and cells. Thus, the biochip with a three-dimensional mesoporous layer according to the present invention has the characteristics of high sensitivity of detection so as it would have a potential to simplify the detection equipments. For example, only data type camera (CCD) would be required instead of complicated imaging technique. Therefore, this present invention does have the economic potential for industrial applications.
Accordingly, the present invention discloses a biochip comprising a substrate and a three-dimensional mesoporous layer on top of the substrate. The surface of the three-dimensional mesoporous layer is chemically modified to recognize labeled DNAs, proteins, peptides, saccharides, and cells. In addition, this invention also discloses a method for preparing the biochip with a three-dimensional mesoporous layer, including a blending process, a heating process, a coating process, a gelation process, a cleaning process, a drying process, and a surface modification process.
What is probed into the invention is a biochip with a three-dimensional structure and a method for forming the same. Detail descriptions of the structure and elements will be provided as followed in order to make the invention thoroughly understood. The application of the invention is not confined to specific details familiar to those who are skilled in the art. On the other hand, the common structures and elements that are known to everyone are not described in details to avoid unnecessary limits of the invention. Some preferred embodiments of the present invention will now be described in greater detail as followed. However, it should be recognized that the present invention can be practiced in a wide range of other embodiments besides those explicitly described, that is, this invention can also be applied extensively to other embodiments, and the scope of the present invention is expressly not limited except as specified in the accompanying claims.
In one embodiment of the present invention, a method for forming a biochip with a three-dimensional structure is disclosed. At first, a precursor solution is provided. The precursor solution comprises an ionic liquid, a catalyzed hydrolysis and/or condensation reagent, and at least one alkoxide monomer and/or aryloxide monomer, where the catalyzed hydrolysis and/or condensation reagent comprises one selected from the group consisting of the following or any combination of the following: alcohol, acidic compound, and alkaline compound. The ionic liquid is used as a template as well as a solvent. The central element of the alkoxide monomer and/or aryloxide monomer comprises one selected from the group consisting of the following elements: Li, Na, K, Rb, Cs, Be, Mg, Ca, Sr, Ba, Ti, Te, Cr, Cu, Er, Fe, Ta, V, Zn, Zr, Al, Si, Ge, Sn, and Pb. Next, a blending process for the precursor solution to hydrolyze and polymerize the at least one alkoxide monomer and/or aryloxide monomer until the viscosity of the precursor solution reaches a specific viscosity more than or equal to 150 cps. Then, setting the precursor solution to have the at least one alkoxide monomer and/or aryloxide monomer continue to undergo hydrolysis and condensation, so as to form a aerogel.
After the aerogel be formed, the extracting process is carried out by a solvent for the aerogel to substitute the ionic liquid in pores of the aerogel. Next, a drying process is carried out to remove the solvent in pores of the aeroge. Then, a grinding process is carried out to grind the aerogel to form a aerogel powder, and the diameter of the aerogel powder ranges about 10 nm to 250 nm. After the grinding process, a modification process is carried out and the internal and external surface of the aerogel powder was modified by a modifier with a specific moiety to form a modified aerogel powder. Finally, a coating process is carried out to coat the modified aerogel powder on a specific region of substrate, so as to form a biochip with a three-dimensional structure. The material of the substrate comprises one selected from the group consisting of the following materials: silicon chip, glass, or polymer.
The above-mentioned coating process described as followed: firstly dispersing the modified aerogel powder in a solution to form a modified solution; next coating the modified solution on a specific region of substrate; and finally performing a baking process to remove the solvent of modified solution and to enhance the adhesive force between the modified aerogel powder and the substrate, so as to form the biochip with a three-dimensional structure. In addition, the temperature of the baking process ranges from 80° C. to 120° C.
The precursor solution also comprises an acidic compound or alkaline compound to catalyze the hydrolysis/polymerization of the alkoxide monomer and/or aryloxide monomer. The method for preparing the precursor solution described as followed: firstly blending the alkoxide monomer and/or aryloxide monomer and the ionic liquid together to form a first mixture; next adding an acidic compound to the first mixture to form a second mixture; and finally adding an alkaline compound to the second mixture to enhance the hydrolysis/polymerization reactions of the alkoxide monomer and/or aryloxide monomer.
The common composition of the aerogel one selected from the group consisting of the following or any combination: SiO2, TiO2, V2O5, and Al2O3. The preferred solvent is the one with a low boiling point (less than or equal to 200° C.). The aerogel being substituted the ionic liquid in pores of the aerogel by the solvent. Preferably, the solvent comprises one selected from the group consisting of the following: nitrile, alcohol, ketone, and water. The average pore diameter of the aerogel ranges about 2 nm to 50 nm. The specific surface area is more than or equal to 100 m2/g and the porosity is 50%-99%.
The aerogel powder was modified by a modifier. The modifier for the modification process is an alkoxide monomer and/or aryloxide monomer with at least one specific moiety. The specific moiety comprises one selected from the group consisting of the following: amine group, hydroxyl group, carboxyl group, and epoxy group. The common modifier comprises N-[3-(trimethoxysilyllpropyl]-1,2-ethanediamine (DAMO), 3-Glycidoxypropyl-trimethoxysilane (GLYMO), 3-Aminopropyltriethoxysilane (APTS), N-(2-Aminoethyl)3-aminopropyltriethoxysilane (TMsen) and so forth. The modified aerogel powder is coated on a specific region of substrate with the coating process, so as to form a biochip with a three-dimensional structure.
According to the first example of the present invention, after the coating process, a converting process is carried out. At first, a converter that comprises a first moiety and a second moiety is provided. Then, the specific moiety of the aerogel powder is bonded with the first moiety of the converter to form a biochip having the second moiety on its surface. For example, when the modifier is N-[3-(trimethoxysilyllpropyl]-1 ,2 -ethanediamine (DAMO), glutaraldehyde can be used as the converter to form the mesoporous layer having aldehyde group on its surface. The converter comprises one selected from the group consisting of the following: antigens, primary antibody, monoclonal antibodies, polyclonal antibodies, nucleic acids comprising monomeric and oligomeric types, proteins, enzymes, lipids, polysaccharides, sugars, peptides, polypeptides, drugs, viruses, microbes, and bioligands.
According to the second example of the present invention, after the converting process, a blocking process is carried out. At first, a blocker that comprises a third moiety is provided. Then, the specific moiety of the aerogel powder is bonded with the third moiety of the blacker to form a biochip having the second moiety on its surface. The third moiety of the blacker reacts with specific moiety which non-reacts with the first moiety of converter.
According to the third example of the present invention, after the blocking process, a specific pairing process is carried out. At first, a pair that comprises a fourth moiety and a fifth moiety is provided. Then, the second moiety of the biochip is bonded with the fourth moiety of the pair to form a biochip having the fifth moiety on its surface. The pair comprises one selected from the group consisting of the following: antigens, primary antibody, monoclonal antibodies, polyclonal antibodies, nucleic acids comprising monomeric and oligomeric types, proteins, enzymes, lipids, polysaccharides, sugars, peptides, polypeptides, drugs, viruses, microbes, and bioligands.
According to the fourth example of the present invention, after the specific pairing process, a labeling process is carried out. At first, a labeling carrier that comprises a sixth moiety and a seventh moiety wherein conjugated with a marker is provided. Then, the fifth moiety of the pair labeling carrier is bonded with the sixth moiety of the labeling carrier to form a biochip having the marker on its surface. The marker comprises one selected from the group consisting of the following: fluorescence substance, phosphorescence substance, luminescence substance, enzyme, radioactive element, quantum dot, nano diamond. The labeling carrier comprises one selected from the group consisting of the following: antigens, primary antibody, labeling primary antibody, secondary antibodies, monoclonal antibodies, polyclonal antibodies, nucleic acids comprising monomeric and oligomeric types, proteins, enzymes, lipids, polysaccharides, sugars, peptides, polypeptides, drugs, viruses, microbes, and bioligands.
In the embodiment, the mentioned ionic liquids are room temperature ionic liquids (RTIL's), and is formed by mixing an organic base with a Lewis acid. When the Lewis acid is halogenated acid, it can form a room temperature ionic liquid but will produce halogen acid if reacting with water. Therefore, the halogenated acid is not suitable for the present invention. The Lewis acid used by the present invention is not halogenated acid so as to prepare a stable ionic liquid in water. In a preferred example, the cationic moiety in the organic base is alkyl or aryl group having the following general equation:
in which R1, R2, R3, and R4 are selected according to the following table.
For example, the common organic cationic moiety comprises one selected from the group consisting of the following: 1-n-butyl-3-methylimidazolium (BMI), 1-octanyl-3-methylimidazolium (OMI), 1-dodecanyl-3-methylimidazolium (DMI), and 1-hexadecanyl-3-methylimidazolium (HDMI). In addition, the anionic moiety in the Lewis acid comprises one selected from the group consisting of the following: BF4−, PF6−, AsF6−, SbF6−, F(HF)n−, CF3SO3−, CF3CF2CF2CF2SO3−, (CF3SO2)2N− [TFSI], (CF3SO2)3C−, CF3COO−, and CF3CF2CF2COO−. When the cationic moiety to be used is determined, the anionic moiety in the Lewis acid can be adjusted to control hydrophilicity/hydrophobicity. For example, BMI-BF4 is hydrophilic and BMI-TFSI is hydrophobic.
For instance, alkyloxide monomer is used as an example. Alkyloxide monomer is hydrolyzed to form hydrophilic silanol (—Si—O—H). Thus, the hydrophilic ionic liquid and silanol are tended to attract to each other and can stabilize the formation of silicon oxide structure so as to obtain more stable three-dimensional silicon oxide mesoporous material. In this embodiment, the weight of the ionic liquid is about 10%-70% weight of the at least alkoxide monomer and preferably about 20%-50%. When the added amount is more than the upper limit, the sol concentration is reduced and the gelation is slow to result in unstable structure.
In this embodiment, the method for forming the biochip with a three-dimensional structure comprises one selected from the type consisting of the following: direct immune, indirect immune, complement fixing immune, sandwich immune.
According to a preferred example of the present invention, the method for forming a biochip with a three-dimensional aerogel layer is provided. The method comprises the following steps.
The Aerogel chemical reaction equation is below:
Calculate the amount of EDC, PEG and sulfo-NHS required. Put the required amount of the compounds in the microtube and record the amount in the microtube. Afterward, according to the amount in the microtube, calculate the volume of buffer needed. The next steps will be to mix the required ratio of quantum dot with the buffer. First of all, put PEG in the buffer and vibrate until it dissolves completely. The next step is to mix EDC with the buffer very quickly and add the quantum dot to neutralize the reaction immediately. Then mix sulfo-NHS with the buffer. Lastly, add the quantum dot into the sulfo-solution and add the PEG solution immediately too.
Vibrate and shake the whole mixture in 4° C. refrigerator for 2 hours. After the reaction is complete, filter the solution by molecular sieve. The residue solution is the branch with quantum dot. The reaction of quantum dot is shown below:
As long as the atom has the spin angular momentum, like if the atom with the odd atomic number or proton number will have the absorption with Nuclear Magnetic Resonance. The common liquid Nuclear Magnetic Resonance (NMR) is usually applied to identify the organic structure. But the detected condition will always be limited by the D solution. Due to advances in solid Magnetic Resonance Imaging (MRI), the solid sample can be detected directly. It's also more understandable of the advance research on chemical structure, bond association and kinematics. We can use the 29Si solid NMR to analyze Si bonding distribution in Aerogel. Based on this study, it clarifies the bonding situation of the net-cross-binding structure in TEOS with dissolved gel-gel reaction. According to the analysis of Silica Aerogel from the 29Si solid NMR spectrum, the particular absorption peak during −99-−102 ppm will appear when the 3nd replacement of Silica happened. The particular absorption peak during −107-−110 ppm will appear when the 4th replacement of Silica happened. The 29Si solid NMR spectrum analysis (
As it's shown in
N2 Adsorption/Desorption Analyzer can measure the property of the material surface. The theory is using Inert Gas to detect the pore size, surface area and pore structure on the material surface physical property. The adsorption degree related to the samples and the property of the used adsorption gas, and can also be the function of the pressure(or concentration) and temperature. The adsorption degree to P/P0 figurer usually is made by the sample (gram) in constant temperature. P0 is the saturated vapor pressure of the analysis gas in experiment temperature. This curve named Adsorption Isotherms. Base one the
In the other way, the dotted line showed the unclean situation of the Silica Aerogel. If there is some residuum on the template, the template would start to crack when the temperature reached to 265° C. . This is also the way to determine if the washing step is completely.
Different sizes of the nano level quantum dot will show the different fluorescence under different UV light.
The reform of the quantum dot is changing the amino base of the hydrophilic surface of the quantum dot. Make this site as an experiment marker for the carboxyl reaction with antibody.
Heavier molecular weight will move slower than the light molecular weight during the electrophoresis process with the same electronic pressure and time. The reformed quantum dot is heavier than the un-reformed one, so the electrophoresis can be the way to detect if the reform is successful.
Adding the buffer solution during the reform process makes the concentration different from the un-reform. Measurement of the concentration again is needed after the reform process. Fix the wave in 527 nm to do the single detection with reformed quantum dot. Measure the absorption and then calculate the concentration afterward.
The concentration calculation function:
C=A/εL
A is the Absorption. E is the constant of the Molar absorption (M−nm)−1. L is the particle diameter (nm). The concentration calculated function below is the sample of
0.04/(77793.9851×0.512)=1.00×10-6
Here we have made the protein biochip. This kind of biochip is made by the specific of special three-dimensional structure with protein-protein, protein-small molecular to detect the particular protein.
As shown in
The fluorescence chip-scanner GenePix 40000B is using dual-laser scanning system to generate the real time ratio image. The ratio image is composed by red, green, blue color with the standard 24 bite. The scanner system default is 635 to 532 nm of laser.
This is the scanned image result of the comparison of the three-dimensional Aerogel chip and two-dimension protein chip which is performed by antibody and antigen specific reaction.
The chip scanner analysis software (GenePixPro6.0) analyzes the particular different signal spot in three-dimensional Aerogel chip and 2-dimension protein chip. The result is listed in Table 1 and Table 2.
Table 1 is the result of the three-dimensional Aerogel chip shown as below:
Table 2 is the result of the 2-dimension protein chip shown as below:
According to Table 2, when the sample concentration reaches 5.0×10−4M in 2-dimension protein chip, the chip signal is around 16756 which is close to light blue background value. The hypothesis is when the sample concentration is to low (10−5), the chip scanner can't detect the signal. When the concentration of three-dimensional Aerogel chip is lower than 1.44×10−6M, the signal intensity is amplified to 26213-31609 because of the Aerogel three-dimensional structure. It is thus evident that the bigger the surface area of three-dimensional Aerogel, the stronger the signal intensity.
Other modifications and variations are possibly developed in light of the above demonstrations. It is therefore to be understood that within the scope of the appended claims the present invention can be practiced otherwise than as specifically described herein. Although specific embodiments have been illustrated and described herein, it is obvious to those skilled in the art that many modifications of the present invention may be made without departing from what is intended to be limited solely by the appended claims.
This is a divisional of U.S. Ser. No. 11/940,417, filed Nov. 15, 2007 by the same inventors, and claims priority there from. This divisional application contains rewritten claims to the restricted-out subject matter of original claims.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11940417 | Nov 2007 | US |
| Child | 13336171 | US |
