US20120141767A1

US20120141767A1 - Biochip array with a Three-Dimensional structure

Info

Publication number: US20120141767A1
Application number: US13/336,171
Authority: US
Inventors: Yui-Whei Chen-Yang; Jui-Chuang Wu; Yen-Kuang Li; Yun-Chu Chen
Original assignee: Chung Yuan Christian University
Current assignee: Chung Yuan Christian University
Priority date: 2007-11-15
Filing date: 2011-12-23
Publication date: 2012-06-07
Also published as: US8735084B2; US20120148725A1

Abstract

The present invention discloses a biochip with a three-dimensional structure. 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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

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.

BACKGROUND OF THE INVENTION

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.

SUMMARY OF THE INVENTION

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.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a picture showing the flowchart for forming a biochip with a three-dimensional aerogel layer according to a preferred example of the present invention;

FIG. 2 is the absorption spectrum of FTIR after and before remove the solvent in three-dimensional aerogel layer according to a preferred example of the present invention;

FIG. 3 shows the analysis result of the unmodified three-dimensional aerogel layer according to a preferred example of the present invention by the ²⁹Si solid-state nuclear magnetic resonance spectrometer;

FIG. 4 shows SEM (scanning electron microscope) images of the surface of the three-dimensional aerogel layer (amplification factor=3000) according to a preferred example of the present invention;

FIG. 5 shows SEM (scanning electron microscope) images of the surface of the three-dimensional aerogel layer (amplification factor=300000) according to a preferred example of the present invention;

FIG. 6 is a N2 Adsorption/Desorption Analyzer curve of the three-dimensional aerogel layer modified by 10% GLYMO according to a preferred example of the present invention;

FIG. 7 is a the needle curve means the pore distribution of the three-dimensional aerogel layer according to a preferred example of the present invention;

FIG. 8 shows the TGA comparison data of the three-dimensional aerogel layer before/after modification according to a preferred example of the present invention;

FIG. 9 shows the digital camera picture of the quantum dot under UV light excitation according to a preferred example of the present invention;

FIG. 10 shows the result of the reformed quantum dot of the Fluoresce Spectrometer according to a preferred example of the present invention;

FIG. 11 shows the electrophoresis photograph of the reformed quantum dot and the un-reformed one according to a preferred example of the present invention;

FIG. 12 shows the flowchart of Sandwich Immunoassay according to a preferred example of the present invention;

FIG. 13 shows the images of the washed blank slide according to a preferred example of the present invention;

FIG. 14 shows the images of two-dimension chip after the amino reforming process according to a preferred example of the present invention;

FIG. 15 shows the scanned image from the three-dimensional structure Biochip with dropping 10 wt % Aerogel dispersal solution;

FIG. 16 shows the images of three-dimension chip after the amino reforming process according to a preferred example of the present invention;

FIG. 17 shows the appearance picture of the slide with three-dimensional Aerogel structure according to a preferred example of the present invention;

FIG. 18 shows the images of three-dimension chip after dropping the detected antibody according to a preferred example of the present invention;

FIG. 19 shows the images of three-dimension chip after the prohibition reaction is applied according to a preferred example of the present invention;

FIG. 20 shows the images of three-dimension chip after doing the prohibition reaction and following by drop the antigen (Human IL6);

FIG. 21 shows the images of three-dimension chip from labeling with the first antibody with the represent quantum dot color in the end; and

FIG. 22 shows the scanned images of two-dimensional protein biochip.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

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 R⁴are selected according to the following table.


R¹	R²	R³	R⁴

CH₃	H	CH₃	H
C₂H₅	H	CH₃	H
C₂H₅	H	C₂H₅	H
CH₃CH₂CH₂CH₂	H	CH₃	H
(CH₃)₂CHCH₂	H	CH₃	H
CH₃CH₂CH₂CH₂	H	C₂H₅	H
CH₃	H	CH₃OCH₂CH₂	H
CH₃	H	CF₃CH₂	H
CH₃	CH₃	C₂H₅	H
CH₃	CH₃	CH₃CH₂CH₂	H
C₆H₆CH₂	CH₃	CH₃CH₂CH₂	H
C₆H₆CH₂	CH₃	CH₃CH₂CH₂CH₂	H
C₆H₆CH₂	CH₃	(CH₃CH₂)(CH₃)CH	H
C₆H₆CH₂	CH₃	CH₃CH₂CH₂CH₂CH₂	H
CH₃	H	C₂H₅	CH₃
C₂H₅	H	C₂H₅	CH₃

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-BF₄is 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.

EXAMPLE

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.

- (1) Wash the slide:
  - Immerse the slide in the NaOH solution and shake by Sonicator for 30 min. Then replace NaOH solution with distilled water and vibrate for 30 min again. Lastly, replace distilled water with Acetone and again vibrate for 30 min. Take the slide out and dry them by oven.
- (2) Aerogel preparation:
  - Mix BMIC-BF4 with formic acid solution first, then mix with TEOS again quickly. Set there and wait till hydrolysis and aging. After it gets well-aging, wash the sample and freeze-dry it. Lastly, grind the sample to become powder then the product is formed and can be used.

The Aerogel chemical reaction equation is below:

- (3) Reform the Amino base in Aerogel:
  - Mix the Aerogel powder with 2% DAMO solution well and keep stirring for 1 hour. Filter the mixture and wash it for 24 hours. Afterward, put the,whole sample in the oven to dry it.
- (4) Synthesis the Aerogel on slide:
  - Prepare the 10% dispersal solution by dispersing the reformed Aerogel in double distilled water and keep stirring that with magnetic bar. Drop 2 ul of dispersal solution on the slide by pippet.
- (5) Reform the quantum dot:
  - Prepare the SBB buffer solution: 3.09 g boric acid+19.07 g sodium borate. Adjust the pH to 9 with NaOH or HCl solution.

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:

- (6) Branch the quantum dot with antibody:
  - PBS buffer is prepared by dissolving 0.24 g KH2PO4, 1.44 g Na2HPO4, 0.2 g KCl and 8 g NaCl with double distilled water. Then adjust the pH to 7.5 with NaOH or HCl solution. Washing buffer is prepared by adding 20.5ul Tween 20 into 580 ml PBS buffer and mix well.
  - Mix the required amount of antibody with the quantum dot. Slightly vibrate the solution and then set the mixture in 4° C. refrigerator overnight. The branch reaction of quantum dot and antibody as shown below:

- (7) Implant antigen and antibody on biochip
  - Drop 1 ul of the different concentration antibody(Mouse anti Human, IL-6) on the scheduled area of the slide. Leave the slide in 37° C. oven for 2 hours reaction. Afterward, wash the sample around 1 minute by vibrating with buffer 3 times.
  - Begin the blocking reaction: spread 1% BSA and PBS on the slide. Immerse the slide in a water tank in 4° C. refrigerator overnight. Afterward, wash the slide around 1 minute by vibrating with buffer 3 times.
  - Drop 1 ul of the antigen (Human IL-6) branched with quantum dot on the same spot on the slide. Leave the slide in 30° C. oven for 1 hour reaction. Then wash the slide around 1 minute by vibrating with buffer 3 times.
  - Drop 1 ul of the antibody (Rabbit anti human, IL-6) branched with quantum dot on the same spot on the slide. Leave the slide in 30° C. oven for 1 hour reaction. Then wash the slide around 1 minute by vibrating with buffer 3 times. Lastly, drop 1 ul of the pure quantum dot on the positive control spot on the slide.
  - After steps (1) to (7) are completed, the slide with Aerogel is formed with the white appearance on the top. This area is the sample evaluation region.

FIG. 2 is the analytic result of the infrared absorption spectrum of Aerogel structure. This result demonstrates that there is a —O—H bonding absorption peak during 3200-3700 cm-1 and 1620-1640 cm-1. Most of the absorption peak during 3200-3700 cm-1 is because of the O—H bond in cm-1H bonding vibration. The absorption peak of the 930-950 cm-1 wave is because of the Si—OH bonding vibration. The other waves like 1000-1200 cm-1, 780-820 cm-1 and 430-460 cm-1 will be the bonding extended vibration of ≡Si—O—Si≡.
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 (FIG. 3) showed the main bonding was the 4th replacement (Q4) of Si at −111 ppm. The sub-bonding was the 3nd replacement (Q3) of Si at −102 ppm. This result appears that Silica Aerogel can stable silica net-cross-binding structure.
As it's shown in FIG. 4 and FIG. 5, according to the micro-observation on Aerogel surface by SEM system, the Silica Aerogel surface displays the sphere conformation (FIG. 4, 30k fold amplifier). Furthermore, the sphere shape is composed by more and tinier silica (FIG. 5, 300k fold amplifier). The silica aggregation size is around 20-30 nm which can approve this is the Nano-grande Aerogel material.
N₂Adsorption/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 FIG. 6, the N2 Adsorption/Desorption Analyzer curve of Silica Aerogel, Silica Aerogel was made with 812 m2/g surface area, 1.97 cm3/g pore volume and 9.4 nm is the average pore size. Besides, FIG. 7 with the needle curve means the pore distribution on Silica Aerogel is very condensed.
FIG. 8 is the TGA curve of Silica Aerogel (detected condition: N2, T=30-1000° C. , temperature increasing rate=10° C./min). The result of the solid line shows that 2.6 wt % of Silica Aergel was lost when the temperature was lower than 100° C. . The loss might be from some humidity from the surface adsorption of the Silica Aerogel. 6.2 wt % of Silica Aergel was lost when the temperature reached to 100° C. The lost might be from some oxyhydrogen-silica base which is not hydrolysis or condensate completely of the surface adsorption of the Silica Aerogel.
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. FIG. 9 showed the digital camera picture of the quantum dot under UV light excitation. The excitation is around green light region.
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. FIG. 10 showed the result of the reformed quantum dot of the Fluoresce Spectrometer. The excitation Wave switch to 540 nm is because of the reformed amino base on quantum dot surface. This result means the reform is successful.
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. FIG. 11, the reformed quantum dot moved slower than the un-reformed one considered the reform is success.
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 FIG. 9 with the right tube of the quantum dot:

- A=0.04
- L=0.512
- ε=77793.9851
- Complete the function,

0.04/(77793.9851×0.512)=1.00×10-6

- The calculated concentration will be 1.00×10-6 M

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 FIG. 12, this experiment is applied with Antibody Sandwich Immunoassay(ELISA). Antibody is the bio-detector and fixed on the biochip, then the antibody will have a specific binding with a particular antigen. Besides, the antibody branched with quantum dot is the target in the experiment. If the particular antigen and antibody aren't bind or the antibody and antigen are not corresponding then the fluorescence image won't show up in the fluorescence scanner screen.
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.
FIG. 13 is the washed blank slide. The background value is extremely low as blue color. Use this background value as the standard to compare the rest of the reform slides are with the good background value. For demonstration of the detected result with the three-dimensional Aerogel chip, the 2-dimenetion chip made by the reformed slides is the comparison corps, so the two-dimension chip with reformed amino base (2% DAMO) would be the first issue need to be demonstrated. The reformed procedures of the slides: immerse the slides in the staining container with 2% DAMO prepared by Ethanol for 4 hours reaction. Wash with double distilled water. Put it into the oven for 20 minute. FIG. 14 showed that most of the background is still navy blue (deep blue) after the amino reforming process. Although some small light blue dots appear, the background value is still acceptable.
FIG. 15 showed the scanned image from the three-dimensional structure Biochip with dropping 10 wt % Aerogel dispersal solution. The round dot is the dropping Aerogel. The image color is light blue which is still included in low background value. FIG. 16 showed turquoise (blue-green color) from the amino reformed Aerogel.
FIG. 17 showed the appearance picture of the slide with three-dimensional Aerogel structure. First of all, the sample evaluation region is formed by steps (1) to (7). Second, the positive control spot is the Aerogel reformed by epoxy group directly branched with amino base reformed quantum dot. On the other hand, the negative control spot is the un-reformed Aerogel. The purpose of the positive control spot is to standardize the different batches of Biochip. It's possible that different batches of the Biochip have uneven quality which causes the different intensities of brightness in the biochip scan results. End up the data is out of comparison. Use the positive control spot to standardize the detection result of the different batches of Biochips. This makes the experiment more meaningful. Additionally, the purpose of the negative control spot is to measure the background value of the Aerogel alone. This can be deducted in the background value in the following data analysis.
FIG. 18 showed the background value is still in blue-green color after dropping the detected antibody (Mouse anti Human IL6). FIG. 19 showed the background value is still in blue-green color after the prohibition reaction is applied. FIG. 20 showed the background decrease slightly to light blue after doing the prohibition reaction and following by drop the antigen (Human IL6). FIG. 21 showed the all orange-yellow color is from labeling with the first antibody (Rabbit anti human IL-6) with the represent quantum dot color in the end. This result means the Antibody Sandwich Immunoassay is successful. FIG. 22 showed the scanned picture of two-dimensional protein Biochip.
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:


sample	concentration	intensity	color

1	1.44 × 10⁻⁶	26213	orange-yellow
2	1.44 × 10⁻⁶	28527	orange-yellow
3	1.44 × 10⁻⁶	29041	orange-yellow
4	1.46 × 10⁻⁶	31609	orange-yellow
5	—	11125	blue

Table 2 is the result of the 2-dimension protein chip shown as below:


sample	concentration(M)	intensity	color

1	1.0	52029	white
2	1.0 × 10⁻¹	23130	red
3	1.0 × 10⁻²	30067	orange
4	1.0 × 10⁻³	22615	green
5	5.0 × 10⁻⁴	16756	blue

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.

Claims

1. A biochip with a three-dimensional structure, comprising a substrate covered with a three-dimensional structure, wherein the surface of the three-dimensional structure layer is formed by a aerogel powder, and the internal and external surface of the aerogel powder was modified by at least one specific moiety and the specific moiety comprises one selected from the group consisting of the following: amine group, hydroxyl group, carboxyl group, and epoxy group.

2. The biochip according to claim 1, wherein the specific surface area of the aerogel powder is more than or equal to 100 m²/g.

3. The biochip according to claim 1, wherein the average pore diameter of the aerogel powder is less than or equal to 20 nm.

4. The biochip according to claim 1, wherein the porosity of the aerogel powder ranges about 50% to 99%.

5. The biochip according to claim 1, wherein the diameter of the aerogel powder ranges about 10 nm to 250 nm.