WO2007139283A1 - Procédé d'élaboration de biocapteur photonique-fluidique à cristaux photoniques fonctionnalisés - Google Patents

Procédé d'élaboration de biocapteur photonique-fluidique à cristaux photoniques fonctionnalisés Download PDF

Info

Publication number
WO2007139283A1
WO2007139283A1 PCT/KR2007/001874 KR2007001874W WO2007139283A1 WO 2007139283 A1 WO2007139283 A1 WO 2007139283A1 KR 2007001874 W KR2007001874 W KR 2007001874W WO 2007139283 A1 WO2007139283 A1 WO 2007139283A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
photonic
biosensor
photonic crystals
functionalized
Prior art date
Application number
PCT/KR2007/001874
Other languages
English (en)
Inventor
Seung-Man Yang
Sang Yup Lee
Tae Jung Park
Seung-Kon Lee
Original Assignee
Korea Advanced Institute Of Science And Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Korea Advanced Institute Of Science And Technology filed Critical Korea Advanced Institute Of Science And Technology
Publication of WO2007139283A1 publication Critical patent/WO2007139283A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
    • G01N21/774—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides the reagent being on a grating or periodic structure

Definitions

  • the present invention relates to a biosensor having a substance capable of binding to biological materials linked to a chemical functional group of functionalized photonic crystals to which a chemical functional group selected from the group of consisting of amine group (-NH 2 ), aldehyde group (-CHO), and carboxyl group (-COOH) is bound, as well as, a method for detecting a target substance without a label using the same.
  • a chemical functional group selected from the group of consisting of amine group (-NH 2 ), aldehyde group (-CHO), and carboxyl group (-COOH) is bound
  • Biosensing materials used for biochips or biosensors are enzymes, antibodies, antigens, lectins, hormones, receptors and so on, which selectively respond and are combined with a specific substance.
  • .methods for detecting a target substance which reacts with or binds to biological materials on a biochip or a biosensor include various ways such as a method using fluorescent materials, a method using cantilever vibration, a method using surface plasmon resonance (SPR), a method using quartz crystal microbalance, or an electrochemical method etc.
  • SPR surface plasmon resonance
  • quartz crystal microbalance or an electrochemical method etc.
  • electrochemical method most common ways are a method using fluorescent materials and an electrochemical method. However, these two methods are based on detecting a signal by attaching a specific type of label to a biological material.
  • reflectance interference spectroscopy is a method of observing a change of interference spectrum by making a lattice structure, which causes interference, and attaching a biological material to the surface of the lattice structure. It is known that sensitivity of up to 10 fM could be achieved and substances of the composition of a lattice structure are various including a high molecular weight hydrogel, silica, and titania and so on. As described above, a lattice structure using the interference of light conceptionally has a lot of similarity to photonic crystals.
  • Photonic crystals are substances prepared by regularly arranging substances having a different refractive index one-, two-, or three-dimensionally, which have an inherent characteristic of selective absorption or reflection of light of a specific wavelength. While both etching and deposition processes used in semiconductor process are mainly utilized in the manufacture of one- or two- dimensional photonic crystal, due to low economic efficiency and limitations for efficient process, self-assembly using nanoparticles is being spotlighted in three- dimensional photonic crystal which controls light three- dimensionally.
  • the elementary principle of colloidal photonic crystals is to selectively control the wavelength of reflected light by regular arrangement of particles having the size corresponding to the wavelength of light.
  • nanoparticles with specific refractive index form a three-dimensional regular crystal structure, it is possible to not only achieve a high reflection rate in any direction by regularly scattering a particular wavelength of light according to arrangement structure and refractive index but also control the wavelength of light reflected or penetrated by regulating microstructure. Assembly of nanoparticles having these characteristics is called photonic crystal since it has regular crystal structure and comes into the spotlight as a new material in every field using light.
  • colloidal photonic crystals can be obtained by crystallizing in the length of light wavelength, through sedimentation or evaporation of colloidal particles of uniform size dispersed in a medium.
  • This structure called colloidal crystals, has a high reflection rate for a specific color among light delivered to crystal surface, which means that light of a specific wavelength can not penetrate the photonic crystals.
  • These characteristics of reflection indicates that light can be trapped or moved in a desired direction when structures with intentional defect are formed inside crystals and thus can be applied to next-generation optical devices having the ability of high integration and high function to replace optical fiber and photonic wave guide which are uncontrollable in complex microstructures, thereby drawing a lot of attention.
  • photonic bandgap which can be changed according to refractive index of materials, the arrangement interval between two particles
  • an effective refractive index change of the material can be calculated by the following formula 1.
  • f s refers to particle volume fraction
  • n s is refractive index of a particle
  • n a i r is refractive index of a filling up material Therefore, when utilizing nanoparticles having the surface characteristic capable of selective immobilization of biological materials such as DNA, RNA, proteins, enzymes, pathogens or viruses, it is advantageous in that it can be applied to a biological detection device by sensing the moving of a photonic bandgap in which a biological material is attached to a photonic crystal surface consisting of nanoparticles to change.
  • the present inventors have owned a patent relating to a method for manufacturing said photonic crystals and a method for manufacturing colloidal crystals with variable forms using a capillary and multi-pore structure (KR 10- 0466250B).
  • KR 10- 0466250B a method for manufacturing colloidal crystals with variable forms using a capillary and multi-pore structure
  • a photonic-fluidic fusion device When using a photonic-fluidic fusion device as a biosensing device, it has various advantages in that analysis can be performed by injecting small amounts of sample (nanoliters per run) due to the use of a microfluidic channel and the amount of a sample used is reduced by 30% because nanoparticles occupy 70% of the volume compared to the existing microfluidic devices.
  • a centrifugal microfluidic device it has advantages in that analysis can be shortened due to an increased crystallization speed compared to the existing process using evaporation, it is possible to analyze various samples at the same time since several chips can be integrated on a CD (compact disk)-type substrate.
  • the aforementioned advantages are far more advanced compared to the existing photonic crystal-based biosensing materials.
  • the existing biosensing materials have disadvantages in that they are dispersed in the fluid in the bulk state or they are in the form of a powder so that it is difficult to have compatibility with a specific device and they require a large amount of samples.
  • a circular photonic crystal is small in size and its change in color is severe depending on the angle of incidence, there are lots of problems in practical use for an accurate diagnosis
  • the present inventors have conducted intensive studies to develop a biosensor which is easy to use, accurate, and easily applicable using photonic crystals, and consequently, paid attention to a photonic crystal functionalized with amine group (-NH 2 ), aldehyde group (-CHO), and carboxyl group (-COOH), and fabricated a biosensor by attaching a substance capable of binding to biological materials to the functionalized photonic crystals to confirm that the biosensor can sense a target substance without a label to detect it, thereby completing the present invention.
  • amine group -NH 2
  • aldehyde group -CHO
  • carboxyl group -COOH
  • a main object of the present invention is to provide a biosensor or a biochip having a substance, which binds to biological materials, linked to a chemical functional group of functionalized photonic crystals to which the chemical functional group selected from the group of consisting of amine group (-NH 2 ), aldehyde group (-CHO), and carboxyl group (-COOH) is bound and a method for fabricating the same.
  • Another object of the present invention is to provide a method for detecting target substances, the method comprising using the biosensor or the biochip.
  • a further object of the present invention is to provide a photonic-fluidic fusion device including the biosensor and a method for detecting biological materials, the method comprising using the photonic-fluidic fusion device.
  • Another additional object of the present invention is to provide CD fluidics on which the photonic-fluidic fusion devices are integrated.
  • the present invention is to provide a method for fabricating a biosensor or a biochip, the method comprising the steps of: (a) injecting a colloidal suspension into a capillary tube; (b) crystallizing colloids by evaporating solvent from the colloidal suspensions; (c) obtaining photonic crystals by removing the capillary tube; (d) preparing functionalized photonic crystals by binding a chemical functional group selected from the group of consisting of amine group (-NH 2 ), aldehyde group (-CHO), and carboxyl group (-COOH) to the obtained photonic crystals; and (e) attaching a substance, which binds to biological materials, to the chemical functional group of the functionalized photonic crystals.
  • the present invention is to provide a biosensor or a biochip prepared by the above method, wherein a substance, which binds to biological materials, is linked to functionalized photonic crystals to which a chemical functional group selected from the group of consisting of amine group (-NH 2 ), aldehyde group (-CHO), and carboxyl group (-COOH) is bound.
  • a chemical functional group selected from the group of consisting of amine group (-NH 2 ), aldehyde group (-CHO), and carboxyl group (-COOH) is bound.
  • the present invention is to provide a method for detecting target substances, the method comprising using the biosensor or the biochip. Furthermore, the present invention is to provide a photonic-fluidic fusion device including the biosensor and a method for detecting biological materials, the method comprising using the photonic-fluidic fusion device.
  • the present invention is to provide CD fluidics on which the photonic-fluidic fusion devices are integrated.
  • FIG. 1 is a scanning electron microscopy (SEM) image of photonic crystals: (A) is photonic crystals formed by regularly arranging silica particles and (B) is photonic crystals formed by regularly arranging polystyrene particles
  • FIG. 2 is a photograph displaying the attachment of functionalized photonic crystals on a substrate using dip-coating.
  • FIG. 3 shows the result of reflection spectra of interaction between a specific nucleic acid of a pathogen (Fusobacterium necrophorum, ATCC25286) causing septicemia and a functionalized photonic crystal to which a probe consisting of 45 bases is bound using a receptor.
  • a pathogen Feusobacterium necrophorum, ATCC25286
  • FIG. 4 is shows the result of reflection spectra of interaction between a specific nucleic acid of a pathogen (Actinetobacter baumannii, KCTC 2771) causing septicemia and a functionalized photonic crystal to which a probe consisting of 45 bases is bound using a receptor.
  • FIG. 5 is a genetic map of plasmid pTrcGBP-SCVme.
  • FIG. 6 shows the result of reflection spectra of interaction between an antibody and a functionalized photonic crystal to which a surface antigen of SARS coronavirus is bound using a receptor.
  • FIG. 7 shows the result of reflection spectra of interaction between a gold (Au) crystal of an average of 5nm diameter and a functionalized photonic crystal to which a gold binding polypeptide is bound using a receptor.
  • FIG. 8 is a SEM image displaying the result obtained by binding an antibody and a target cell using a functionalized photonic crystal.
  • FIG. 9 is a schematic view of crystallization at the end by centrifugal force as a reaction occurs for the detection of biological materials by functionalized photonic crystals inside a channel of a photonic-fluidic fusion device according to the present invention
  • (a) is each part of a centrifuge-based photonic-fluidic fusion device chip
  • (b) is the actual photograph
  • (C) is a schematic view of a channel end of the centrifuge-based photonic-fluidic fusion device chip
  • (d) is an illustration of crystallization at the channel end of the photonic-fluidic fusion device chip.
  • photonic crystals were prepared by a method of the patent (KR 10-0466250B) owned by the present inventors. That is, after injecting a colloidal suspension into a capillary tube, colloids were crystallized by evaporating solvent from the colloidal suspension and then photonic crystals were prepared by removing the capillary tube.
  • the above-mentioned patent application did not contain examples of detecting target substances by the fabrication of a biosensor using photonic crystals.
  • the prepared photonic crystals were linked to a chemical functional group selected from the group consisting of amine group (-NH 2 ), aldehyde group (- CHO), and carboxyl group (-COOH) to obtain functionalized photonic crystals.
  • a chemical functional group selected from the group consisting of amine group (-NH 2 ), aldehyde group (- CHO), and carboxyl group (-COOH)
  • biosensing materials are immobilized by a linker shown in FIG. 6.
  • biological materials are attached to photonic crystals without a linker by covalent or non-covalent bonds, which is different from the method of KR 10-2006-0007830. That is, methods for immobilizing biological materials on the surface of photonic crystals include physical adsorption, chemical binding, electrochemical binding, electrostatic binding, hydrophobic binding and hydrophilic binding.
  • Chemical binding refers to a binding by a chemical functional group such as amine group, carboxyl group, aldehyde group, thiol group, phosphate group, nickel group, acidic group, alkane group, alkene group, alkyne group, aromatic group, alcohol group, ether group, ketone group, esther group, amide group, amino acids, nitro group, nitryl group, carbohydrate group, lipid group, phospholipid group, steroid group etc.
  • photonic crystal surface having the chemical functional group is able to not only immobilize different kinds of linkers but also attach biological materials without these linkers.
  • the surface of photonic crystals having amine group or aldehyde group can be used to bind DNA or proteins covalently or non-covalently without a linker.
  • silica surface is treated with acid to release active silanol groups on the surface.
  • the surface silanol groups are modified with silane derivatives such as (3-aminopropyl) triethoxysilane to obtain a surface layer having reactive amine or carboxyl groups, which enables covalent or non- covalent bonds with biological materials.
  • silane derivatives such as (3-aminopropyl) triethoxysilane to obtain a surface layer having reactive amine or carboxyl groups, which enables covalent or non- covalent bonds with biological materials.
  • a biosensor or a biochip in which a substance capable of binding to biological materials is linked to a chemical functional group of the functionalized photonic crystals, was fabricated.
  • a biosensor or a biochip according to the present invention has a merit of detecting target substances without a label by functionalizing photonic crystals.
  • the present invention relates to a method for fabricating a biosensor or a biochip which comprises the steps of: (a) injecting a colloidal suspension into a capillary tube; (b) crystallizing colloids by evaporating solvent from the colloidal suspension; (c) obtaining photonic crystals by removing the capillary tube; (d) preparing functionalized photonic crystals by binding a chemical functional group selected from the group consisting of amine group (-NH 2 ), aldehyde group (-CHO), and carboxyl group (-COOH) to the obtained photonic crystals; and (e) linking a substance, which binds to biological materials, to a chemical functional group of the functionalized photonic crystals, and a biosensor or biochip prepared by the above method, in which a substance capable of binding to biological materials is linked to functionalized photonic crystals to which a chemical functional group selected from the group consisting of amine group (-NH 2 ), aldehyde group (-CHO), and carboxyl group (-
  • the substance which binds to biological materials is preferably selected from the group consisting of nucleotides, proteins, peptides, carbohydrates, fatty acids, lipids, ligands, cells, and metals
  • the proteins are preferably enzymes or antibodies
  • the ligands are preferably selected from the group consisting of compounds which bind to one or more of nucleotides, proteins, peptides, carbohydrates, fatty acids, lipids, cells, and metals.
  • the present invention in another aspect, relates to a method for detecting target substances, the method comprising the use of the biosensor or the biochip.
  • the method preferably comprises detecting target substances without a label, and the target substances are preferably selected from the group consisting of nucleotides, proteins, ligands, compounds, carbohydrates, fatty acids, lipids, cells, and metals.
  • the inventive biosensor or biochip having a substance capable of binding to biological materials linked to functionalized photonic crystals can be used by attaching it to a dip-coated glass substrate or a photonic-fluidic fusion device.
  • the present invention in another further aspect, relates to a photonic- fluodic fusion device containing the biosensor and a method for detecting biological materials, the method comprising utilizing a photonic-fluidic fusion device.
  • NIL Nanoprint lithography
  • Example 1 Manufacture of photonic crystals
  • FIG. l(A) illustrated a SEM image of the manufactured silica photonic crystals.
  • Example 2 Manufacture of silica photonic crystals added with amine (-NH 2 ) group
  • Silica photonic crystals prepared in Example 1 were dispersed into ethanol to 0.5- 1.5% by weight and were thoroughly mixed for 10 minutes after adding with ammonia (NH 4 OH) solution (5-8% by volume). 0.3 vol% of silica/ethanol dispersion was dropwised into a solution which were diluted with 1-2 mi ethanol per 1 g of (3-aminopropyl)triethoxysilane. Tetraethoysilane corresponding to 2-3 volumes of the dilute solution of (3-aminopropyl) triethoxysilane was dropwised and allowed to react with slow stirring for over 48 hours.
  • NH 4 OH ammonia
  • Silica photonic crystals prepared in Example 1 were surface treated with (3- aminopropyl) triethoxysilane and dried in a vacuum. N,N-dimethylformamide 4 times heavier than the weight of silica photonic crystals and 30-40 wt% of poly(acrylic acid)) were well-mixed to be dispersed and subjected to heating in a 140 0 C oil bath for 2 hours, followed by substituting methanol for the solvent, thus collecting photonic crystals by drying it in a vacuum oven.
  • Example 4 Attachment of functionalized photonic crystals on a substrate using dip-coating.
  • the substrate was exposed to O 2 plasma (oxygen plasma, LFE plasma Etcher, LFE corporation, Massachusetts, USA), and then functionalized 200-300 nm of monodisperse silica photonic crystals were immersed in a solution dispersed in distilled water or ethanol to a concentration of 0.5-1 wt%.
  • O 2 plasma oxygen plasma, LFE plasma Etcher, LFE corporation, Massachusetts, USA
  • the suspension was slowly stirred at a speed at which the interface was not destroyed.
  • the glass substrate was vertically immersed in the functionalized silica photonic crystal dispersion and slowly pulled up at a velocity of 0.2-0.5 ⁇ m to coat colloidal crystals in the form of a thin film, thus manufacturing functionalized photonic crystals attached on the substrate.
  • the coating was performed according to process variables such as a three-phase contact angle (air, substrate, and solution) and evaporation velocity of a solution etc.
  • process variables such as a three-phase contact angle (air, substrate, and solution) and evaporation velocity of a solution etc.
  • complex forces such as gravity, buoyant force, evaporation, and capillary force are applied.
  • FIG. 2 illustrated a photograph of the chip-substrate produced by a self-assembly method of monodisperse nano-photonic crystals.
  • Example 5 Fabrication example 1 of a biochip for detecting nucleic acids using functionalized photonic crystals
  • Photonic crystals functionalized with amine (-NH 2 ) group, manufactured in Example 2 were attached on a substrate using the same method as described in Example 4.
  • a probe DNA capable of specifically detecting Fusobacterium necrophorum ATCC25286, one of pathogens causing septicemia in blood vessels, was attached to the photonic crystals to bond it with F. necrophorum- specific DNA, thus manufacturing a biochip capable of detecting F. necrophorum.
  • the probe DNA (SEQ ID NOs: 1 and 2) specific to F. necrophorum DNA was attached to an amine group on the surface of photonic crystals by ionic bonds to immobilize on the photonic crystal surface.
  • SEQ ID NO: 1 S'-GTAGTTTTCTTGCGCTGTAT-S' (Probe DNA-Fne2-20)
  • SEQ ID NO:2 5'-GAAAGTCCTGTATTGGTAGTTTTCTTGCGCTGTA TCTC TTCTCCC-3' (Probe DNA-Fne2-45)
  • Photonic crystals replaced with the amine groups were placed into PBS (Phosphate Buffered Saline, 137 mM sodium chloride, 10 mM phosphate, 2.7 mM potassium chloride, pH 7.4) buffer and 1 ⁇ M of probe DNA was added, followed by allowing to react at 25°C for 1 hour. After the reaction, in order to facilitate the binding of the probe DNA and target DNA, UV light was irradiated to induce crosslinking. In order to remove the remaining oligonucleotides, the photonic crystals were washed three times with PBS buffer (pH 7.4).
  • PBS Phosphate Buffered Saline, 137 mM sodium chloride, 10 mM phosphate, 2.7 mM potassium chloride, pH 7.4
  • the complementary double helix DNA fractions obtained from PCR was subjected to agarose gel electrophoresis to isolate about 815 bp of DNA fragments.
  • the reactant was converted to single stranded DNA by enzymatic treatment.
  • the obtained single stranded DNA and reactants including probe- bound photonic crystals were reacted in PBS buffer (pH 7.4) at 3O 0 C for 6 hours.
  • the PCR was performed as follows: first denaturation at 94°C for 5 minutes 1 time, 30 cycles consisting of second denaturation at 94 0 C for 1 minute, annealing at 56 0 C for 1 minute and extension at 72 0 C for 1 minute; and final extension at 72 0 C for 5 minutes 1 time.
  • a control group-01 is a photonic crystal to which only probe DNA is bound
  • a control group-02 is a negative control group in which a probe-bound photonic crystal is bound to about 900 bp of DNA (derived from Klebsiella pneumoniae ATCC 700603) which is different from the sequence and size of the target DNA.
  • Example 6 Fabrication example 2 of a biochip for detecting nucleic acids using functionalized photonic crystals
  • Photonic crystals functionalized with amine (-NH 2 ) group, manufactured in Example 2 were attached on a substrate using the same method as described in Example 4.
  • a probe DNA capable of specifically detecting Actinetobacter baumannii (KCTC 2771), one of pathogens causing septicemia, was attached to the photonic crystals to bond it with A. baumannii-specific DNA, thus manufacturing a biochip capable of detecting A. baumanni.
  • the probe DNA (SEQ ID NOs:3-5) specific to A. baumanni DNA was attached to an amine group on the surface of photonic crystals by ionic bonds to immobilize on the photonic crystal surface.
  • SEQ ID NO:3 5 I -AGGGCACACATAATG-3 I (probe DNA- Acti23 SOl-15)
  • SEQ ID NO:4 5'-ACGAAAGGGCACACATAATG-S'
  • SEQ ID NO:5 5'-AACGTAGAGGGTGATATTCCCGTACACGAA AGGGCACACA TAATG-3 1 (probe DNA-Acti23S01-45) 1585Fw and 23BR (Keum, et ah, MoI. Cell Probes., 20:42-50, 2006) as a primer set were used to make target DNA.
  • a probe binding species-specifically to them was designed such that base sequences of 23 S rDNA and 16S-23S rDNA intergenic spacer region (ISR) of A. baumannii (KCTC 2771) were aligned by a multiple sequence alignment method (Keum, et al., MoI. Cell Probes., 20:42-50, 2006).
  • Photonic crystals replaced with the amine groups were placed into PBS (pH 7.4) buffer and 1 ⁇ M of probe DNA was added, followed by allowing to react at 25°C for 1 hour. After the reaction, in order to facilitate the binding of the probe DNA and target DNA, UV light was irradiated to induce crosslinking. In order to remove the remaining oligonucleotides, the photonic crystals were washed three times with PBS buffer (pH 7.4).
  • the complementary double helix DNA fractions obtained from PCR was subjected to agarose gel electrophoresis to isolate about 1239 bp of DNA fragments.
  • the reactant was converted to single stranded DNA by enzymatic treatment.
  • the obtained single stranded DNA and reactants including probe- bound photonic crystals were reacted in PBS buffer (pH 7.4) at 3O 0 C for 6 hours.
  • the PCR was performed as follows: first denaturation at 94°C for 5 minutes 1 time, 30 cycles consisting of second denaturation at 94°C for 1 minute, annealing at 56 0 C for 1 minute and extension at 72°C for 1 minute; and final extension at 72 0 C for 5 minutes 1 time.
  • a control group-01 is a photonic crystal to which only probe DNA is bound
  • a control group-02 is a negative control group in which a probe-bound photonic crystal is bound to about 900 bp of DNA (derived from K. pneumoniae ATCC 700603) which is different from the sequence and size of the target DNA.
  • Example 7 Fabrication of a biochip for detecting antigen-antibody-reaction using functionalized photonic crystals
  • GBP Gold Binding Polypeptide
  • SARS coronavirus in a fused form using a recombinant plasmid pTrc-SCVe-SBD (Lee, SJ. et al, Anal. Chem., 77:5755, 2005) as a template DNA primers of SEQ ID NOs:6-8 were used.
  • the digestion site of restriction enzyme Ncol was inserted into the primer of SEQ ID NO: 6 and the digestion site of a restriction enzyme HmdIII was inserted into the synthesized primer of SEQ ID NO: 8
  • SEQ ID NO:7 S'-CGCAAGCCACGTCTGGTACGATTCAATCTATGCAC
  • SEQ ID NO:8 5'-CGCGCGGGATCCTTATTAAACCAGCAGGTCCGGAA
  • PCR was performed using the above pTrc-SCVe-SBD as a template and the primers of SEQ ID NOs:7 and 8, PCR was performed using the amplified DNA products as a template and primers of SEQ ID NOs: 6 and 8, thus obtaining PCR products in a fused form of surface antigens of GBP and SARS coronavirus.
  • PCR was performed as follows: first denaturation at 94°C for 5 minutes 1 time,
  • FIG. 5 is a genetic map illustrating the recombinant plasmid pTrcGBP- SCVme.
  • a GBP protein is fused at the N-terminal end of the surface antigen of SARS coronavirus. From the point of the present invention, although GBP protein is fused at the C-terminal end of it, it is obvious to a person skilled in the art that the same results can be obtained.
  • E. coli XLl -Blue (Stratagene, USA) was transformed with the recombinant plasmid pTrcGBP-SCVme and gene expression was induced by the treatment of isopropyl- ⁇ -thiogalactoside (IPTG).
  • IPTG isopropyl- ⁇ -thiogalactoside
  • the E. coli XLl -Blue transformed by pTrcGBP-SCVme could produce a part of the surface antigen (SCVme) which was fused with gold binding polypeptide, and was inoculated into a 50OmL flask containing 100 ml of LB media and cultured at 37°C.
  • the recombinant plasmid pTrcGBP- SCVme contains a trc promoter
  • 1 mM IPTG was added to induce the gene expression when the optical density at a 600 nm wavelength reached 0.6.
  • the culture broth was centrifuged at 4°C and 6,000 rpm for 5 minutes and the supernatant was discarded.
  • the resulting precipitate was washed with 100 ml of TE buffer (Tris-HCL 1OmM; EDTA ImM, pH8.0). The washed substance was centrifuged at 4°C and 6,000 rpm for 5 minutes, and then suspended in 100 ml of TE buffer.
  • the resulting cell was disrupted in an ultrasonicator (Branson Ultrasonics Co., USA).
  • the disrupted solution was centrifuged at 4 0 C and 6,000 rpm for 30 minutes, and the supernatant was collected and filtered through a 0.2 /an filter with discarding undissolved proteins. Proteins contained in the filtrate were quantified by the Bradford protein assay (Bradford, M.M., anal. Biochem., 72:248-54, 1976), and then diluted with PBS buffer (pH 7.4) to the concentration of 1 mg/ml.
  • Example 2 were attached on a substrate using the same method as described in
  • Example 4 Functionalized photonic crystals were fixed in the prepared solution at 37°C for 30 minutes, and washed 3 times with PBS buffer (pH 7.4) at ambient temperature. Protein-immobilized photonic crystals were reacted with rabbit anti-SARS coronavirus surface protein antibody at 37 0 C for 30 minutes and washed 3 times with PBS buffer (pH 7.4) at ambient temperature to analyze photonic spectra, thus detecting the reaction (FIG. 6). As a result, photonic crystals as a control group showed a peak reflectance at about 570 nm wavelength, whereas photonic crystals to which the surface antigen protein of SARS coronavirus is specifically bound showed a peak reflectance at about 580 nm.
  • the surface antigen of SARS coronavirus was exemplified as a target protein.
  • a target protein it is obvious to a person skilled in the art that it is possible to use various proteins or ligands as a target protein.
  • Example 8 Fabrication of a biochip for detecting metals using functionalized photonic crystals
  • Photonic crystals functionalized with amine (-NH 2 ) group, manufactured in Example 2 were attached on a substrate using the same method as described in Example 4. Then, the functionalized photonic crystals were fixed with gold binding polypeptide, manufactured by a method in KR 10-2005-0104830 owned by the present inventors, at ambient temperature for 30 minutes and washed with PBS buffer (pH 7.4) 3 times. GBP-immobilized photonic crystals were allowed to react with a suspension of a gold colloid with an average 5-nm of diameter at ambient temperature for 30 minutes and washed 3 times with PBS buffer (pH 7.4) at ambient temperature to analyze photonic spectra, thus detecting the reaction (FIG. 7).
  • photonic crystals as a control group showed a peak reflectance at about 558 nm wavelength
  • photonic crystals to which GBP is specifically bound showed a peak reflectance at about 565 nm
  • gold crystals specifically reacted with GBP showed a peak reflectance at about 569 nm
  • a gold crystal was exemplified as a target metal substance.
  • a gold crystal was exemplified as a target metal substance.
  • various metal substances as a target metal substance.
  • An antibody (cat no. ab8273) made by Abeam (Cambridge, MA, USA) was used as a monoclonal antibody for detecting Salmonella, which is mouse monoclonal antibody to Salmonella core antigen.
  • Photonic crystals functionalized with carboxyl (-COOH) group, manufactured in Example 3 were attached on a substrate using the same method as described in Example 4. Then, the Salmonella antibody were mixed well with the functionalized photonic crystals and immobilized at 37°C for 30 minutes and then washed 3 times with PBS buffer (pH 7.4) at ambient temperature. After the antibody-immobilized photonic crystals were allowed to react with S.
  • Predictive example 1 Introduction of a functionalized photonic crystal into a photonic-fluidic fusion device and application to a biosensor crystallizing photonic crystals using centrifugal force
  • a chip-substrate was manufactured by the self-assembly method of a monodisperse nano-photonic crystal of the manufactured silica photonic crystals.
  • FIG. 9(a) photonic-fluidic fusion device capable of utilizing centrifugal force can be prepared. The detailed schematic diagram is illustrated in FIG. 9(c).
  • colloidal suspensions of photonic crystals which are functionalized with reaction groups, are injected into reservoirs located at the upper part of each channel and then each probe and target samples are allowed to flow into the channels, thereby inducing the reaction.
  • the oxygen plasma treatment makes the channel surfaces more hydrophilic and thus increases the capillary forces of fluids. If CD is rotated using the centrifugal force, the colloids (each sample and colloidal suspension) which are reacted at the upper part of each channel migrate into the ends of the channels. As shown in FIG. 9(d), since the colloidal particles which are arrived at the ends of the channels by the centrifugal force are dried by exposure to the ambient air, the colloidal particles are crystallized.
  • the degree of crystallization can not only maintain the shape of colloidal particles depending on controlling the effect of drying time, temperature, and humidity and so on, but also make the accurate measurement of optical spectrum to be obtained.
  • the reservoirs have small holes to have effects of evaporation prevention and crystallization facilitation by controlling the input of air.
  • the fluidic channels are rotated at 400-1600 rpm for 15-60 minutes.
  • the solvent could be removed by high speed centrifugation at 4000 rpm for 15 minutes. Also, the remaining solvent can be removed in the vacuum.
  • the photonic-fluidic fusion device and CD fluidics having the following features; use as a colloidal photonic crystal based biosensing device, analytical feature using microfluidic devices, and manipulating feature of small amounts of samples.
  • optical spectra were measured by actually reacting a probe substance with a target substance on a substrate on which functionalized colloidal crystals are dip-coated, instead of a fluidic device.
  • the present invention is effective to provide a biosensor or a biochip in which a substance capable of binding to biological materials is linked to a chemical functional group of functionalized photonic crystals and a method for fabricating the same.
  • the present invention is also effective to provide a method for detecting a target substance without a label using the biosensor or a photonic-fluidic fusion device including the biosensor.
  • the biosensor can detect various target substances in a more stable manner with higher efficiency than the existing biosensors and has high productivity and economic efficiency due to simple manufacturing process thereof.

Landscapes

  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plasma & Fusion (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

Biocapteur dans lequel une substance capable de liaison avec des matériaux biologiques est liée à un groupe chimique fonctionnel de cristaux photoniques fonctionnalisés auxquel est lié le groupe chimique fonctionnel pouvant appartenir au groupe des éléments suivants: groupe amine (-NH2), groupe aldéhyde (-CHO), et groupe carboxyle (-COOH). Également, dispositif de fusion photonique-fluidique comprenant le biocapteur, et procédé de détection de substances cibles sans élément de marquage par le biais du biocapteur ou de ce dispositif de fusion.
PCT/KR2007/001874 2006-05-26 2007-04-17 Procédé d'élaboration de biocapteur photonique-fluidique à cristaux photoniques fonctionnalisés WO2007139283A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2006-0047807 2006-05-26
KR20060047807 2006-05-26

Publications (1)

Publication Number Publication Date
WO2007139283A1 true WO2007139283A1 (fr) 2007-12-06

Family

ID=38778778

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2007/001874 WO2007139283A1 (fr) 2006-05-26 2007-04-17 Procédé d'élaboration de biocapteur photonique-fluidique à cristaux photoniques fonctionnalisés

Country Status (1)

Country Link
WO (1) WO2007139283A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101285820B (zh) * 2008-04-30 2011-07-27 中国科学院化学研究所 反蛋白石结构膜的用途
WO2012078351A3 (fr) * 2010-11-29 2012-08-16 President And Fellow Of Harvard College Manipulation de fluides dans des structures photoniques poreuses tridimensionnelles ayant des propriétés de surface comprenant des motifs
DE102012219643A1 (de) * 2012-10-26 2014-04-30 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Sensorelement mit einer photonischen kristallanordnung
CN110987888A (zh) * 2019-12-16 2020-04-10 南京工业大学 自组装光子晶体毛细传感器及其制备方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040028360A (ko) * 2002-09-30 2004-04-03 한국과학기술원 구형의 콜로이드 결정, 다공성 구조체의 제조방법 및 이에사용되는 전기수력학적 분무장치
US20040134414A1 (en) * 2002-07-31 2004-07-15 Lewis John South Layered photonic crystals
KR100466250B1 (ko) * 2002-09-30 2005-01-14 한국과학기술원 다양한 형태의 콜로이드 결정 및 다공성 구조체의 제조방법
US6946086B2 (en) * 2000-12-01 2005-09-20 Clemson University Chemical compositions comprising crystalline colloidal arrays
US20050250158A1 (en) * 2004-03-03 2005-11-10 Atul Parikh Arrays of colloidal crystals
KR20060007830A (ko) * 2004-07-22 2006-01-26 주식회사 엘지화학 생체감지물질이 고정된 광결정구 및 이의 제조방법 및이용방법

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6946086B2 (en) * 2000-12-01 2005-09-20 Clemson University Chemical compositions comprising crystalline colloidal arrays
US20040134414A1 (en) * 2002-07-31 2004-07-15 Lewis John South Layered photonic crystals
KR20040028360A (ko) * 2002-09-30 2004-04-03 한국과학기술원 구형의 콜로이드 결정, 다공성 구조체의 제조방법 및 이에사용되는 전기수력학적 분무장치
KR100466250B1 (ko) * 2002-09-30 2005-01-14 한국과학기술원 다양한 형태의 콜로이드 결정 및 다공성 구조체의 제조방법
US20050250158A1 (en) * 2004-03-03 2005-11-10 Atul Parikh Arrays of colloidal crystals
KR20060007830A (ko) * 2004-07-22 2006-01-26 주식회사 엘지화학 생체감지물질이 고정된 광결정구 및 이의 제조방법 및이용방법

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101285820B (zh) * 2008-04-30 2011-07-27 中国科学院化学研究所 反蛋白石结构膜的用途
WO2012078351A3 (fr) * 2010-11-29 2012-08-16 President And Fellow Of Harvard College Manipulation de fluides dans des structures photoniques poreuses tridimensionnelles ayant des propriétés de surface comprenant des motifs
US9279771B2 (en) 2010-11-29 2016-03-08 President And Fellows Of Harvard College Manipulation of fluids in three-dimensional porous photonic structures with patterned surface properties
DE102012219643A1 (de) * 2012-10-26 2014-04-30 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Sensorelement mit einer photonischen kristallanordnung
DE102012219643B4 (de) * 2012-10-26 2014-09-25 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Sensorelement mit einer photonischen kristallanordnung
CN110987888A (zh) * 2019-12-16 2020-04-10 南京工业大学 自组装光子晶体毛细传感器及其制备方法

Similar Documents

Publication Publication Date Title
Inan et al. Photonic crystals: emerging biosensors and their promise for point-of-care applications
CA2490413C (fr) Conjugues polyanioniques de nanoparticules et methodes d'utilisation de ces conjugues pour detecter des substances a analyser
Phillips et al. Recent advances in surface plasmon resonance based techniques for bioanalysis
Guo Surface plasmon resonance based biosensor technique: a review
US7709243B2 (en) Biochip and biomolecular detection system using the same
TWI519774B (zh) 表面增強拉曼散射用金屬粒子及分子感測、以及其製造方法
EP2878950B1 (fr) Biocapteur comprenant des nanoparticules métalliques
Salva et al. Methods for immobilizing receptors in microfluidic devices: A review
Jang et al. Fabrication of protein chips based on 3-aminopropyltriethoxysilane as a monolayer
BRPI0621413A2 (pt) método de processamento de uma amostra biológica e/ou quìmica
JP2017503502A (ja) 調節可能な移動性を有するプローブと結合された支持型脂質二重層を含む人工細胞膜及びそれを用いて分子間の相互作用を分析する方法
JP5315351B2 (ja) 試料中の選択された種類の分子を検出するための方法
Satija et al. A dendrimer matrix for performance enhancement of evanescent wave absorption-based fiber-optic biosensors
Albers et al. Surface plasmon resonance on nanoscale organic films
Chiu et al. Graphene oxide based surface plasmon resonance biosensors
Drozd et al. Recent advancements in receptor layer engineering for applications in SPR-based immunodiagnostics
WO2007139283A1 (fr) Procédé d'élaboration de biocapteur photonique-fluidique à cristaux photoniques fonctionnalisés
Hiep et al. RNA aptamer-based optical nanostructured sensor for highly sensitive and label-free detection of antigen–antibody reactions
Bischler et al. Development of a fully automated in-vitro diagnostics system based on low-Q whispering gallery modes in fluorescent microparticles
ITTO20060883A1 (it) Procedimento e microdispositivo a trasduzione ottica per l'identificazione e/o quantificazione di un analita in un campione biologico
US20090305230A1 (en) Real Time Detection of Molecules, Cells and Particles Using Photonic Bandgap Structures
JP2003014765A (ja) センサーおよびそれを用いた物質の反応の検出方法
JP2007093355A (ja) 標的物質の光学的検出方法と検出システム
KR20140097679A (ko) 금 나노입자를 이용한 ｄｎａ 혼성화 반응의 단일 부정합 검출방법
Isoda et al. Development of a Silica Surface Modified with Reactive Amino Group as an Immobilized Carrier for Use as Biosensor Material

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07746037

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07746037

Country of ref document: EP

Kind code of ref document: A1