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WO2005040094A1 - Nouveau compose dendrimere et biopuce utilisant celui-ci - Google Patents

Nouveau compose dendrimere et biopuce utilisant celui-ci Download PDF

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Publication number
WO2005040094A1
WO2005040094A1 PCT/KR2003/002261 KR0302261W WO2005040094A1 WO 2005040094 A1 WO2005040094 A1 WO 2005040094A1 KR 0302261 W KR0302261 W KR 0302261W WO 2005040094 A1 WO2005040094 A1 WO 2005040094A1
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Prior art keywords
substrate
dendrimer
chemical formula
amine
compound
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PCT/KR2003/002261
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English (en)
Inventor
Joon Woon Park
Young Seo Choi
Chang Won Yoon
Hae Dong Lee
Bong Jin Hong
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Pohang University of Science and Technology Foundation
Posco Holdings Inc
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Posco Co Ltd
Pohang University of Science and Technology Foundation
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Application filed by Posco Co Ltd, Pohang University of Science and Technology Foundation filed Critical Posco Co Ltd
Priority to AU2003273108A priority Critical patent/AU2003273108A1/en
Priority to PCT/KR2003/002261 priority patent/WO2005040094A1/fr
Priority to US10/917,601 priority patent/US9201067B2/en
Priority to EP04774642A priority patent/EP1664341B1/fr
Priority to KR1020067007462A priority patent/KR101125787B1/ko
Priority to RU2006108114/13A priority patent/RU2326172C2/ru
Priority to CA002539510A priority patent/CA2539510C/fr
Priority to JP2006526832A priority patent/JP4499727B2/ja
Priority to CN200480034008.4A priority patent/CN1882701B/zh
Priority to AU2004272465A priority patent/AU2004272465B8/en
Priority to PCT/KR2004/002383 priority patent/WO2005026191A2/fr
Publication of WO2005040094A1 publication Critical patent/WO2005040094A1/fr
Anticipated expiration legal-status Critical
Priority to US12/102,802 priority patent/US9671396B2/en
Priority to JP2010025682A priority patent/JP5095764B2/ja
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/003—Dendrimers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures

Definitions

  • the present invention relates to novel dendrimer compounds and a method for mesospacing amine density of substrate surface by fixing the dendrimer compound on the substrate surface of biochip. Further, real-time analysis of specific reaction between probing molecules such as biotin immobilized on the substrate surface and target molecules such as streptavidin through surface plasmon resonance (SPR) spectroscopy confirmed the applicability of substrate mesospacing on small molecule chip or protein chip.
  • SPR surface plasmon resonance
  • Protein chip is a chip with tens or hundreds of proteins immobilized on the solid substrate surface and it can be utilized not only for the basic science such as study of protein expression pattern and characteristic analysis, but also for the development of new drugs and disease diagnosis. Protein chip possesses more various applicable areas compared to DNA chip and its application is based on four characteristics including screening, quantification, kinetic measurement, and mass spectrometric analysis.
  • the key technology in protein chip can be exemplified by protein immobilization which enables stable, uniform and high density immobilization of proteins on substrate surface and detection of protein interaction which enables effective analysis of protein-protein interactions.
  • small molecule chip is a chip with high density new drug candidate materials, which are expected to combine with proteins, immobilized on the chip, and can be very useful for development of new drugs. Immobilization of proteins such as enzyme, antigen, and antibody on the chip surface is widely used not only for analysis of a specific protein but also as a way of detection for cancer or specific disease. Usual proteins can be immobilized on the chip even without linker since they possess amine or carboxylic acid which can react with several active groups that are applied on the solid chip.
  • the method of conventional random protein immobilization employing the mentioned active groups cannot achieve functional preservation of the active site in biomolecules and uniform high density immobilization at the same time. It also has a problem of low analytical sensitivity or dissolution due to the excessive adsorption of non-specific proteins or ligands. Therefore, the first thing to consider in immobilizing proteins on a particular chip is that the immobilized proteins must maintain its original characteristics and for this, the proteins should be immobilized so that the linking parts are exposed well.
  • current research is concentrated on development of techniques to immobilize proteins on the substrate so that the active site of biomolecules can be preserved, and also high S/N ratio and high density can be achieved.
  • Dendrimers are three-dimensional molecules monodispersed with regular polybranch. In the beginning, dendrimers were studied for chemical or physical characteristics in solution. However, since the size and structure can be controlled in the process of synthesis, various active groups can be attached inside or at terminals. Due to this particular structure and characteristics, now dendrimer is applied in surface chemistry such as chemical or physical sensor, LCD display, organic light emitting diode, electro-optic film, surface treatment of MEMS device, and photoresists in nonolithography. Within the research of density control using dendrimer, to permit immobilization with enough spacing for physiological materials, Whitesell et al.
  • the following describes (1) dendrimer compound shown in chemical formula 1 , the intermediate product, and their production method, (2) method to immobilize the dendrimer compound on the substrate and substrate-dendrimer compound obtained therefrom, (3) method of immobilizing functional material such as biotin on the substrate and substrate-dendrimer-biotin compound obtained therefrom, (4) biochip with the immobilized dendrimer and its production method, and (5) biochemical, bioengineered analytical method and diagnostic method using the biochip and a device for them.
  • Dendrimer Compounds The present invention provides a dendrimer represented by Chemical Formula 1 shown below. [Chemical Formula 1]
  • X is amine protective group removed by base
  • L is-R-NH-C(O) as a spacer
  • R is substituted or unsubstituted linear or branched alkenyl.
  • the protective group X is amine protective group which prevents exposure of amine and it is removed in basic condition exposing the amine.
  • any materials with such characteristics may be used as X, use of 9- fluorenylmethoxycarbonyl (Fmoc), 1 ,1 ,-dimethyl-2-cyanoethoxycarbonyl is desirable.
  • Spacer L maintains certain length between amine and surface of the substrate when physiological materials are attached onto the exposed amine after X is removed from the immobilized dendrimer on the surface, thus increasing reactivity of amine.
  • R is not limited and its length or structure can be controlled depending on the kind of chemicals which is attached to focal site of dendrimer.
  • R is proper to design R with length of more than one CH 2 .
  • alkyl chain is too long, there is possibility that the terminal amine might not be exposed into the solution where the protein and the target protein interact each other and alkenyl with proper length must be chosen.
  • linear alkenyl is more appropriate than substituted or unsubstituted brancehed alkenyl.
  • the dendrimer compound in Chemical Formula 1 is hyperbranced molecule with conical shape possessing one amine group and nine carboxylic acid functional group.
  • Nine carboxylic acids form multiple ionic bonding or multiple covalent bonding by amide coupling reaction with amine on the substrate surface and conclusively amine density on the surface is decreased.
  • Amine reactive group at the termini is protected and monolayer can be generated when dendrimer layer was formed on the substrate surface.
  • protective group is stable in acidic condition, carboxylic termini of the dendrimer can be easily exposed by hydrolysis process in organic synthesis process. After immobilized on the substrate and treated with acetic anhydride to remove extra amine, acetic acid is produced but has no effect on the stability of protective group.
  • Chemical Formula 1a is an example of the present invention of dendrimer composition.
  • Amine protective group X is 9-fluorenylmethoxycarbonyl (Fmoc) and spacer is -(CH 2 )n-NH-C(O).
  • Amine protective group Fmoc in the above Chemical Formula 1a (1) absorbs UV and it is convenient to confirm progress degree of reaction using thin layer chromatography and to separate during the purification process by using column chromatography during organic synthetic process, (2) is very stable to acidic condition (not cleaved even after 24 hours under 96% formic acid) and quantitative amount can be obtained when exposing carboxylic acid termini of dendrimer by treating with acid during the synthesis process, (3) is stable to acetic acid that is formed when treated with acetic acid to remove the rest of the amine after being immobilized on the substrate and enable even dispersal of amine, (4) have advantage of very fast deprotection time (it takes 6 seconds under 20 % piperidine when the Fmoc protected amino acid (ex.
  • Dendrimer of Chemical Formula 1 is produced as following: (a) Step for producing the spacer composition azidoalkylamine of Chemical Formula 2 shown below; (b) Step for producing the composition of Chemical Formula 4 by reacting the composition of Chemical Formula 2 with composition of Chemical Formula 3 and triphosgene; (c) Step for producing the composition of Chemical Formula 5 by reducing azide group of the composition of Chemical Formula 4 to amine; (d) Step for producing the composition of Chemical Formula 6 by introducing protective group X to the amine termini of the composition of Chemical Formula 5; and (e) Step for producing the composition of Chemical Formula 1 by hydrolyzing the above Chemical Formula 6.
  • Y is protective group for carboxylic acid and is a group that is removed by acid.
  • Chemical Formula 6 becomes Chemical Formula 1.
  • Y can be any substitute group which can be removed by acid and for example, t-butyl, tetrahydrofuranyl, methoxymethyl can be Y. Among these, t-butyl, which is easily removed in weak acid condition (pH 2-4) and the synthesized material can easily be analyzed by NMR spectrum, is most appropriate.
  • Step (a) can be performed in the following steps: (a-1) Step for producing diazidoalkane of Chemical Formula 2b by substituting bromide of dibro oalkane of Chemical Formula 2a with azide; and (a-2) Step for producing azidoalkylamine of Chemical Formula 2 by reducing azide group of only one side of diazidoalkane of Chemical Formula 2b.
  • step (a-2) for the synthesized diazidoalkane, reduce only one azide by adding reducing agent such as triphosphine in the mixed solution (ether/ethyl acetate/5% HCL). Under this condition, since initially formed azidoamine complex migrate to aquatic phase by becoming hydrogenated by acid, transformation into diamine complex by overreduction becomes limited. After discarding the organic phase, wash the aqueous phase with CH 2 CI 2 and remove residual triphosphine oxide and the starting material.
  • DMF/water mixed solution and b is reacting for 24 hours at room temperature by adding 1 equivalent amount of triphenylphophine into 1 :1 :1.6 (v/v/v) (diethyl ether/ethylacetate/5%HCI) mixed solution.
  • Step (b) can be performed in the following steps: (b-1) Step for preparing the composition of Chemical Formula 3; and (b-2) Step for producing composition of Chemical Formula 4 by reacting the composition of Chemical Formula 2 with composition of Chemical Formula 3.
  • Step (b-1) can be achieved by already know synthesis method, Lin's amine synthesis method (Newkome et al.; Macromolecules vol 24, p 1443 (1991)) or
  • Gawley method (Gawley et al.; J. Org. Chem. Vol 67, p1411 (2002)).
  • Gawley reference describes a method to produce the second generation dendrimer by using relatively cheap tris(hydroxymethyl)aminomethane (Tris) and the composition of Chemical Formula 3 according to the present invention can be synthesized using tris ⁇ [2-(tert-butoxycarbonly)ethoxy]methyl ⁇ methylamine as basic repetitive unit according to the above Gawley method.
  • Reaction Formula 2 shows an example of reaction in step (b-1). [Reaction Formula 2]
  • step (b-2) is processed by reacting the composition of Chemical Formula 3 synthesized according to the methods of Gawley with a spacer group composition, azidoalkylamine of Chemical Formula 2. To link these two molecules, first produce isocyanate complex by reacting azidoalkylamine with triphosgene in CH 2 CI 2 solvent.
  • isocyanate group easily reacts with nucleophile and to inhibit the secondary reaction, first make a dilute solution of triphosgene in CH 2 CI 2 and then proceed with the reaction by slowly injecting the solution in which diisopropylethylamine, which increases the reactivity of amine of azidoalkylamine, is dissolved. Confirm the progress degree of the reaction by TLC and after covering the reactive solution on NaCI as thin layer, analyze the IR spectrum. Isocyanate formed shows peculiar absorbance at 2095 cm "1 .
  • the composition of Chemical Formula 5 is produced by reducing azide group present at focal site of composition of Chemical Formula 4 by hydrogenation.
  • the additional purification process such as extraction or column chromatography is necessary and the yield is not so good. Therefore, considering the convenience of purification process after the reaction, it is appropriate to process the reaction by using Pd/C and hydrogen. The progress degree of the reaction can be confirmed by observing the disappearance of the starting material through TLC and it is appropriate to use ethanol as reaction solvent rather than methanol, which is commonly used, to prevent cleavage of t-butoxide. For purification, remove the catalyst by using syringe filter.
  • the reaction in step (d) is a process of protecting the terminal amine of the composition of Chemical Formula 5 with the protective group. If amine termini are protected by the protecting group that is removed in basic condition, formation of monolayer molecules is possible in substrate reaction using multiple covalent bonding or multiple ionic bonding. Besides, since it is also stable in acidic condition, it is stable in acetic anhydride treatment process to inactivate the residual amine after immobilization of the dendrimer on the substrate. In basic condition, since the reversal of protection is very fast reaction within several seconds, the advantage is that the protective group can be removed easily.
  • the protective group can be anything as long as it can satisfy such condition and particularly, 9-fluorenylmethoxycarbonyl (Fmoc) belongs to the group. It is proper to process the reaction of Step (d) under CH 2 CI 2 condition and to increase the reactivity of amine, diisopropylethylamine is added. The completion of the reaction can be confirmed by formation of materials that are UV-reactive
  • a corresponds to the reaction of step (b-1) and reaction occurs by adding triphosgene, diisopropylethylamine, and CH 2 CI 2
  • b corresponds to the reaction of step (c) and the reaction lasts for 12 hours with supply of hydrogen gas after adding 10% Pd/C and EtOH
  • c corresponds to the reaction of step (d) and the reaction lasts for 3 hours at room temperature after adding the amine protective group X, diisopropylethylamine, and CH 2 CI 2
  • d corresponds to the reaction of step (e) and the reaction lasts for 18 hours at room temperature after adding 96% formic acid.
  • Amine protective group X is identical as the one mentioned in Chemical Formula 1.
  • the present invention immobilizes the dendrimer of Chemical Formula 1 by methods described below: (a) Step for bringing amine group to substrate surface; (b) Step for forming multiple ionic bonding or multiple covalent bonding between amine of the substrate surface and carboxylic acid of dendrimer by reacting the dendrimer of Chemical Formula 1 on the above-mentioned substrate surface; (c) Step for exposing the terminal amine of the dendrimer by removing amine protective group X of the dendrimer which is bound to the above-mentioned substrate.
  • one more step for capping an inactivating amine, which did not bond with carboxylic acid of the dendrimer of Chemical Formula 1 , on the substrate surface can be included by treating with the substrate surface with acetic anhydride.
  • amine group can be introduced to the surface by aminosilanization and when the substrate is gold thin film, amine can be introduced by treating aminoalkylthiol complex.
  • Substrate is not particularly limited and it includes glass substrate and metal substrate such as gold and silver.
  • glass substrate glass thin film such as silicone wafer, silica plate, slide glass, and fused silica plate can be used and for the metal substrate, it includes gold and silver.
  • reagent for aminosilanization of the glass substrate 3- aminopropyl)diethoxymethysilane, N-trimethysilylpropyl-N,N,N,- trimethylammoniumchloride or (N,N-dimethylaminopropyl)trimethoxysilane can be used.
  • Fig. 2 shows the condition of the surface of the glass substrate with amines group when three kinds of aminosilanizing reagent have been immobilized above.
  • (3-aminopropyl)diethoxymethysilane is used as aminosilanizing reagent in Fig.
  • N-trimethysilylpropyl-N,N,N,-trimethylammoniumchloride is used in Fig. 2B
  • (N,N-dimethylaminopropyl)trimethoxysilane is used in Fig. 2C.
  • aminoalkylthiol compound to bring amines group to gold thin film substrate 11-mercaptoundecylamine: MA, as indicated in Fig. 3, can be used.
  • Fig. 3 shows the process of immobilization of dendrimer compound in the present invention onto the gold thin film substrate.
  • the amine brought to the surface is primary amine, both multiple covalent bonding and multiple ionic bonding are possible and when there is no primary amine as N-trimethysilylpropyl-
  • N,N,N,-trimethylammoniumchloride in Fig. 2B or (N,N- dimethylaminopropyl)trimethoxysilane in Fig. 2C only multiple ionic bonding is possible.
  • Fig. 3 when self assembly of aminoalkylthiol compound on the surface is completed, wash the substrate with solvent and dry it. Afterward, to immobilize the dendrimer on the substrate surface by multiple ionic bonding, immerse the substrate with the amines exposed into the solution including the dendrimer (X-NH-L-[1]amine-[9]acid) and conducting the reaction in atmosphere, allowing the dendrimer to form self-assembled monolayer on the substrate surface through multiple ionic bonding.
  • the dendrimer X-NH-L-[1]amine-[9]acid
  • the reaction at room temperature for about 24 hours is appropriate.
  • the dendrimer compound is immobilized on the substrate through multiple covalent bonding, conduct reaction with carboxyl termini of the dendrimer, EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide), and sulfo-NHS (N- hydroxysulfosuccinimide) to activate the carboxyl termini. Then run the reaction for
  • the dendrimer When the dendrimer is immobilized on the substrate where the primary amine is exposed, control is necessary for even distribution of active amines that are exposed on top of the substrate. While the active amines located at focal site of the dendrimer is evenly distributed with proper density, the unreacted amines of aminosilane or amonoalkylthiol are unevenly distributed and their reactivity is different from the active amines located at the focal site of the dendrimer, to obtain proper active amine density, even distribution, and constant reactivity, the unreacted amines of aminosilane or amonoalkylthiol must be removed. To remove such unreacted amines, substrate surface is treated with capping agent such as acetic anhydride. The amine termini of the dendrimer is protected by protective group X which is stable in acidic condition, and then the unreacted amines on the substrate surface can be selectively eliminated.
  • capping agent such as acetic anhydride
  • the process of removing the protective group is necessary to expose the amine group to the substrate surface.
  • the removal of the protective group is carried out by treating the substrate with basic compound.
  • the protective group X is 9-fluorenylmethoxycarbonyl: Fmoc
  • secondary amine such as piperidine can be used as the agent for removing the protective group.
  • Piperidine is usually used by diluting with DMF solvent and although the concentration of piperidine in DMF is not limited, when the dendrimer of the present invention is applied to the substrate, the removal of the protective group X was more rapidly processed at concentration of 5 ⁇ 20 vol%, more rapidly at concentration of about 5 vol% for the same reaction time. (See Fig.
  • biotin-streptavidin binding is used to analyze the interaction between ligand and protein.
  • the substrate is gold thin film
  • the interaction with proteins of the substrate where biotin (probe compound) is immobilized is analyzed in situ by through SPR spectroscopy.
  • biotin and streptavidin by immersing (glass substrate-dendrimer-biotin) complex into solution with Cy3- labeled streptavidin.
  • Fig. 1 describes the structure of dendrimer according to the present invention.
  • Figs. 2A-2C show the surface condition when the glass substrate surface is treated with 3 different kinds of amino(or ammonium)silane compound.
  • Fig. 3 shows the process of bringing the dendrimer to the surface of aminoalkylthiolized gold thin film substrate.
  • Fig. 4A is a fluorescence image showing the binding between biotin and streptavidin when biotin is immobilized directly on aminosilanized glass substrate and subsequently fluorescence-tagged streptavidin is bound.
  • Fig. 1 describes the structure of dendrimer according to the present invention.
  • Figs. 2A-2C show the surface condition when the glass substrate surface is treated with 3 different kinds of amino(or ammonium)silane compound.
  • Fig. 3 shows the process of bringing the dendrimer to the surface of aminoalkylthiolized gold thin film substrate.
  • Fig. 4A is a fluorescence image
  • FIG. 4B is a fluorescence image showing the nonspecific binding between biotin and streptavidin when (glass substrate-dendrimer) complex is obtained by the method of Example 2 and then treated with biotin and bound with streptavidin without removing the amine protective group X.
  • Figs. 4C-4E are fluorescence images showing binding between biotin and streptavidin when (glass substrate-dendrimer) complex is obtained by the method of Example 2, and then the amine protective group X is removed with 1 vol%, 5 vol%, and 20 vol% piperidine in DMF, respectively, that complex is treated with biotin and bound with streptavidin.
  • Fig. 5 shows the process of introducing the biotin to (substrate-dendrimer) complex.
  • FIG. 6A-6C shows a molecular structure of three kinds of biotin derivative used in the production of biotin immobilized mixed SAM of Comparative Examples 1 ⁇ 5, and Figs. 6D-6F is a molecular structure of three kinds of alkanethiol used in the production of mixed SAMs of Comparative Examples 1 ⁇ 4.
  • Fig. 7 is a graph showing the interaction between biotin and streptavidin on mixed SAMs of Comparative Examples with varying density and on substrate complex of Practical Example according to the present invention by SPR (surface plasmon resonance) spectroscopy.
  • Chemical Formula 1b The structure of the following Chemical Formula 1b is an example of the dendrimer compounds according to the present invention and the synthetic method of the dendrimer compounds according to the present invention is described by using the synthetic method of the following Chemical Formula 1b.
  • the rest of the compounds in Chemical Formula 1 can be synthesized by the same method as Fmoc-NH-hexyl-[1]amine-[9]acid compound or the Chemical Formula 1b except for the choice of the starting material. [Chemical Formula 1 b]
  • Step (a-2) Synthesis of Azidohexylamine 1.0 equivalents of 1 ,6-diazidohexane was dissolved in diethyl ether and 5.0 % aqueous HCI mixed in a volume ratio of 1 :1.6 (v/v) was added. Then the mixture was cooled to 0°C and 1.0 equivalents triphenylphosphine in a same volume of diethyl ether and ethyl acetate was added dropwise for 4 h. The reaction mixture was allowed to reach room temperature and left stirring for 24 h. After removal of the organic layer, the aqueous layer was washed with CH 2 CI 2 and basicified with 1 M NaOH to pH 10 at 0°C.
  • Step (b-1) Synthesis of [1]amine-[9]ester of Chemical Formula 3 according to Gawley method
  • Step (b-2) Synthesis of the Compound of Chemical Formula 4 Synthesis of ⁇ /-(6-Azidohexyl)- ⁇ /'-trisr(2-(f(tris ⁇ 2-(terf-butoxycarbonyl)ethoxyl- methyl)methyl)aminolcarbonyl)ethoxy)methvnmethylurea.
  • Triphosgene (58.2 mg, 0.20 mmol) was dissolved in anhydrous CH 2 CI 2 (5.0 mL).
  • DIEA N,N-diisopropylethylamine
  • aminosilane and aminoalkylthiol compound for glass substrate and gold surface, respectively.
  • aminosilane compound such as (3-aminopropyl)diethoxymethysilane solution in toluene (A), N-trimethysilylpropyl-N,N,N,-trimethylammoniumchloride solution in acetone solution (B), and (N,N-dimethylaminopropyl)trimethoxysilane solution in toluene (C) can be used.
  • the clean silica substrate was dried under 30mTorr vacuum. After adding (3-aminopropyl)diethoxymethysilane solution in toluene (10 "3 M) (A) to Teflon reaction container, immersed the dry silica substrate and reacted at room temperature to aminosilanize the glass substrate surface as shown in Fig. 2A. After completion of silanization reaction, washed with toluene and baked for
  • capping process was done for block of extra amine exposed to the substrate surface by immersing the substrate into 5 vol% acetic anhydride solution in CH 2 CI 2 . Afterward, the glass substrate was put into 5 % piperidine DMF solution and agitated for 20 minutes at room temperature to remove Fmoc functional group. After the reaction, the substrate was sequentially immersed into DMF and CH 2 CI 2 to wash with sonicator for 1 minute and then vacuum dried. (See Fig. 2A)
  • the clean silica substrate was dried under 30mTorr vacuum. After adding N-trimethysilylpropyl-N,N,N,-trimethylammoniumchloride solution in acetone solution (B) to Teflon reaction container, immersed the dry substrate and reacted at room temperature to aminosilanize the substrate surface as shown in Fig. 2B. After completion of silanization reaction, washed with acetone and dried for 30 minutes in 120°C oven. After cooling down the substrate to room temperature, washed the substrate for 3 minutes each with ultrasound under acetone, 1 :1 (v/v) acetone/methanol, and methanol, respectively.
  • the clean silica substrate was dried under 30mTorr vacuum. After adding (N,N-dimethylaminopropyl)trimethoxysilane solution in toluene
  • the method to self-assemble the dendrimer on the gold thin film by multiple ionic bonding is similar to the method to form the dendrimer molecular film on the glass substrate and is described in Fig. 3.
  • Example 5 Incorporation of Dendrimer onto Gold Thin Film Substrate 2: Multiple Covalent Bonding.
  • the procedure for reacting the dendrimer with high density amine on the surface by multiple covalent bonding is same as dendrimer incorporation method by multiple ionic bonding of the Example 4 except that reacting the dendrimer of Chemical Formula 6 with EDAC(0.1 M) and N-hydroxysulfosuccinimide (0.1 M, sulfo-NHS) to incorporate succinimide, which is amine reactive form, onto 9 carboxyl groups of the dendrimer and then reacting it with amine which is present on the substrate surface to accomplish the amide bond between the high density amine and the dendrimer.
  • [9]acid (5.0 mM) mixture for about 2 hours.
  • Put this solution into Teflon reaction container immerse and incubate the gold substrate with aminoalkylthiol molecular thin film which is produced from step (B) for 3 hours, and then after the completion of the reaction, wash it with flowing water. Subsequently, provide capping to the extra amine by the same method as in the Example 4 and perform deprotection of Fmoc.
  • Example 1 the surface-bound 9-anthraldehyde was hydrolyzed in water and the fluorescence intensity of the separated anthraldehyde was measured.
  • the amine density of the dendrimer molecular layer which is self-assembled on aminosilane molecular layer was 0.05 / nm 2 . Because the amine density of aminosilane-treated substrates is 3.1-4.2 amines/nm 2 for
  • Example 4 and 5 was respectively 0.04 ea/nm 2 and 0.08ea/nm 2 . Because the amine density of aminoalkylthiol layer on gold was reported as 4.8 ea/ nm 2 (Mrksich et al. J.
  • the present invention give a effect of reduction of amine density up to 120 times and 60 times each. This fact imply that the formed dendrmer layer can supply a efficient surface for interaction between immobilized probe and biomolecular target, which is derived from reduction of amine density.
  • Probe is a molecule binding specifically with a target in solution, including DNA, protein, carbohydrate and small molecule.
  • Biotin is used as probe molecule in the case of streptavidin as target, GSH (glutathione) is used in the case of glutathione S-transferase and complementary DNA is used in the case of single strand DNA.
  • GSH glutathione
  • complementary DNA is used in the case of single strand DNA.
  • Example 6 Glass substrate-Dendrimer-Short biotin Complex 1 After generating dendrimer monolayer, biotin was immobilized on that layer through microarrayer. For immobilizing biotin, produce the spotting solution of succinimidyl D-biotin (1.0mg Fig. 6A) in 1mL sodium bicarbonate buffer 50 mM and DMSO (6:4 v/v). After the arraying and the incubation for 90 min in a humidified chamber ( ⁇ 75 % humidity), the biotin microarrays were subsequently washed by succinimidyl D-biotin (1.0mg Fig. 6A) in 1mL sodium bicarbonate buffer 50 mM and DMSO (6:4 v/v). After the arraying and the incubation for 90 min in a humidified chamber ( ⁇ 75 % humidity), the biotin microarrays were subsequently washed by
  • Compound 2 is same as the method of Example 6 except for using glass substrate acquired from Example 2 instead of one from Example 1.
  • Compound 3 is same as the method of Example 6 except for using glass substrate acquired from Example 3 instead of one from Example 1.
  • Example 9 Gold substrate-lonically bound dendrimer- Biotin Complex
  • Example 10 (Gold substrate-Covalently bound dendrimer- Biotin) Complex
  • the procedure for generating (Gold substrate-Covalently bound dendrimer- Biotin) Compound is same as the method of Example 9 except for using substrate acquired from Example 5 instead of one from Example 4.
  • [Comparative Example 1] Gold substrate-12:1 Mixed SAM-Short biotin) Complex A. Preparation of Mixed SAMs composed of 11-mercaptoundecanol (11-MUOH) and 11- mercaptoundecanoic acid (11-MUA) in a 12:1 ratio (v/v). For the comparison, gold coated substrates of Example 4B step was dipped in an ethanolic solution dissolving 11-mercaptoundecanol (11-MUOH) and 11- mercaptoundecanoic acid (11-MUA) in a 12:1 ratio (v/v).
  • Fig. 6C in 50 mM triethanolamine (TEA) aqueous solution (pH 8.0, 0.7 mg/mL) and, in result, the substrate without a spacer between 11-MUOH and biotin was acquired. After immobilization of biotin, the substrate was placed in bicarbonate buffer solution (0.1 M, pH 9.5) for 30 min in order to hydrolyze the remaining ester group to make acid group.
  • TAA triethanolamine
  • the mixed SAM was reacted with (+)-biotinyl-3,6,9- trioxaundecanediamine (EZ-biotin-linker-LC-PEO-amine, long biotin) in 50 mM triethanolamine aqueous solution (pH 8.0, 1.2 mg/mL) and, in result, a distance between MUOH and biotin is so far to reduce a steric hindrance in protein binding.
  • the substrate was then placed in bicarbonate buffer solution (0.1 M, pH 9.5) for 30 min in order to hydrolyze the remaining ester group.
  • the procedure for generating (Gold substrate-100:1 Mixed SAM-Long biotin) Complex is same as the method of Comparative Example 3 except that the ratio of 11-MUOH and 16-MHA was altered from 12:1 to 100:1.
  • B. Immobilization of Biotin Subsequently, immobilized biotin on a mixed SAM by the method which is exactly same as Comparative Example 3. After removal of an active amine, the substrate was placed in bicarbonate buffer solution (0.1 M, pH 9.5) for 30 min in order to hydrolyze the remaining ester group to make acid group..
  • Comparative Example 1 > MA SAM (Comparative Example ⁇ ) > multiple ionically bound dendrimer layer (Example 9) > 100:1 Mixed SAM (long biotin, Comparative Example 2) > 12:1 Mixed SAM (short biotin, Comparative Example 3) > 100:1 Mixed SAM (short biotin, Comparative Example 4) Campbell and coworkers showed that two surface biotins bound two biotin sites on the same streptavidin on 12:1 mixed SAM (long biotin) and only one surface biotin bound one of two biotin sites on one streptavidin on 100:1 mixed SAM (long biotin) (Langmuir vol.16, p9421 (2000)).
  • the binding level of streptavidin reached the maximum on 12:1 mixed SAM (long biotin) and reduced as the concentration of biotin was increased.
  • the covalently bound dendrimer layer generated by the present invention enhanced the binding level of streptavidin about 60% in comparison with 12:1 mixed SAM (long biotin).
  • the density of biotin in12:1 mixed SAM is 0.37 ea/nm 2 whch is corresponding to 1/13 of the density of a general self-assembled aminoalkylthiol on gold (4.8ea/nm 2 ) and the density of biotin in covalently bound dendrimer layer is 0.08 ea/nm 2 which is the same amount as the amine density of dendrimer layer measured in Experimetal Example 1.
  • the high efficiency of biotinylated dendrimer layer than one of mixed SAM in despite of low ligand density is originateted from the structural feature of the dendrimer monolayer such as the surface exposure of derivatized biotin ligand which is optimally spaced and is able to avoid phase separation appearing in mixed SAM case.
  • the mesospacing of ligand renders a high binding affinity toward protein.
  • Binding level of streptavidin on biotinylated MA layer is similar to one of streptavidin on 12:1 mixed SAM in spite of relatively high biotin density, which is different from Campbell's result.
  • the nonspecific binding level is minimal 7 times larger than to the dendrimer layer or mixed SAM. Therefore, it is presumed that the seemingly high binding level of streptavidin on biotinylated MA layer came from this nonspecific binding.
  • the difference of binding affinity between covalently bound dendrimer layer (Example 10) and ionically bound dendrimer layer (Example 9) is originated from the amine density of dendrimer layer. So to speak, as shown in
  • the binding level of streptavidin of each complex is measured by scanning microarrayed spot of each complex through ScanArray ® Lite (GSI Lumonics) at the laser power and the gain of detector adjusted to 80% and analyzing the fluorescence intensity through quantitative microarray analysis software, ImaGene 4.0 (BioDiscovery, Inc.). Each intensity was 4.3 X 10 4 , 4.7X 10 4 and 3.5 X 10 4 for 1 , 5 and 20 % piperidine condition. This results shows that deprotecion in 5% DMF is optimal condition showing high efficiency.
  • Biochip> (Substrate-dendrimer-Probe molecule) Complex generated by the present invention can be applied to detection of a special molecule, which means that it can be used for diagnosis and analysis of disease, and various experiment and analysis etc.
  • a biochip containing (Substrate-dendrimer-Probe molecule) Complex acquired by the present invention is generated and is react with biosubstance, such as blood, cell or tissue derived from animal or plant, we can identify the existence of target molecule from binding level.
  • biosubstance such as blood, cell or tissue derived from animal or plant
  • a method for diagnosis of disease or analysis of special molecule contains (i) production of biochip including (Substrate-dendrimer) Complex or (Substrate- dendrimer-Probe molecule) Complex followed by the present invention, (ii) step for reacting the biochip with a biomaterial in interest. Also, after immobilizing a new drug on substrate complex to generate small molecule chip, we can find a target protein.
  • a method for immobilizing a probe molecule onto (Substrate-dendrimer) Complex contains a microarraying, a spotting by micropipette or a dipping in reaction solution. By reacting oligo-DNA, cDNA, genomic DNA, protein, carbohydrate, polymer, nanoparticle and cell with this substrate, we can identify a target molecule through detection method containing fluorescence intensity, electric signal etc.
  • the biochip manufactured by a dendrimer of the present invention (i) can give a mesospacing between a probe molecule on any substrate, (ii) can prevent a phase separation in contrast with mixed SAM, (iii) can efficiently immobilize various kinds of functional molecule, which is derived from a enhanced activity of an amine of dendrimer, (iv) can supply a excellent biochip showing high binding affinity for DNA or protein, which is derived from a uniform distribution of a functional molecule.

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Abstract

L'invention concerne des composés dendrimères qui sont représentés par la formule chimique 1, leur utilisation sur des complexes du type substrat-dendrimère et substrat-dendrimère-molécules cibles, et l'utilisation de ces composés sur une biopuce. Dans la formule chimique 1, X représente un groupe protecteur amine qui est éliminé par une base, L représente R-NH-C(O) comme espaceur, et R représente alcényle substitué ou non substitué.
PCT/KR2003/002261 2001-09-05 2003-10-24 Nouveau compose dendrimere et biopuce utilisant celui-ci Ceased WO2005040094A1 (fr)

Priority Applications (13)

Application Number Priority Date Filing Date Title
AU2003273108A AU2003273108A1 (en) 2003-10-24 2003-10-24 Novel dendrimer compound and a biochip using the same
PCT/KR2003/002261 WO2005040094A1 (fr) 2003-10-24 2003-10-24 Nouveau compose dendrimere et biopuce utilisant celui-ci
US10/917,601 US9201067B2 (en) 2003-03-05 2004-08-12 Size-controlled macromolecule
JP2006526832A JP4499727B2 (ja) 2003-09-18 2004-09-17 サブストレート、製造方法、診断システム及び検出方法
KR1020067007462A KR101125787B1 (ko) 2003-09-18 2004-09-17 분자 크기 제어된 거대분자
RU2006108114/13A RU2326172C2 (ru) 2003-09-18 2004-09-17 Макромолекула регулируемого размера
CA002539510A CA2539510C (fr) 2003-09-18 2004-09-17 Macromolecule a taille regulee
EP04774642A EP1664341B1 (fr) 2003-09-18 2004-09-17 Macromolecule a taille regulee
CN200480034008.4A CN1882701B (zh) 2003-09-18 2004-09-17 大小受控的大分子
AU2004272465A AU2004272465B8 (en) 2003-09-18 2004-09-17 Size-controlled macromolecule
PCT/KR2004/002383 WO2005026191A2 (fr) 2003-09-18 2004-09-17 Macromolecule a taille regulee
US12/102,802 US9671396B2 (en) 2001-09-05 2008-04-14 Solid substrate comprising array of dendrons and methods for using the same
JP2010025682A JP5095764B2 (ja) 2003-09-18 2010-02-08 サブストレート、製造方法、診断システム及び検出方法

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JP2001108683A (ja) * 1999-10-14 2001-04-20 Fuji Photo Film Co Ltd Ｄｎａ断片固定固相担体、ｄｎａ断片の固定方法および核酸断片の検出方法
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JP2001108683A (ja) * 1999-10-14 2001-04-20 Fuji Photo Film Co Ltd Ｄｎａ断片固定固相担体、ｄｎａ断片の固定方法および核酸断片の検出方法
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CN113970592A (zh) * 2020-07-23 2022-01-25 南京大学 一种酸性磷酸酶定量检测的质谱传感芯片及其制备方法

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