WO2005016869A1

WO2005016869A1 - Novel dendrimer compound, a biochip using the same and a fabricating method thereof

Info

Publication number: WO2005016869A1
Application number: PCT/KR2003/001913
Authority: WO
Inventors: Joon Woon Park; Bong Jin Hong; Soon Jin Oh; Young Seo Choi; Kwan Yong Choi; Tae One Youn; Sung Hong Kwon
Original assignee: Postech Foundation; Posco
Priority date: 2003-08-19
Filing date: 2003-09-18
Publication date: 2005-02-24
Also published as: AU2003263632A1

Abstract

The present invention relates to novel dendrimer compounds and method of producing a bio chip using the dendrimer. The present invention relates to the dendrimer that is represented by Chemical Formula 1, a substrate where the dendrimer is fixed, and the bio chip utilizing these compounds. [Chemical Formula I] wherein X is amine protecting group that is separated by acid, L is a spacer or -R-C(O)-NH- and R is substituted or unsubstituted hydrocarbon.

Description

NOVEL DENDRIMER COMPOUND, A BIOCHIP USING THE SAME AND A FABRICATING METHOD THEREOF

BACKGROUND OF THE INVENTION a) Field of the Invention The present inventions relates to a novel dendrimer compound and a method of producing a bio chip using the dendrimer compound. b) Description of the Related Art Substrate surface with molecular layer containing amine group has been applied to fixation of physiological molecules such as enzymes and antibodies, fixation of inorganic catalysts, modification of electrodes, chromatography, and affinity column; and it has also been used in many areas such as ionic polymer, nonlinear optical chromophore, chemical/biological sensor, photoresist, and corrosion passivation. The chemical, physical characteristics of molecular layer, which is formed on the substrate surface and containing amine group, is very important because this affects the form of the molecule that is fixed or self- assembled and surface density, and also is a factor determining the structure and characteristics of finally formed functional thin layer. It has been known that the number of amine groups when forming amine groups on the solid substrate surface is 1-10 per 100A². The solid substrate with amine groups on the surface can be used as plates for producing DNA chip or bio chip. However, the substrate with density of 1~10 amine groups per 100 A² on the surface cannot accommodate fixation of DNA or various biomolecules due to large steric hindrance between molecules. For example, in case of DNA chip, the efficiency of the chip can be increased only if hybridization of single stranded DNA that is fixed on the substrate surface and to achieve this, the single stranded DNA that is fixed on the surface must be dispersed evenly maintaining certain distance. To provide such condition on the substrate surface, Tarlov et al. have reported their research result demonstrating control of density by decreasing the concentration of self-assembled molecules that are required for surface reaction

(Rastislav Levickey et al., "Using self-assembly to control the structure of DNA monolayers on gold", J. Am. Chem. Soc. 120, pp9787-9792 (1998)). However, such method is modification by controlling the concentration of self-assembled molecules rather than direct modification of the surface and results in cohesion of molecules with functional groups, causing irregular spreading on the substrate surface. Therefore, it is difficult to control the distance between single stranded DNAs evenly. Thus, it is very important to find the optimal condition for hybridization efficiency and the concentration. Also difficulty of incorporation of single stranded DNA with more than certain concentration has been pointed out. Okahata et al. incorporated DNA on the surface by using the binding between biotin and avidin (Kenichi Niikura et al., "Direct monitoring of DNA polymerase reactions on a quartz-crystal microbalance", J. Am. Chem. Soc. 120, pp8537-8538 (1998)). They covered the QCM (Quartz Crystal Microbalance) surface with gold and synthesized compound having thiol and avidin as functional groups before incorporating single stranded DNA with biotin at the termini. In this method, signal is the frequency of QCM that changes according to the progress of hybridization. However, this method is not direct control of the structure on the surface, but indirectly using biotin-avidin binding. Also there are several shortcomings due to the use of QCM. Not only in the case of DNA, but also in the case of protein, extra space is necessary to achieve helical structure. Whitesell et al. placed monolayer of aminotrithiol on gold surface and achieved formation of the helical structure by polyalanine. Also in case of poly(phenylalanine), monolayer was not enough to achieve the helical structure due to insufficient space. Therefore, the helical structure was achieved by forming double layer and increasing the extra space (James K. Whitesell et al., "Directionally aligned helical peptides on surfaces", Science Vol. 262, pp73-76 (July 2, 1993)). This method, incorporating proteins by changing the surface directly, might seem to be possible to apply to DNA case, but since the double helice of DNA are larger than double helice of proteins (A type is 25.5A and B type is 23.7 A), a dendrimer larger than the one used in this study must be incorporated. Also since sulfur is incorporated into the dendrimer used here, it can only be applied to gold surface. SUMMARY OF THE INVENTION The present invention provides the following to overcome the problems of prior arts described above: a novel dendrimer compound enabling uniform low density dispersion of amine group on the substrate surface and method of synthesizing it; the substrate where the dendrimer compound is incorporated and method of producing it; bio chip using the substrate and method of producing it; and biochemical analytic and diagnostic methods by using the bio chip. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the process of fixing the dendrimer compound according to the present invention onto a substrate; Fig. 2 shows the process of separating the amine protecting group from the dendrimer compound which is fixed to the substrate; Fig. 3 shows the process of incorporating a linker compound onto the surface of the substrate of Fig. 2; Fig. 4 shows the process of incorporating probe DNA onto the surface of the substrate of Fig. 3; Fig. 5 shows the process of hybridizing target DNA with the substrate of Fig. 4; Fig. 6 shows UV absorption spectrum measured before and after the incorporation of the dendrimer in the present invention onto the surface of the substrate including hydroxy group and after separation of amine protecting group; Fig. 7 shows AFM (Atomic Force Microscope) images taken before and after the incorporation of the dendrimer compound in the present invention onto the surface of the substrate including hydroxy group; Fig. 8(a) shows fluorescent signal detection data resulted when the probe DNA and the target DNA are hybridized complementarily on the substrate produced according the present invention, and Fig. 8(b) shows fluorescent signal detection data resulted due to a single mismatched base (T-T mismatch) between the probe DNA and the target DNA nucleotide sequences on the substrate produced according to the present invention; Fig. 9(a) is identical to Fig. 8(a), and Fig. 9(b)-Fig. 9(d) individually show fluorescent signal detection data when one of the bases at terminal regions is mismatched between the probe DNA and the target DNA on the substrate produced according to the present invention; Fig. 10(a)-Fig. 10(b) show comparative experimental results of the hybridization when APDES, rather than the dendrimer compound in the present invention, is fixed to the substrate. Fig. 10(a) shows fluorescent signal detection data when the probe DNA and the target DNA are complementarily hybridized and Fig. 10(b) shows fluorescent signal detection data resulted due to a single mismatched base (T-T mismatch) between the probe DNA and the target DNA nucleotide sequences on the substrate produced according to the present invention; Fig. 11(a)-Fig. 11(d) show comparative experimental results of the hybridization when APDES, rather than the dendrimer compound in the present invention, is fixed to the substrate. Fig. 11 (a) shows fluorescent signal detection data resulted when the probe DNA and the target DNA are hybridized complementarily and Fig. 11(b)-Fig. 11(d) individually show fluorescent signal detection data when one of the bases at terminal regions is mismatched between the probe DNA and the target DNA on the substrate produced according to the present invention. DETAILED DESCRIPTION OF THE INVENTION The Dendrimer Compound The following is the chemical formula for the dendrimer compound: [Chemical Formula 1]

The X term denotes the amine protection group, which can be de-protected by acid; the L stands for the spacer, -R-C(O)-NH-, and the R can be either a substituted or non-substituted hydrocarbon group. The X mentioned above can be 1) bezyloxycarbonyl 2) Benzyloxycarbonyl, in which the benzyl is metathesized into a functional group in which the hydrogen attracts electrons. 3) 9-anthrylmethoxycarbonyl 4) t-butoxycarbonyl, etc. The functional group that attracts the electron in the 2) group above include nitrogen group, halogen group, and the cyano group. To go into detail, the amine protection group for group 2) can be 2-nitrobenzyloxycarbonyl, 3-nitrobenzyloxycarbonyl, 4- nitrobenzyloxycarbonyl, 2-fluorobenzyloxycarbonyl, 3-fluorobenzyloxycarbonyl, 4- fluorobenzyloxycarbonyl, 2- chlorobenzyloxycarbonyl, 3- chlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl. The "R" of spacer L can be: Substituted or non-substituted, linear or branched chain alkanyl Substituted or non-substituted, linear or branched chain alkenyl

Substituted or non-substituted, linear or branched chain alkinyl Substituted or non-substituted, cycloalkanyl Substituted or non-substituted, cycloalkenyl substituted or non substituted aromatic compound substituted or non substituted polyalkenylglycol.

The desirable length of R would be at least 5 to 15 . Spacer L increases the reactivity of the terminal amine by providing space between the dendrimer and the terminal amine, thus minimizing the steric hindrance within the dendrimer. The spacer's chemical structure prevents it from exhibiting any steric hindrance within itself. The dendrimer compound illustrated in Chemical Formula 1a is a trigonal hyperbranch molecule that contains one amino group and nine carboxylic acid groups. The nine carboxylic acid groups decrease the amine density present on substrate surface (and lower the steric hindrance present on the surface) by forming multiple bonds with the amine or hydroxide on the surface; lower density of amine increases the reactivity of amine, and bio chips using substrates fixated with such dendrimers exhibit enhanced detection capability. As mentioned above, the spacer present in the dendrimer, which separates the main body with the amine end point, reduces the steric hindrance produced by the body, and improves the bonding ability of amine. Furthermore, the dendrimer's amine tip is shielded by a protection group, which enables monolayer arrangement of dendrimers on the substrate surface, and was designed to be easily removable. Chemical Formula 1a below is one structural example of the novel dendrimer compound. The amine protection group X represents anthrylmethoxycarbonyl, and the spacer L is composed of -(CH₂)₃-C(0)-NH-.

[Chemical Formula 1a]

(9-anthrilmethyl N-([tris(2-[(tris(2- carboxyethoy)methyl)methylamino)carbonyl]ethoxymethyl)methyl]aminocarbonyl)pro pylcarbamate) The amine endpoint of Chemical Formula 1a is protected by an anthrylmethoxycarbonyl group. The protected amino group is not easily damaged by other molecules during surface reactions during which the X-NH-spacer-[1]amine- [9]acid is fixed onto the substrate surface. It also minimizes the subsidiary reactions that occur during the synthesis of X-NH-spacer-[1]amine-[9]acid dendrimer. Anthrylmethoxycarbonyl groups can be conveniently removed, allowing them to turn back into primary amine groups after the reaction; due to the anthrylmethoxycarbonyl group's strong UV absorbance capacity; the process of removal can be easily traced. The propyl selected for the spacer, which provides extra space, enhances the reactivity of the primary amine group produced during the removal process that occurs after the dendrimer is inserted into the substrate. As for the repeating unit used in synthesizing the dendrimer presented in Chemical Formula 1a, tris[(methoxycarbonyl)ethoxy]methylaminomethane can be used; the compound is widely used in dendrimer synthesis processes.

Dendrimer production The dendrimer compound presented in Chemical Formula 1 can be produced following the proceduree outlined below: a) Spacer compound is prepared for preparation of Chemical Formula 2 b) Compound in Chemical Formula 2 is reacted with that of Chemical Formula 3 to produce the product presented in Chemical Formula 4 c) Base is applied to the compound from Chemical Formula 4 to separate the carboxyl protection group Y, resulting in Chemical Formula 5 d) The compound from Chemical Formula 5 is reacted with the chemical from Chemical Formula 3 to produce the compound in Chemical Formula 6 e) Base is applied to the chemical compound of Chemical Formula 6 to separate the carboxyl protection group Y and attain the final dendrimer compound of Chemical Formula 1

[Chemical Formula 2] X — m — — COOH

[Chemical Formula 3]

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6] X-NH-Spacer[1]amine-[9]ester

Again, the terms X and L designate the amine protection group and spacer, respectively. Y symbolizes the carboxyl protection group, which is a substitution group that is separable by acids and bases. Y can be any chemical compound that fits the description above. A few examples would be -CH₃, -CH₂CH₃, -CH₂C₆H₆. The compound presented in Chemical Formula 2 can be attained by reacting the compound presented in Chemical Formula 2a with either 2b or 2c. [Chemical Formula 2a] H2M^"-R-£QQH. [Chemical Formula 2b]

[Chemical Formula 2c] XCl In addition, steps B and D can both be executed in acetonitrile, dimethylformamide, and methylene chloride solvents, under the presence of 1-[3- (Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (represented as EDC) and 1-hydroxybenzotriazole( represented as HOBT). Steps C and E can be carried out by applying a base such as NaOH, which will separate the carboxyl protector Y, and expose the carboxylgroup. The reaction formula below schematizes the synthesis of dendrimer. [Reaction Formula 1]

Producing substrates with fixed dendrimers

Example 1 The process of dendrimer fixation onto substrate surface undergoes three basic steps: a) Insertion of hydroxyl onto the substrate surface b) Processing the substrate surface with the Chemical Formula 1 dendrimer; the hydroxyl group forms multiple covalent bonds with the carboxyl group of the dendrimer c) De-protecting the dendrimer and exposing the amine end point. After step B, a supplementary step can be added in which the substrate surface is treated with aceticanhydride to eliminate unreacted hydroxide groups. The steps are further explained in detail below.

Step A: Insertion of hydroxyl onto the substrate surface

To enable the dendrimer and substrate to form covalent bonds, hydroxyl groups are inserted into the substrate surface. Substrates can range from, but are not limited to, (i) glassy thin films including silicon wafer, glass slide, silica and fused silica (ii) metallic thin films such as gold or silver (iii) metallic or nonmetallic beads with a diameter of a few hundred nanometers (iv) metallic or nonmetallic nanoparticles with a diameter under one hundred nanometers. Nonmetallic beads and nanoparticles include glassy beads and nanoparticles. Nanoparticle substrates include those mentioned in many dissertations; the following papers have been studied extensively in producing the novel dendrimer compound presented in this paper.

1. Robert Elghanian et al., "seletive colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles", Science, Vol.277, pp1078-1081 (AUG 22, 1997)

2. Mark A. Osborne et al., "Single-molecule analysis of DNA immobilized on microsheres", Analytical Chemistry, Vol.72, No. 15, pp3678-3681 (AUG 1 , 2000)

3. Dustin J. Maxwell et al., "Self-assembled nanopaticle probes for recognition and detection of biomolecules", J.AM.CHEM.SOC.2002,124,pp9606-9612 (JUL 17, 2002)

4. Sheila R. Nicewarner Pena et al., "Hybridization and enzymatic etension of Au nanoparticle-bound oligonucleotides", J.AM.CHEM.SOC.2002, 124, pp7314-7323 (JUN 1 , 2002)

5. YunWei Cao et al., "DNA-modified core-shell Ag/Au Nanoparticles", J.AM.CHEM.SOC.2001 ,123, pp7961-7962 (JUL 2, 2001) 6. Lin He et al., "Colloidal Au-enhanced surface pasmon resonance for ultrasensitive detection of DNA hybridization", J.AM.CHEM.SOC.2000,122, pp9071-9077 (AUG 9, 2000)

7. Linette M. Demers et al., "A fluorescence-based method for determining the srface coverage and hybridization efficiency of thiol-capped oligonuclotides bound to gold thin films and nanoparticles", Anal. Chem. 2000,72, pp5535-5541 (OCT 21 ,

2000)

8. T. Andrew Taton et al., "Scanometric DAN array detection with nanoparticles probes", SCIENCE Vol.289 pp1757-1760 (SEP 8, 2000)

9. So-jung Park et al., "Array-based electrical detection of DNA with nanoparticle probes", SCIENCE, Vol 295 p1503-1506 (FEB 22, 2002)

10. T Andrew Taton et al., "Two-color labeling of oligonucleotide arrays via size- selective scattering of nanoparticle probes", J.AM.CHEM.SOC. 2001 ,123, pp5164- 5165 (MAY 5 2001 ) In the process of hydroxyl induction, epoxy-silanizing agent is dissolved in a solution, and the substrate is submerged in the solution for a set time to carry out an epoxy-silanization reaction. Toluene, ethanol and 95% ethanol can be used as the solvent. Once the reaction is fully carried out, the substrate is then exposed to polyethyleneglycol under the presence of acid. To go into further detail, after the silanization reaction, the substrate is washed and dried, then submerged in a polyethylene glycol solution at 100 degrees Celsius for approximately ten hours; the final dry product is a substrate with hydroxyl groups inserted on its surface. As for the epoxy silanization agent, (3-glycidoxypropyl)methyldiethoxysilane, (3-glycidoxypropyl)triethoxysilane, (3- glycidoxypropyl)dimethylethoxysilane, (3- glycidoxypropyl)methyldimethoxysilane, (3-glycidoxypropyl)trimethoxysilane, (3- glycidoxypropyl)dimethylmethoxysilane, 5,6-epoxyhexyltriethoxysilane, 5,6- epoxyhexylmethyldiethoxysilane, 5,6-epoxyhexyldimethylethoxysilane can be used. TPU (N-(3-triethoxysilylpropyl)-o-polyethylene oxide urethane) and TPH (N- (3-triethoxysilyl)-propyl)-4-hydroxybutylamide) can both be used as silan compounds containing hydroxyl functional groups, while mercaptoalkanol and mercaptopolyethyleneglycol can be used as thiol compounds containing hydroxyl groups. Hydroxyl compounds can be dissolved in solvents such as toluene, ethanol, and 95% ethanol.

Step B: Processing the substrate surface with the Chemical Formula 1 dendrimer

When the hydroxyl containing substrate is exposed to DCC, DMAP, or A-[9]- acids for a set time, the hydroxide groups on the substrate surface form covalent bonds with the carboxyl end groups of dendrimer compounds. Since one dendrimer compound contains one amine end group and nine carboxyl end groups, compared to when amine end groups are directly injected onto the surface, the covalent bonds formed between the carboxyl end groups and the hydroxide groups dramatically decrease the amine group density. After three days of exposure, the substrate is washed and dried, resulting in the final form represented in Chemical Formula 6, in which X-NH-Spacer-[1]-amine-[9]-acid atomic layers are fixed onto the substrate surface.

Step C: De-protection

Since amine end groups fixed on the substrate surface are protected, a de- protection process is required to expose the amine group. The amine protection group X can be separated by applying acid onto the dendrimer; for instance, if X represents anthrylmethoxycarbonyl, it can be detached from the dendrimer by immersing it in a 1 M TFA (trifluoroacetic acid) solution for three hours. The de-protection process is also necessary for removing extraneous substances physically absorbed into the substrate surface, such as TFA and DIPEA.

In such cases, the substrate can be exposed to 10% diisopropylethylamine solution for 10 minutes, and then rinsed with affluent amount of solvent, such as dichloromrthane and methanol. As it can be seen in Fig. 1 , the dendrimer, X-NH-Spacer-[1]-amine-[9]-acid, and the substrate are held together tightly by the covalent bonds between the hydroxyl groups present on the substrate surface and the carboxyl group present in the dendrimer, thus exhibiting high stability high temperatures and various pH settings. In addition, as Fig. 2 shows, a) the exposed amine groups on the top substrate layer are primary amine groups, therefore showing much higher reactivity, b) the amine groups are spatially combined with the larger dendrimer instead of the substrate surface itself, enabling the amine groups to evenly distribute among themselves at lower densitys, c) the amine groups are not directly connected to the main body, but are connected with a spacer in between, thus dramatically minimizing the steric hindrance that comes from the main body. The amine density resulting from the above steps were experimentally determined to be around 0.1-0.2 amines/nm², with each of the amine groups sufficiently spaced in between. Such distributional nature of the amine groups, along with the low steric hindrance the dendrimer compound presents, are among the many positive attributes DNA chips using such substrates would exhibit.

Inserting probe DNA into the substrate

In order to affirm the novel dendrimer compound's advantages in producing bio chips, Probe DNA can be inserted onto the substrate surface in the following steps: a) Carry out a reaction on the substrate surface with linker chemicals, causing the dendrimer's terminal amines to bond with the linker compounds b) Execute a second reaction in which the probe chemicals bond with the linker compounds. Step a involves immersing the substrate in a solution containing the linker compounds for some time, then rinsing (with solvents) and drying it. Different linker compounds can be used for the procedures above, including, but not limited to: DSC(disuccinimidyl carbonate), DSG(disuccinimidyl glutarate), DSO(disuccinimidyl oxalate), PDITC(phenylendiisothiocyanate), DMS(dimethylsuberimidate), SMB(succinimidyl-4-maleimido butyrate), or

SSMCC(sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate).

In step b, solutions containing the probe DNA is spotted onto designated spots layed out by a microarrayer onto the substrate resulting from step a; the substrate is given time to react in a humidity chamber, then immersed in a buffer solution. Probe DNA that have failed to bond with dendrimers are later rinsed out during a rinsing process.

Hybridization

Treat the processed substrate surface with a hybridization solution of reasonable target compound (such as target DNA) density, oligonucleotide, cDNA, and genomic DNA can all be applied as target DNA. Rinse out the remaining unreacted target DNA with buffer solution. Dry the resulting substrate, and measure the fluorescent signal strength of the substrate using instruments such as a fluorescence microscope or fluorescence laser scanner.

Examples The process of hybridization includes, but is not limited to, the detailed explanation below.

Synthesizing Examples 1 through 5 yields the final dendrimer compound presented in Chemical Formula 1a.

Synthesizing Example 1 (step 1) : Synthesis of 9-anthrylmethyl N-(3- carboxypropyl)carbamate

After dissolving 4-aminobutyric acid (0.5g, 4.85mmol, 1eq) in DMF, mix the solution with 1.5 grams of triethylamine at 50 °C. Add 9-anthrylmethyl p- Nitrophenyl carbonate (1.81g, 4.85 mmol) slowly and mix over a period of two hours. The progression of the reaction can be checked using thin film chromatography. Once the reaction is completed, remove the DMF solvent and add 0.5 N sodium hydroxide until the reaction solution turns basic. Use water and ethyl acetate to then extract the sodium hydroxide and form a water layer. Treat the water layer with diluted hydrochloric acid at 0°Cuntil it reaches pH1~2; extract the ethyl acetate, then dry with Anhydride magnesium sulfate and filter out the solvent through vacuum distillation. The final product is a viscous yellow fluid (1.06g, percent yield 64.8%)

9-anthrylmethoxycarbonate, which was selected as the amine protection group X, is easily de-protected in an acidic environment, while it exhibits high stability in basic surroundings; thus the compound is not damaged during the process of synthesizing A-NH2-spacer-[1]-amine-[9]-acid. Its high absorbance capacity of UV rays also enable UV ray spectroscopes to observe the progression of the reaction process, and easily detect the presence of 9-anthrylmethoxycarbonate during the de-protection process.

Analysis of the synthesized chemical compound yielded the data listed in table 1 below. The table shows the13C NMR peak analysis data for anthryl and carbamate groups, which ranged from124 ppm to 132 ppm for anthryl and was a solid157ppm for carbamate.

Table 1

Synthesizing Example 2 (step 2): synthesis of 9-anthrylmethyl N-

[Tris[2-(methoxycarbonyl)ethoxy]methylmethyl]amino]carbonylpropylcarbamate Dissolve 1.2 chemical equivalent of N-(3-carboxypropyl)carbamate (1.06g,

3.14 mmol) attained from synthesis step 1 into a solvent of acetonitrile, and mix with 1.5 chemical equivalent of EDC and HOBT. Combine the solution with 1 chemical equivalent of a second solution composed of tris[(methoxycarbonyl)ethoxy]methylaminomethane dissolved in acetonitrile (0.99g, 2.62mmol) for 12 hours at a high temperature. DCC can be substituted for EDC in the reaction; however DCC produces dicyclohexylurea as a side product in the reaction, which complicates the extraction of the desired product, and lowers the reaction yield. Once it is confirmed through thin film chromatography that the reaction has ended, eliminate the acetonitrile solvent and dissolve the product in ethyl acetate.

Rinse with 1 N of hydrochloric acid and saturated sodium bicarbonate, then dry using Anhydride magnesium sulfate and condense the product through vacuum distillation. The pure product (1.32g, percent yield 72.1%) can be obtained from the yellow, viscous fluid through column chromatography (using an unfolding solvent such as ethyl acetate: hexane = 5:1 (v/v)).

13C NMR analysis results of the products are listed in table 2. As it can be seen from table 2, the two characteristic peaks emitted by the ester group were at 172.7 ppm and 173.2 ppm, while the peak from the methoxyl group was 52.1 ppm.

Table 2

Synthesizing Example 3 (step 3): hydrolysis : synthesis of 9-anthrylmethyl N- [(tris[(2-carboxyethoxy)methyl]methylamino)carbonyl]propylcarbamate Dissolve the 9-anthrylmethyl N-[Tris[2 (methoxycarbonyl)ethoxy]methylmethyl]amino]carbonylpropylcarbamate compound obtained from step 2 into a solution of acetone and 0.2 N NaOH for 36 hours at high temperature. After the completion of the reaction is confirmed through thin film chromatography, remove the acetone solvent and extract the product using water and ethyl acetate to form a water layer. Treat the water layer with diluted hydrochloric acid at 0°Cuntil it reaches pH1~2; extract the ethyl acetate, then dry with Anhydride magnesium sulfate and filter out the solvent through vacuum distillation. Once the product goes through re-crystallization using acetone and ether, yellow, crystalline solids are obtained. (0.54g, percent yield 88.0%) NMR analysis results are listed on table 3. The disappearance of the characteristic 52.1 ppm peak caused by the methoxyl group confirms that the product has undergone hydrolysis. Table 3

Synthesizing Example 4 (Step 4): synthesis of 9-anthrylmethyl N-

[(tris[2-[(tris[2-(methoxycarbonyl)ethoxy]methylmethyl)amino]carbonyl]propyl carbonate

Dissolve 1 chemical equivalent of 9-anthrylmethyl N-[(tris[(2- carboxyethoxy)methyl]methylamino)carbonyl]propylcarbamate (0.54g, 0.82 mmol) obtained from step 3 in an acetonitrile solvent, along with 3.5 chemical equivalents of EDC and HOBT; Then combine 3 chemical equivalents of solvent comprised of tris[(methoxycarbonyl)ethoxy]methylaminomethane (0.93g, 2.46 mmol) dissolved in acetonitrile, and mix the solvents for 36 hours. DCC can be used in lieu for EDC in the reaction; however DCC produces dicyclohexylurea as a side product in the reaction, ipso facto complicating the extraction of the desired product, and lowering the reaction yield. After the completion of the reaction is confirmed through thin film chromatography, remove the acetonitrile solvent, dissolve again in ethyl acetate, and then rinse with 1 N of hydrochloric acid and saturated sodium bicarbonate. Thoroughly dry the product using Anhydride magnesium sulfate and condense it through vacuum distillation. The title chemical (1.26g, percent yield 87.8%) can be extracted from the yellow, viscous fluid through column chromatography (using an unfolding solvent such as ethyl acetate: hexane = 5:1 (v/v)).

13C NMR results are listed in table 4, which show characteristic peak values of 173.3 ppm -172.5 ppm, 171.6 ppm, and 52,1 ppm for amide, ester, and methoxyl, respectively.

Table 4

Synthesizing Example 5 (Step 5) : hydrolysis: synthesis of 9-anthrylmethyl N-[(tris[2-[(tris[2- (carboxyethoxy)methyl]methylamino)carbonyl]ethoxymethyl]methyl)aminocarbonyl)p ropylcarbamate ( which represents the final dendrimer compound, A-NH-spacer-[1]- amine-[9]-acid)

Dissolve the 9-anthrylmethyl N-[(tris[2-[(tris[2- (methoxycarbonyl)ethoxy]methylmethyl)amino]carbonyl]propyl carbonate (0.60g,

0.34 mmol) obtained from step 4 in acetone and 0.2N NaOH at a high temperature for 48 hours. After the completion of the reaction is confirmed through thin film chromatography, remove the acetone solvent and extract the product using water and ethyl acetate to form a water layer. Treat the water layer with diluted hydrochloric acid at 0°Cuntil it reaches pH1~2; extract the ethyl acetate, then dry with Anhydride magnesium sulfate and filter out the solvent through vacuum distillation to obtain the viscous, yellow, liquid product.(0.37g, percent yield 67.6%)

NMR analysis results are listed on table 5 below; the disappearance of the characteristic 52.1 ppm peak caused by methoxyl groups confirms that the product has undergone hydrolysis.

Table 5

9-anthrylmethyl N- IH NMR(DMSO)

({[tris({2-[({tris[(2- δ 13.00-11.00(br, CH2COOH, 9H), 8.66(s, C14H9CH2, 1 H), carboxyethoxy)methyl] 8.42(d, C14H9CH2, 2H, ), 8.13 (d, C14H9CH2, 2H), 7.62(t, methyl}amino)carbonyl] C14H9CH2, 2H), 7.54(t, C14H9CH2, 2H), 7.12(t, ethoxy}methyl)methyl]a OCONHCH2.1 H), 7.10(s, OCH2CH2CONHC, 3H), 7.06(s,

Example 1: Production of substrate with fixated dendrimers and the confirmation of its characteristics

Thoroughly rinse three different types of glassy thin films, silicon wafer, glass slide, fused silica, and dry under a 30mTorr vacuum. Immerse the three films in a toluene solution containing (3- (glycidoxypropyl)methyldiethoxysilane (30mM) at a high temperature. Following the completion of the silanization reaction, rinse the substrates with toluene and expose the three to an oven at 120°C for 30 minutes. Give time for the substrates to cool down at a high temperature, then immerse them in toluene and methanol solution for 3 minutes; rinse the three with ultrasonic waves and dry again. After applying two drops of sulfur in pure ethylene glycol, submerge the silanized substrates in the solution for 8 hours at a temperature between 80 - 100 °C. Once the reaction is complete, rinse the substrates with ultrasonic waves while immersed in an ethanol-methanol solution, and dry.

After resolving A-NH-spacer-[1]-amine-[9]-acid (2mM) (1eq.) in a small amount of DMF solvent, dilute it in dichloromethane solvent. Add DCC(dicyclohexylcarbidiimide) (9.3eq.) and DMAP(4-(dimethylamino)pyridine) (0.93eq.) and stir. After soaking the ethylene glycol-modified substrates in the above solution for 3 days, rinse the substrates with ultrasonic waves while immersed in methanol and deionized water solution, and dry.

Experimental Example 1 : Sensing changes on the dendrimer fixed substrate surface

Measure the change in thickness of the silicon wafer resulting from the induction of A-NH-spacer-[1]-amine-[9]-acid through an ellisometer. Changes were within 10 to 13 ^' .

UV ray absorbtion spectrum data obtained from fused silica films showed that the anthracene portion of the dendrimer displayed high absorbtion peaks in response to lights emitted at a wavelength of 257 nm. This verifies that the dendrimers have been effectively fixed onto the substrate surface.

Experiment al Example 2: Measuring the density of reactive amine groups

9-anthraldehyde is a substance that has 6 times the molar absorbtivity than that of, which has been conventionally used. Density calculation with 4- nitrobenzaldehyde is virtually impossible for surfaces including the molecular layers of A-NH-spacer-[1]-amine-[9]-acid, due to the extremely low density of amine groups. The amine density is only obtainable by measuring the fluorescence signals emitted from 9-anthraldehyde. The process is as follows: Immerse the substrate in a 1 M solution of TFA dissolved in dichloromethane for 3 hours; immerse the substrate in a dichloromethane solution containing 20% DIPEAfor 10 minutes. Rinse the substrate with supersonic waves while immersed separately in dichloromethane and methanol. Combine 9-anthraldehyde with the amine groups and perform hydrolysis in water. The fluorescent spectrum is obtained from the resulting solution. Experimental data revealed that the amine density was 0.1-0.2 amines/nm².

Experimental Example 3: Analyzing surface form Atomic Force Microscope observations indicated that no noticeable structural changes had occurred subsequent to the reaction, neither were any clusters of any kind observed. Such results indicate that the A-NH-spacer-[1]-amine-[9]-acid molecules have formed an even monolayer on the substrate surface.

Probe DNA insertion and the analysis of its fixation capability

Example 4 (substrate/A-NH-spacer-[1]-amine-[9]-acid/probe DNA)compound

Immerse the substrate produced from the steps above in a 1 M solution of

TFA dissolved in dichloromethane for 3 hours and in a dichloromethane solution containing 20% DIPEA for 10 minutes to remove the amine protection group of A- NH-spacer-[1]-amine-[9]-acid. Rinse the substrate with supersonic waves while immersed separately in dichloromethane and methanol and dry. UV spectroscopy is used confirm that the protection groups have been separated. UV absorption spectrum data showed that the 257 nm absorption peak disappeared after de-protection. Dissolve 25 mM of DSC (dissuccinimidylcarbonate) in acetonitrile and add catalytic amounts of DIPEA (diisopropylethylamine) to form a homobifunctional linker solution. Expose the de-protected substrate to the solution under a nitrogenous atmosphere. After given four hours of reaction time, retrieve the substrate and submerge it in a DMF solvent under a nitrogenous atmosphere for another hour. Rinse the substrate with sufficient amounts of methanol and dry. Other compounds, such as DSG, DSO, DMS, and PDITC can be used in place of DSC.

Probe DNA dissolved in 50mM sodium bicarbonate buffer solution (pH 8.5, 10% DMSO(dimethylsulfoxide)) is then spotted on a DSC induced substrate surface in a 95% humidity chamber. The structure of the probe DNA used was 5'-Cy3- ACAAGCACAGTTAGG-C7-aminolink-3^', with an attached fluorescent title compound (Cy3). The spotting process was carried out with a Microsys 5100 microarrayer (Cartesian Technologies, Inc., USA). After 10 hours of reaction time is given, remove the unreacted probe DNA by immersing the substrate in 2x SSPE (SALINE-SODIUM PHOSPHATE-EDTA) (Saline-Sodium Phosphate-EDTA) and 0.2% SDS (sodium dodecysulfate) buffer solution (pH7.4) at 37 for three hours, then expose it to boiling water for 5 minutes and dry in nitrogen gas.

Comparative Example 1 : (substrate/APDES/probe DNA) compound

The reactivity of substrates with fixed A-NH-spacer-[1]-amine-[9]-acid compounds and probe DNA, along with the hybridization capacity between probe and target DNA molecules are evaluated and compared through the following experiment: Prepare a substrate with APDES (3-aminopropylmethyldiethoxysilane, commonly used in amino silanization reactions) compounds fixed on the surface. To do this submerge the substrate in a toluene solution with 30mM APDES. Once the silanization reaction is complete after approximately three hours, rinse the substrate with toluene and dry at 120°C for thirty minutes. Cool the substrate to normal air temperature, then rinse again with ultrasonic waves in toluene and methanol solution for three minutes. Insert probe DNA onto the substrate surface following the procedureseof Example 4 mentioned previously. To insert homobifunctional linker compounds, add catalytic amounts of DIPEA to acetonitrile solution with 25mM DSC, and immerse the APDES-modified substrate in the solution under a nitrogenous atmosphere. Given four hours, submerge the substrate in a DMF solution for an additional hour, then rinse it with methanol and dry. Probe DNA (20 M), dissolved in 50mM sodium bicarbonate buffer solution (pH 8.5 10%) DMSO(dimethylsulfoxide)) is spotted onto the surface in a 95% humidity chamber using the same process and instruments used previously. After ten hours, the unreacted probe DNA is removed by exposing the substrate to 2x SSPE (SALINE-SODIUM PHOSPHATE-EDTA) and 0.2% SDS (sodium dodecysulfate) buffer solution (pH 7.4) at 37°C for three hours and in boiling water for 5 minutes. The probe DNA used is identical to the one used for the dendrimer- modified substrate.

Comparative Example 2 (substrate/dendrimer compound lacking a spacer/probe DNA) compound

Instead of A-NH-spacer-[1]-amine-[9]-acid, fix the compound N-Cbz-[1]amine-[9]acid in Chemical Formula 7. (N-(benzyloxycarbonyl)-tris[((N'-(carbonyl)- tris((carboxyethoxy)methyl)methylamino)ethoxy)methyl]aminomethane) onto the substrate surface, and insert probe DNA the same way as mentioned in Comparative Example 1

[Chemical Formula 7]

First, fix the N-Cbz-[1]amine-[9]acid onto the glass substrate, and remove the amine protection group Cbz using trifluouracetic acid, as explained in Korean public patent no. WO02/020469 . Follow Example 4 to insert probe DNA on the substrate surface; dissolve 25 mM of DSC (dissuccinimidylcarbonate) in acetonitrile to form a homobifunctional linker solution and add catalytic amounts of DIPEA to the solution, then immerse the

N-[1]amine-[9]acid substrate for four hours. Retrieve the substrate and submerse it in DMF solution under a nitrogenous atmosphere for at least an hour. Probe DNA (20 μM), dissolved in 50mM sodium bicarbonate buffer solution (pH 8.5, 10% DMSO(dimethylsulfoxide)) is therein spotted onto the surface of the substrate in a 95% humidity chamber using the same process and instruments used previously.After ten hours, the unreacted probe DNA is removed by exposing the substrate to 2x SSPE (SALINE-SODIUM PHOSPHATE-EDTA) and 0.2% SDS (sodium dodecysulfate) buffer solution (pH 7.4) at 37°C for three hours and in boiling water for 5 minutes. The probe DNA used is identical to the one used for the dendrimer fixed substrate.

Experimental Example 4: Comparison of the fixation capacity of probe DNA on the two substrate surfaces Dry the substrates obtained from Example 4 and Comparative Examples 1 and 2, and analyze fluorescence signal emission using a laser scanner. The detected fluorescence signals were examined (Fig. 8) using the software Imagene 4.0(Biodiscovery). Figure 8a shows the scan data for the substrate from oneExample. The laser intensity was 90, and the fluorescent signature was 25,000 with the detector gain settings set at 80. The fluorescence intensity was the calculated average value, and all fluorescence data was obtained under same conditions. Fig 8b presents the scan data for the substrate from Comparative Example

1. The laser intensity was 90, and the detected intensity was 40,000 with the detector gain settings set at 80. The signal intensity, which is roughly twice as stronger than that of 8a, means that there is nearly as twice the number of probe DNA fixed on the substrate surface. Fig 8c presents the scan data for the substrate from Comparative Example

2. The intensity was too small to be measured under the given settings above, so the laser intensity was adjusted to 100, and the detected intensity was 3,000 with the detector gain settings set at 90. Even under the stronger laser intensity settings, this value is signfinicantly lower than that obtained from 8a. Such differences in fluorescent signal strength in fact show the probe DNA's surface bonding strength; as can be seen from the data tables, dendrimers lacking a spacer exhibit significantly lower surface fixation rates due to the steric hindrance coming from the molecular compound itself.

Target DNA insertion and the investigation of target DNA hybridization Example 5: (substrate/A-NH-spacer-[1]-amine-[9]-acid/probe DNA/target DNA)compound

Prepare five glass slides with a A-NH-spacer-[1]-amine-[9]-acid fixed surface. Remove the amine protection group A by immersing the substrate in a 1 M solution of TFA dissolved in dichloromethane for 3 hours; then immerse the substrate in a dichloromethane solution containing 20% DIPEA for 10 minutes. Rinse the substrate with supersonic waves while immersed separately in dichloromethane and methanol. The removal of the amino protection group can be confirmed through UV ray spectroscopy; data should show that the 257 nm wavelength absorption peak has disappeared from the de-protection absorption spectrum. Dissolve 25 M of DSC (dissuccinimidylcarbonate) in acetonitrile and add a catalytic amount of DIPEA (diisopropylethylamine) to form a homobifunctional linker solution. Expose the de-protected substrate to the solution under a nitrogenous atmosphere. After given four hours of reaction time, retrieve the substrate and submerge it in a DMF solvent under a nitrogenous atmosphere for another hour. Rinse the substrate with sufficient amounts of methanol and dry. Other compounds, such as DSG, DSO, DMS, and PDITC can be used in place of DSC. Five types of probe DNA listed below(20 M), dissolved in 50mM sodium bicarbonate buffer solution (pH 8.5, 10% DMSO(dimethylsulfoxide)) are spotted onto the surface in a 95% humidity chamber using the same process and instruments used previously. After ten hours, the unreacted probe DNA is removed by exposing the substrate to 2x SSPE (SALINE-SODIUM PHOSPHATE-EDTA) and 0.2% SDS (sodium dodecysulfate) buffer solution (pH 7.4) at 37°C for three hours and in boiling water for 5 minutes. 5'-Cy3-TGGACACTCGGAATG-3^' was fixed onto the dendrimer surface as target DNA, Cy3 being a fluorescent title substance. As for the five probe DNA types, the following were used: 1. 5'-C6-aminolink-CATTCCGAGTGTCCA-3^' (complementary binding) 2. 5^'-C6-aminolink-CATTCCGTGTGTCCA-3^' (T-T mismatch at the middle position of a strand)

3. 5^'C6-aminolink-CATTCCGAGTGTCCT-3^' (T-T mismatch at the middle position of a strand)

4. 5^'-C6-aminolink-CATTCCGAGTGTCCG-3^' (T-G mismatch at the middle position of a strand)

5. 5^'-C6-aminolink-CATTCCGAGTGTCCC-3^' (T-C mismatch at the middle position of a strand)

Both probe and target DNA were purchased from metabion, and the spotting process was executed via Mixrosys 5100 microarrayer (Cartesian Technologies, Inc.,

USA).

Experimental Example 5: Investigation of the hybridization efficiency of substrates (Example 5).

The fluorescence signal emission of the dried substrate from Example 5 was measured using a laser scanner (ScanArray Lite, GSI Lumonics). The detected fluorescence signals were analyzed (Fig. 9 and 10) using the software Imagene 4.0 (Biodiscovery).

Fig. 9a shows a completely complementary hybridization between target and probe DNA (5'-C6-aminolink-CATTCCGAGTGTCCA-3') Fig. 9b shows the fluorescent signal strength resulting from a nucleotide mismatch at the end position between probe DNA (5^'-C6-aminolink- CATTCCGTGTGTCCA-3') and target DNA during hybridization The fluorescent signal strength of 9a was 22,500, wherein 9(b) was less than 500, resulting in a ratio of 1 :0,022 (laser intensity was set at 90, detector grain at 80). Using thermodynamic equations, the theoretical difference in extent of hybridization for 9(b) within the solution at 50 ^°C can be obtained, which turns out to be 1 :0.016 (matching signal: mismatching signal). It can be seen that the theoretical ratio and the actual ratio (the difference in degree of hybridization observed from the substrate surface, which contains evenly distributed, yet low density molecular layers of A-NH-spacer-[1]-amine-[9]-acid) closely resemble each other. Substrates containing A-NH-spacer-[1]-amine-[9]-acid layers on its surface provide sufficient free space between probe DNA molecules, closely simulating the hybridization environment in a solution. Thus thermodynamic functions applicable to solutions can also be applied to the substrate surface. Due to such characteristics, it is possible to apply thermodynamic equations to the production and utilization of DNA chips, which produces predictable results and higher reliability among DNA chips. In addition, the fluorescence intensity of a substrate fixed only with probe DNA turned out to be 25,000 (Fig. 8a), and the intensity decreased to 22,500 after the target DNA was complementarily hybridized, thus showing 90% hybridization efficiency. Substrates with A-NH-spacer-[1]-amine-[9]-acid layers formed on its surface, thus allowing the DNA molecules to evenly space themselves, provide unrivaled efficiency when compared to other methods of DNA fixation such as the light transcription process developed by Affimetrix Co. which yielded an efficiency rate below 10% (Neoces B. Leontis and John SantaLucia, Jr. " Molecular Modeling of Nucleic Acids" chapter 13 American Chemical Society, Washington, DC) Fig. 10 presents the data on fluorescence intensity difference during a complementary bonding reaction (10a-10b) and a reaction in which the end strands of probe DNA and target DNA are mismatched (10c-10d). 5'-C6-aminolink-CATTCCGAGTGTCCA-3'_ was used as probe DNA for the reaction in Fig. 10a, while 5'-C6-aminolink-CATTCCGAGTGTCCT-3' was used as target DNA in reaction 10b. In Fig. 10c, 5'-C6-aminolink-CATTCCGAGTGTCCG-3' was used as target DNA (T-G mismatch); for Fig. 10d, 5'-C6-aminolink- CATTCCGAGTGTCCC-3' was used as target DNA (T-C mismatch). The obtained fluorescence signatures were 22,500 for 10a, 7,900 for 10b, 8100 for 10c, and 9,000 for 10d. The theoretical fluorescence intensity resulting from a mismatch at the end site of strands during hybridization in a solution at 50°C were as following: 1:0.51 (T-

T mismatch) 1:0.26 (T-G mismatch), 1:0.37 (T-C mismatch). The actual data closely resembled the predicted values: 0.35 (T-T mismatch) 0.36(T-G mismatch), 0.40 (T- C mismatch). Similarly, when a mismatch occurs among the middle site of probe and target strand DNAs, the actual and theoretical difference in the extent of hybridization during both complementary and non-complementary reactions closely match each other as long as the same substrate with X-NH-spacer-[1]-amine-[9]- acid layers is used.

Comparative Example 3 : (substrate/APDES/probe DNA/target DNA) compound Instead of using the five probe DNA types with the fluorescent compounds, Cy3, attached to them, Cy3 was detached from all the compounds. The probe DNA types remained the same.

1. 5^'-C6-aminolink-CATTCCGAGTGTCCA-3' (complementary binding) 2. 5'-C6-aminolink-CATTCCGTGTGTCCA-3^' (T-T mismatch at the middle position of a strand)

3. 5^'C6-aminolink-CATTCCGAGTGTCCT-3^' (T-T mismatch at the middle position of a strand) 4. 5^'-C6-aminolink-CATTCCGAGTGTCCG-3^' (T-G mismatch at the middle position of a strand)

5. 5'-C6-aminolink-CATTCCGAGTGTCCC-3^' (T-C mismatch at the middle position of a strand)

Hybridization process was carried out as outlined in Example 5. The five substrates were immersed separately in 2x SSPE and 0.2% SDS buffer solution (pH 7.4) containing the target DNA (with Cy3 attached to it). Following the completion of the hybridization process, the substrates were rinsed with 2x SSPE and 0.2% SDS buffer solution (pH 7.4).

Experimental Example 6: comparison of the hybridization efficiency of the substrates from Comparative Example 3.

The fluorescence signal emission of the dried substrate from Comparative Example 3 was analyzed using a laser scanner (ScanArray Lite, GSI Lumonics).

The detected fluorescence signals were examined (Fig. 11 and 12) using the software Imagene 4.0(Biodiscovery). Fig 11 data was obtained during a completely complementary hybridization. In 11b, a T-T mismatch occurred at the middle position of the probe DNA and target strand DNAs. The fluorescent intensity was 10,500 for 11a and 3000 for 11 b under the same condition as in the above examples. The fluorescent intensity ratio for fig. 11 was 1 :0.29, which is substantially lower in selectivity when compared to the glass substrate modified with A-NH- spacer-[1]-amine-[9]-acid (1 :0.022). The fluorescent intensity of the substrate when only the probe DNA had been inserted was 40,000. Following a complementary hybridization, the fluorescent intensity dropped to 10,500, resulting in 26% hybridization efficiency, which is substantially lower than the hybridization efficiency of a dendrimer-modified substrate. The lower percentage is due to the substrate's failure to maintain even spacing between probe DNA's and increasing the steric hindrace. A-NH-spacer-[1]- amine-[9]-acid maximizes the hybridization efficiency by maintaining equal space between DNA molecules.

Fig 12a, like that of 10a, is a fluorescent spectroscopy of a complementary hybridization process. The fluorescent intensity was 10500, 5600, 6300, and 7300 for 12 a, 12b, 12c, and 12d, respectively. The fluorescent intensity ratio for an end mismatch case was 1 :0.54-.70, which lacks selectivity when compared that of the A- NH-spacer-[1]-amine-[9]-acid fixed substrate. Adding to these shortcomings is the fact that there are large differences in fluorescent intensity between spots; in other words, the coefficient variance value was large (20-40%; 10-20% for substrates with fixed A-NH-spacer-[1]-amine-[9]- acid), and the fluorescent variance was irregularly distributed even within the same spot (Fig. 11, 12) These findings indicate that the probe DNA have not been evenly distribute and instead have condensed among themselves. If a substrate with A-NH- spacer-[1]-amine-[9]-acid is reacted with DSC, the distance between the amine functional groups, which is longer than the length of the DSC linker compound, allows only one side of the DSC compound to bond to the substrate surface, while the rest is left to react with the amine functional group of probe DNA. On the other hand, in compounds that cannot guarantee harmonious hybridization such as APDES, the length between the amine functional groups is shorter than the DSC linker compound, and thus both sides of the linker compounds are free to bond with the amine functional groups. The freely bonding DSC linker compounds then bond irregularly onto the surface, often forming concentrated clusters. Probe DNA inserted into such settings, thus also become concentrated at certain points. Consequently, the reason why A-NH-spacer-[1]-amine-[9]-acid provides higher hybridization efficacy and selectivity along with lower CV values over APDES lies in the fact that the dendrimer compound guarantees optimized space for hybridization and prevents probe DNA molecules from condensing among themselves.

Comparative Example 4 (substrate/dendrimer compound lacking spacer/probe DNA/target DNA) compound Excluding the usage of the following probe DNA groups in which the fluorescent title substances (Cy3) have been removed (listed below), Example was followed exactly as outlined in Comparative Example 1 to obtain (substrate/dendrimer lacking spacer/probe DNA) compound. 1. 5'-C6-aminolink-CATTCCGAGTGTCCA-3^' (complementary binding)

2. 5'-C6-aminolink-CATTCCGTGTGTCCA-3^' (T-T mismatch at the middle position of a strand)

5. 5^'-C6-aminolink-CATTCCGAGTGTCCC-3^' (T-C mismatch at the middle position of a strand) Target DNA introduction, hybridization process were carried out exactly as outlined in Comparative Example 5 to obtain (substrate/dendrimer compound lacking spacer/probe DNA/ target DNA) compound.

Experimetal Exapmle 7: Comparison of the hybridization efficiency of the substrates obtained from Comparative Example 4

The fluorescence emission signal of the dried substrate obtained from Comparative Example 4 was measured using a laser scanner (ScanArray Lite, GSI Lumonics). The detected fluorescence signals were analyzed (Fig. 13) using the software Imagene 4.0(Biodiscovery).

Experimental Example 4 revealed that the fluorescent signature of N-Cbz-

[1]amine-[9]acid substrate was too small to be detected under conventional settings, so the he laser intensity was adjusted to 100, and the detector gain settings set at 90. Even under the stronger laser intensity settings, this intensity value, 3000, was signfinicantly lower than that obtained from the novel dendrimer compound. Hence, it is safe to conclude that N-Cbz-[1]amine-[9]acid containing substrates have considerably low DNA surface insertion rations due to the steric hindrance caused by the molecules, and consequently remains largely unreactive to target DNA during hybridization. Such low fluorescent strength values were obtained in both complementary and non-complementary reactions; their extremely small values rendered it impossible to discern between the two reactions by looking at the data alone. As it can be observed from the various experiments above, substrates and

DNA chips utilizing the novel dendrimer compound presented in this paper exhibits superior absorption capacity towards probe compounds and has a high hybridization efficiency for target compounds. The compound can produce bio chips with enhanced detection sensibilities and higher reliability, especially by enabling the application of thermodynamic functions to the process of hybridization on the substrate surface, which can assist in making theoretical diagnosis & analysis of bio chips. Substrates that make use of the dendrimer compound accept, as probe and target compounds, not only DNA, but protein, carbohydrates, polymers, nanoparticles, and cells. Such is relevant in the scientific world today in which the application of bio chips to protein and carbohydrates are undergoing research. Bio compounds can be fixed onto the substrate through both linker compounds and direct reaction with the amine functional group. As for the linker compounds,

homobifunctional linker compounds or heterobifunctional linker comounds such as DSG(disuccinimidylglutarate),

DSO(disuccinimidyloxalate), PDITC(phenylendiisothiocyanate), DMS(dimethylsuberimidate), SMB(succinimidyl-4-maleimido butyrate), SSMCC(sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and proteins such as biotin and avidin can all be used. When not using linker compounds, the amine group endpoint of the dendrimer compound can be made to form covalent bonds with the carboxylic acid group in the bio compound. The technique has been frequently used in various studies. When inserting the bio compounds in a chip format, the compound can be spotted onto the dendrimer fixed substrate surface through a microarrayer, or manually through the micro-pipette (when producing less compact bio chips). The substrate then undergoes one lass step of hybridization, in which it is hybrid with target compounds (oligonucleotide, cDNA, genomic DNA, protein, carbohydrate, high molecules, nano particles, and cells), and is then analyzed for the title chemical's fluorescent signatures (by inserting fluorescent dyes) or its electric signals. Perfected biochips can be used for diagnostic purposes by detecting the presence of bio substances that cause certain illnesses.

The effects of X-NH-spacer-[1]amine-[9]acid:

The novel dendrimer compound X-NH-spacer-[1]amine-[9]acid exhibits the following advantages:

1. Decreases the density of amine groups and minimizes the steric hindrance emitted from the substrate surface and the dendrimer body by maintaining a constant distance between amine groups. 2. Providing sufficient free space between probe DNA molecules and maximizing the hybridization efficacy. Such contributes to enhancing the accuracy of bio chips because fluorescence spectroscope data shows easily noticeable differences between complementary and non-complementary reactions. 3. An ideal hybridization environment is created on the substrate surface due to the optimized spacing between probe DNA molecules, which simulates the environment within a solution; this allows the application of thermodynamics functions to the substrate surface, which enables theoretical calculations and result predictions in bio chips. Such advantages the dendrimer compound provides will play in important role in furthering the development of many bio chips. As an example, the lower the functional group density is, the easier it is to fix bio molecules onto the substrate surface, which will lead to improvements in the quality of the chip. The dendrimer can also become an important element in surface research (in which molecules are fixed to the substrate surface and their properties are investigated) as a surface substrate. The optimized spacing the dendrimer presents will enable individual raw molecules to be used as effective sensors.

Claims

WHAT IS CLAIMED IS

1. A dendrimer composition represented by Chemical Formula 1 :

[Chemical Formula 1]

wherein X is amine protecting group which is separated by acid, L is a spacer, which is -R-C(O)-NH-, and R is substituted or unsubstituted hydrocarbon.

2. The composition according to Claim 1 , wherein the X in Chemical Formula 1 is (1) benzyloxycarbonyl, (2) benzyloxycarbonyl of which hydrogen of the benzene group is substituted with a functional group which attract electrons, (3) 9- anthrylmethoxycarbonyl or (4) t-butoxycarbonyl.

3. The composition according to Claim 2, wherein the functional group of benzyloxycarbonyl, of which hydrogen of the benzene group is substituted with the functional group, is nitro, halogen, or cyano group.

4. The composition according to Claim 2, wherein the benzyloxycarbonyl, of which the hydrogen of the benzene group is substituted with the functional group, is

2-nitrobenzyloxycarbonyl, 3-nitrobenzyloxycarbonyl, 4-nitrobenzyloxycrbonyl, 2- fluorobenzyloxycarbonyl, 3-fluorobenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl, 2- chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl, 2- bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 2-iodobenzyloxycarbonyl, 2-iodobenzyloxycarbonyl, 4-iodobenzyloxycarbonyl, 2- cyanobenzyloxycarbonly, 3-cyanobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl.

5. The composition according to Claim 1 or Claim 2, wherein the length of R is 5-15 A.

6. The composition according to Claim 1 or Claim 2, wherein the R is selected from a group comprising substituted or unsubstituted linear or branched alkanyl; substituted or unsubstituted linear or branched alkenyl; substituted or unsubstituted linear or branched alkinyl; substituted or unsubstituted cycloalkanyl; substituted or unsubstituted cycloalkenyl; substituted or unsubstituted aromatic composition group; and substituted or unsubstituted polyalkylenglycol.

7. The composition according to Claim 1 or Claim 2, wherein the R is unsubstituted linear alkyl of C3-C9.

8. The composition according to Claim 1 , wherein the dendrimer is represented by Chemical Formula 1a.

[Chemical Formula 1a] 9-anthrilmethyl N-([tris(2-[(tris[(2- carboxyethoxy)methyl]methylamino)carbonyl]ethoxymethyl)methyl]aminocarbonyl)pr opylcarbamate.

9. A composition represented by Chemical Formula 6. [Chemical Formula 6]

wherein X is amine protective group which is separated by acid, L is a spacer, which is -R-C(O)-NH-, R is substituted or unsubstituted hydrocarbon, and Y is carboxy protecting group which is separated by base.

10. The composition according to Claim 9, wherein Y is methyl.

11. A method of producing the dendrimer represented by Chemical Formula 1 comprising: (i) preparing the composition of Chemical Formula 6; and (ii) reacting the composition of Chemical Formula 6 with base and separating carboxyl protecting group Y to expose the carboxyl group.

[Chemical Formula 1]

[Chemical Formula 6]

wherein X is amine protecting group that is separated by acid, L is a spacer, which is -R-C(O)-NH-, R is substituted or unsubstituted hydrocarbon, and Y is carboxyl protecting group, which is separated by base.

12. The method according to Claim 11 , wherein step (i) is comprising (a) preparing compound of the following Chemical Formula 2 with spacer compound; (b) producing the compound of Chemical Formula 4 by reacting the compound of Chemical Formula 2 and the compound of Chemical Formula 3; (c) yielding the compound of Chemical Formula 5 by reacting with the compound of Chemical Formula 4 with base and separating carboxyl protecting group Y; and (d) producing the compound of Chemical Formula 6 by reacting the compound of Chemical Formula 5 and the compound of Chemical Formula 3. [Chemical Formula 2] X — HN— R — C0OH

[Chemical Formula 3]

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

13. The method according to Claim 12, wherein the step (a) includes a step to yield the compound of Chemical Formula 2 by reacting the compound of Chemical Formula 2a with one or more from Chemical Formula 2b compound and Chemical Formula 2c compound. [Chemical Formula 2a]

[Chemical Formula 2b]

14. The method according to Claim 12, wherein the steps (b) and (d) are carried out in acetonitryl, dimethylformamide, or Chloromethane solvent.

15. The method according to Claim 12, wherein the steps (b) and (d) are carried out in the presence of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydochloric acid salt and 1-hydroxybenzotriazol.

16. The method according to Claim 12, wherein the base group in steps (c) and (ii) is NaOH.

17. The method according to Claim 12, wherein the above method is carried out as in the following Reaction Formula 1.

[Reaction Formula 1]

wherein X is amine protecting group that is separated by acid, L is a spacer, which is -R-C(O)-NH-, R is substituted or unsubstituted hydrocarbon, Y is carboxyl protecting group that is separated by base, a is reacting for 1-3 hours at 40~60°C after adding triethylamine and DMF, b is reacting for 6-24 hours at 15-40 °C after adding EDC, HOBT, the compound of Chemical Formula 3 and acetonitril, c is reacting for 6-48 hours at 15-40 °C after adding NaOH, d is reacting for 6-24 hours at 15-40 °C after adding EDC, HOBT, the compound of Chemical Formula 3 and acetonitril, and e is reacting for 6-48 hours at 15-40 °C after adding NaOH.

18. The method according to Claim 11 or Claim 12, wherein the X is 9- anthrylmethoxycarbonyl, R is propyl, and Y is methyl.

19. A method of producing dendrimer-substrate compound comprising (a) incorporating hydroxyl group onto the surface of the substrate; and (b) reacting the dendrimer of Chemical Formula 1 of Claim 1 to form multiple covalent bonds between the hydroxyl group on the surface of the substrate and carboxyl group of the dendrimer.

20. The method according to Claim 19, wherein step (c), a step to expose amine termini by adding base to the dendrimer-substrate compound and separating the amine protecting group, is added after step (b).

21. The method according to Claim 19, wherein the substrate is (i) glass thin film, (ii) metallic thin film with metal oxide, (iii) metallic or non-metallic bead or (iv) metallic or non-metallic nanoparticle.

22. The method according to Claim 19, wherein the substrate is glass thin film, glass bead, or glass nano particle and the step (a) is carried out by treating the substrate with epoxysilanization agent to induce epoxysilaniation reaction on the substrate surface.

23. The method according to Claim 19, wherein the substrate is glass thin film, glass bead, or glass nanoparticle and the step (a) is carried out by treating the substrate with silan compound with hydroxyl group.

24. The method according to Claim 23, wherein the silan compound with the hydroxyl group is TPU(N-(3-triethoxysilylpropyl)-o-polyethylene oxide urethane) or TPH(N-(3-triethoxysilyl)-propyl)-4-hydroxybutylamide).

25. The method according to Claim 19, wherein the substrate is metallic thin film, metallic bead, or metallic nano particle and the step (a) is carried out by treating the substrate with thiol compound with hydroxyl group.

26. The method according to Claim 25, wherein the thiol compound with hydroxyl goup is mercaptoalkanol or mercapto(polyethylene glycol).

27. The dendrimer-substrate compound produced by the method according to Claim 19.

28. The dendrimer-substrate compound produced by the method according to Claim 20.

29. A dendrimer-substrate compound comprising a substrate and the dendrimer molecular layer of Chemical Formula 1 , which is multiple covalently bonded to the substrate surface.

[Chemical Formula 1]

30. The dendrimer-substrate compound according to Claim 29, wherein the dendrimer molecular layer is actually formed as monolayer.

31. The dendrimer-substrate compound according to Claim 29, wherein the dendrimer molecular layer is 5-15A thick.

32. The dendrimer-substrate compound according to Claim 29, wherein the amine density of the substrate surface is 0.2/nm² or less.

33. The dendrimer-substrate compound according to Claim 29, wherein the amine density of the substrate surface is 0.1~0.2/nm².

34 The dendrimer-substrate compound according to Claim 29, wherein the substrate is (i) glass thin film, (ii) metal thin film, (iii) metallic or non-metallic bead, or

(iv) metallic or non-metallic nanoparticle.

35. The dendrimer-substrate compound according to Claim 34, wherein the glass thin film of (i) is silicone wafer, silica, fused silica, or glass slide plate.

36. The dendrimer-substrate compound according to Claim 34, wherein the metal thin film of (ii) is gold thin film or silver thin film.

37. The dendrimer-substrate compound according to Claim 34, wherein the metallic or non-metallic bead of (iii) has diameter of several hundred nm or more and is glass bead, gold bead, or silver bead.

38. The dendrimer-substrate compound according to Claim 34, wherein the metallic or non-metallic nonoparticle has diameter of less than one hundred nm and is glass bead, gold bead, or silver bead.

39. A dendrimer-substrate compound comprising a substrate and the dendrimer molecular layer of Chemical Formula 6, which is multiple covalently bonded to the substrate surface. [Chemical Formula 6]

wherein X is amine protecting group which is separated by acid, L is a spacer, which is -R-C(O)-NH-, R is substituted or unsubstituted hydrocarbon, and Y is carboxy protecting group, which is separated by base.

40. The dendrimer-substrate compound according to Claim 39, wherein the dendrimer molecular layer is actually formed as monolayer.

41. The dendrimer-substrate compound according to Claim 39, wherein the amine density of the substrate surface is 0.2/nm² or less.

42. The dendrimer-substrate compound according to Claim 39, wherein the substrate is (i) glass thin film, (ii) metallic thin film with metal oxide, (iii) metallic or non-metallic bead or (iv) metallic or non-metallic nanoparticle.

43. The method of producing a bio chip comprising: (a) preparing the dendrimer-substrate compound according to Claim 29 and (b) fixing probe compound on the surface of dendrimer-substrate compound by reacting the dendrimer-substrate compound with probe compound.

44. The method according to Claim 43, wherein an additional step is added before the step (b) to link the termini of the dendrimer and the linker compound by treating the surface of the dendrimer- substrate compound with linker.

45. The method according to Claim 44, wherein the linker compound is homobifunctional linker, heterobifunctional linker, biotin, or avidin.

46. The method according to Claim 43, wherein the probe compound is DNA, protein, carbohydrate, polymer, nonoparticle, or cells.

47. A bio chip manufactured according to the method of Claim 43.

48. A bio chip comprising substrate, the dendrimer molecular layer of Chemical

Formula 1 according to Claim 1 , which is multiple covalently bonded to the substrate surface, and probe compound, which is bound to amine on the termini of the dendrimer molecular layer.

49. The bio chip according to Claim 48, wherein the termini amine of the dendrimer molecular layer and the probe compound is linked by linker compound.

50. The bio chip according to Claim 48, wherein the probe compound is DNA, protein, carbohydrate, polymer, nonoparticle or cells.

51. A method of detecting a target compound by using the bio chip according to Claim 48.

52. A method of diagnosis by using the bio chip according to Claim 48.

53. A method of diagnosis comprising a step to hybridize by treating the bio chip according to Claim 48 with target compound and a step to analyze the degree of hybridization from the hybridized substrate.

54. The method according to Claim 53, wherein the target compound is DNA, protein, carbohydrate, polymer, nonoparticle, or cells.

55. The method according to Claim 53, wherein the target compound is oligonucleotide, cDNA, or genomic DNA.