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1 Surface Chemistry for Biochip and Bioanalysis 박준원포항공과대학교

2 Biochip 의탄생 Light-directed, spatially addressable parallel chemical synthesis Fodor, S. P. et al. Science 251(4995), , 1991 US Pat.#: , , Conventional method southern blotting

주요회사의기술 Affymetrix Market leader, Mask 를이용한 GeneChip

Illumina Bead-based technology 보유, 3μm 크기의공을이용한진단

보유 ( 자료 ) Affymetrix photolithography ( 자료 ) Agilent

3 주요회사의기술 Affymetrix Market leader, Mask 를이용한 GeneChip System 을 1996 년상업화하여시장지배 Agilent 잉크젯프린터를이용한마이크로어레이제작 Illumina Bead-based technology 보유, 3μm 크기의공을이용한진단 Nimblegen Maskless Array Synthesizer (MAS) technology 보유 ( 자료 ) Affymetrix photolithography ( 자료 ) Agilent Inkjet기술도해 ( 자료 ) Illumina bead array ( 자료 ) Nimblegen MAS기술도해

4 DNA 칩시장 DNA 칩시장은높은성장세를기록중 ( 단위 : $ Millions) * Global Industry Analysis Inc. "Biochips" 2004 년 4

5 KFDA 바이오칩승인현황 BAC 칩, 마크로젠 태아대상유전자검사, 기존검사법인양수검사, 융모막검사에비 해검사결과를신속하게도출 HPV 칩, 바이오메드랩, 마이진, 굿젠등 3 사 자궁경부암, 곤지름등을유발하는 HPV(Human Papilloma Virus; 인체유두종 ) 진단 ( 자료 ) 마이진 HPV Chip Kit

6 CYP 450 칩 (Roche, AmpliChip TM) 1998 년, 미국에서만 100,000 명이상이 ADR(Adverse Drug Reaction) 을원인으로사망 CYP 450 chip 으로약물대사관련유전자변이진단 UM(Ultrarapid metabolizer) ㅏ PM(Poor metabolizeer) 판별 진통제, 항우울제, 항히스타민제, 심혈관질환치료제의특수처방

7 DNA Chip 의응용 We now believe that the only disease not having some genetic component is trauma. M.D. Collins Francis (Director of the US National Human Genome Research Institute, Bethesda, MD, USA) Gene Expression Single Nucleotide Polymorphism (SNP) Gene DNA Diagnostics Chip

8 Gene Diagnostics DNA Chip 은여러가지병의진단에이용될수있음 Heart disease, Cancer, HIV/AIDS, Tuberculosis, Cytomegalovirus, Hypertension, steoporosis, Infertility, Alzheimer s disease 등등

9 Gene Expression Gene 의발현상태를밝혀냄

10 Gene Expression 의다양성

11 C-DNA Chip for Gene Expression DNA clones test reference laser1 excitation laser2 Reverse transcription PCR amplification purification label with fluor dyes emission Robotic printing Hybridize target to microarray Computer analysis

12 Single Nucleotide Polymorphism (SNP) Nucleotide Polymorphism: Difference in DNA sequence among individuals 개개인의 Genome 중 99.9 % 의염기서열은서로동일, 단지 0.1 % 만이개인마다다름 이 0.1 % 의차이로인해사람마다서로다르게특정약 (drug) 에대해 반응한다. 아울러각개인의유전자특성을포괄적으로확인하는것이 경제적으로가능하다. Pharmacogenetics

13 Pharmacogentics 의예 Drug Metabolism pathway Disease pathway Drug Metabolism pathway Disease pathway Drug Metabolism pathway Disease pathway

14 Personalized Medicine 시대의도래 몇방울의피와같은소량의샘플로걸리기쉬운병과진행중인병을동시에 알아낼수있음 Personalized medicine 시대를가능케함 21 세기초반에는실현가능 인간의평균수명이 100 세정도까지연장될것이다.

15 The Core Technique of Bio Chip Photochemistry Integration Surface chemistry Data mining Data processing Bio Chip Analysis - Fluorescence, electrochemistry

16 The Various Biochips DNA chip oligo, c-dna, aptamer Protein chip Carbohydrate chip Cell chip

sensitivity, detection limit, 낮은 variation 등이중요 - personalized

17 바이오칩기술의전망 Whole genome analysis - Affymetrix, Agilent 등고집적화기술을보유한회사에유리 - 새로운기술이떠오르기전까지안정적 Human diagnosis - 신뢰도, sensitivity, detection limit, 낮은 variation 등이중요 - personalized medicine 시대의도래 Point-of-care product - 의료, 식품, 농수산, 애완동물 - 짧은분석시간이관건 Blood glucose meter

18 범용화를위한과제 Price of each chip, detector Detection limit -PCR 과정없이소량의샘플만으로도분석이가능? FDA 인증전체분석에걸리는시간 의사, 소비자들의신뢰및선택편의성 - lab-on-a-chip의성공 FDA 인증의사, 소비자들의신뢰확보 fm? or am?! hours? or mins?!!

NanoCone Technology NH NH NH 2 NH NH NH 2 NH 2 2 2 NH 2 NH2 2 NH 2 2 NH NH NH NH 2 2 2 NH 2 NH 2 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 Aminosilane Surface (2D) Gel-like

19 NanoCone Technology NH NH NH 2 NH NH NH 2 NH NH 2 NH2 2 NH 2 2 NH NH NH NH NH 2 NH 2 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 Aminosilane Surface (2D) Gel-like Surface (3D) NSB Amine Surface NanoCone Competing technologies do not achieve homogeneity of surface-immobilized biomolecules. Severe steric hindrance and high nonspecific binding result in low accuracy and low reproducibility eventually. Control of regular spacing between surface-immobilized biomolecules provides homogeneity and results in high accuracy and reproducibility. Minimized steric hindrance and low nonspecific binding allow biomolecules to mimic solution-phase behavior.

20 Controlled Spacing up to 10 nm H H H NH H H H H H H H H H H NH NH H H NH NH H H HH H NH H H H H H H H H H H H NH H H N N H H H N N H H H H H H H NH H H H H H H H H H H H H H H H [9]-acid [18]-acid [27]-acid, [81]-acid ~ 3 nm Spacing ~ 10 nm

21 Immobilization of Gold Nanoparticles at the Dendron Surface d: 1.4 nm Langmuir, 21, 4257 (2005).

Spacing between Dendron Molecules on Surface SEM Image Distance between Dendron Molecules 80 70 60 Population 50 40 30 20 10 0 1.0 1.5 2.0 2.5 3.0 3.5 4.

22 Spacing between Dendron Molecules on Surface SEM Image Distance between Dendron Molecules Population Particle number / area : 130 ea / 50 x 50 nm 2 Density: ea/nm 2 Distance (nm) Average distance: 3.2 nm Standard deviation: 0.4 nm

23 DNA Microarray on the Dendron Surface Improves Significantly Detection of Single Nucleotide Variations

24 Fabrication of DNA Microarray on Dendron-Modified Surface Substrate Dendron PM site MM site Nucleic Acids Research, 33(12), e106 (2005).

Comparison of Discrimination Efficiency (I) NSB Slide Competitor Slide Slide PM EndA Deletion 2 nd C Int A Inser 2 nd mismatch GG GT GA Internal mismatch TT TG TC NSB 100 10 0.4 0.1 0.4 0.4 0.3 0.

25 Comparison of Discrimination Efficiency (I) NSB Slide Competitor Slide Slide PM EndA Deletion 2 nd C Int A Inser 2 nd mismatch GG GT GA Internal mismatch TT TG TC NSB Competitor Probe oligonucleotide IMM: 5 -NH2-C6-CAT TCC GXG TGT CCA-3 2nd MM: 5 -NH2-C6-CAT TCC GAG TGT CYA-3 Insertion: 5 -NH2-C6-CAT TCC GAAG TGT CCA-3 Target: 5 -Cy3-TGG ACA CTC GGA ATG-3 Hybridization: 1 nm target at 45 o C for 1 h, 1 min washing at 50 o C Deletion Int: 5 -NH2-C6-CAT TCC G_G TGT CCA-3 Deletion 2nd: 5 -NH2-C6-CAT TCC GAG TGT C_A-3 Deletion End: 5 -NH2-C6-CAT TCC GAG TGT CC_-3

26 Fast & Highly Specific Hybridization 1 min 5 min 10 min 30 min Fluorescence Signal (a.u.) Hybridization Time (min) Fluorescence Signal (a.u.) Hybridization Time (min) PM-TA MM-TT MM-TG MM-TC Detection of single nucleotide variation in model system depending on hybridization time. All probe spots are repeated 10 times horizontally and four spots in the first column from the left are position markers which indicate each position of spotted probes.

27 Short Washing Time Dendron Modified Surface Generic Amine Surface Fluorescence Intensity (a.u.) Washing time (min) TC MM TG MM TT MM TA PM Fluorescence Intensity (a.u.) TC MM TG MM TT MM TA PM Washing time (min) Hybridization: at 45 o C for 1 h, washing at 37 o C Nucleic Acids Research, 33(12), e106 (2005).

28 Application of Streptavidin-Dyes Conjugate Detection limit: 50 fm Biosensors and Bioelectronics, 22(7), 1532 (2007).

29 DNA Microarray on the Dendron Surface Improves Significantly Detection of Single Nucleotide Variations in p53 Gene

30 Simultaneous Detection of 7 Hotspots of p53 Gene (3.1) 7.0 (2.1) 4.1 (1.9) (2.3) 8.9 (2.8) 2.7 (1.1) (2.6) 8.5 (1.7) 5.1 (1.3) 15.9 (5.2) (1.5) 2.5 (1.7) (4.2) 9.5 (2.3) 9.6 (1.9) (2.5) 6.8 (2.5) 2.5 (1.2) 9.1 (2.2) 14.8 (3.2) 8.2 (1.7) Codon G A Relative 0 20 Intensity (%) C T Single Nucleotide Intensity less than 16% was observed for the all mutations. Nucleic Acids Research, 33(10), e90 (2005).

31 NanoCone Surface Validation for Gene Expression DNA Microarray NSB9 Amine Slides Vs Corning Slides (UltraGAPS TM ) * The following data were acquired in-house and through a contract research organization

32 Extremely High Sensitivity 50 µg total RNA 10 µg total RNA 1 µg total RNA NSB9 Amine Slide 50 µg total RNA 10 µg total RNA 1 µg total RNA Corning UltraGaps slide 293 RNA = Cy3, HeLa RNA = Cy5 Strong background signal Lots of missing data

No Blocking Process No blocking, 42 o C with

expression analysis with NanoCones slides, blocking

33 No Blocking Process No blocking, 42 o C with formamide Blocking, 42 o C with formamide PSTECH slide; No blocking, 65 o C High quality image No blocking, 65 o C Blocking, 65 o C For gene expression analysis with NanoCones slides, blocking agent was not needed, while use of BSA was required for Corning slides.

PRDUCTS Surface functional group Lateral spacing between surface functional groups 3-4 nm 6-7 nm Amine Epoxy Aldehyde NSB9 Amine Slide NSB9 Epoxy Slide NSB9 Aldehyde Slide NSB27 Amine Slide NSB27

34 PRDUCTS Surface functional group Lateral spacing between surface functional groups 3-4 nm 6-7 nm Amine Epoxy Aldehyde NSB9 Amine Slide NSB9 Epoxy Slide NSB9 Aldehyde Slide NSB27 Amine Slide NSB27 Epoxy Slide NSB27 Aldehyde Slide NSB9 Amine Slide SPECIFICATIN Ideal surface for oligonucleotide microarrays for SNP genotyping or gene expression profiling The outer surface is functionalized with amino group Lateral spacing between amino groups: 3-4 nm Density of amino groups: ea/nm 2 Size: 1" x 3 FEATURES ptimized spacing for DNA hybridization Minimized steric hindrance & electrostatic repulsion High specificity & sensitivity Fast DNA hybridization High hybridization efficiency Low background signal Small amount of total RNA needed NSB27 Amine Slide SPECIFICATIN Applicable to microarrays with cdna, protein, peptide, aptamer, etc. The outer surface is functionalized with amino group Lateral spacing between amino groups: 6-7 nm Density of amino groups: ~ 0.01 ea/nm 2 Size: 1" x 3" FEATURES Larger spacing for big biomolecules Minimized steric hindrance & electrostatic repulsion High specificity & sensitivity Fast bimolecular interaction High binding efficiency Low background signal

Microarray 기초 및 응용

Microarray 기초 및 응용 Microarray 기초및분석 이바이오젠 목 차 마이크로어레이기초 정의 / 분류 / 제작기술 / 원리 Gene Expression 실험분석 시료준비 RNA QC 실험분석과정 데이터분석 -2- 바이오칩 & 마이크로어레이 - 정의 1. 유리, 실리콘, 플라스틱등의매체위에생체 분자를집적하여만든것 2. 현재 DNA, 단백질, 화학물질, 유기물질등바 이오소재를집적하여만든