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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.37 No.4 pp.181-188

Surface Polarity Dependent Solid-state Molecular Biological Manipulation with Immobilized DNA on a Gold Surface

Jeong Hee Kim*, Jiyoung Lee
Department of Oral Bioc-hemistry and Molecular Biology, School of Dentistry, Kyung Hee University
received October 5, 2012 ; revised November 5, 2012 ; accepted December 11, 2012


As the demand for large-scale analysis of gene expres-sion using DNA arrays increases, the importance of the surface characterization of DNA arrays has emerged. We com-pared the efficiency of molecular biological applications on solid-phases with different surface polarities to identify the most optimal conditions. We employed thiol-gold reactions for DNA immobilization on solid surfaces. The surface polarity was controlled by creating a self-assembled monolayer (SAM) of mercaptohexanol or hepthanethiol, which create hydrop-hilic or hydrophobic surface properties, respectively. A hyd-rophilic environment was found to be much more favorable to solid-phase molecular biological manipulations. A SAM of mercaptoethanol had the highest affinity to DNA mole-cules in our experimetns and it showed greater efficiency in terms of DNA hybridization and polymerization. The opti-mal DNA concentration for immobilization was found to be 0.5 mM. The optimal reaction time for both thiolated DNA and matrix molecules was 10 min and for the polymerase reaction time was 150 min. Under these optimized condi-tions, molecular biology techniques including DNA hybri-dization, ligation, polymerization, PCR and multiplex PCR were shown to be feasible in solid-state conditions. We de-monstrated from our present analysis the importance of surface polarity in solid-phase molecular biological appli-cations. A hydrophilic SAM generated a far more favorable envi-ronment than hydrophobic SAM for solid‐state molecular techniques. Our findings suggest that the conditions and met-hods identified here could be used for DNA‐DNA hybri-dization applications such as DNA chips and for the further development of solid-phase genetic engineering applicatio-ns that involve DNA-enzyme interactions.


 In recent years, biotechnology related to the manipulation of  solid-phase DNA has  expanded  dramatically. These de-velopments have paralleled the increased demands of human genome sequencing and large-scale analysis of gene expres-sion  using DNA  arrays  and RNA  array  [1-3]. DNA  chips contain array of DNA fragments an orderly pattern at which the DNA fragments are available for next step of molecular biological approach  [4]. These DNA chips can be used  for the gene expression monitoring, detection of polymorphism and mutations, sequencing the human genome and DNA diag-nostics [5-7]. Recently, further advanced form of DNA chips including production of protein array from DNA microarray and an aptamer chip have been tried [8-10].

 While DNA array based  technology  is a promising area, not much attention was paid  to  the surface characterization of DNA arrays. The environment of  the  immobilized DNA fragments  at  the  solid  surface  depends  upon  the mode  of immobilization and can differ  from  that experienced  in  the bulk solution. Thus, in order to be functional after immobi-lization of DNA fragments on the solid surface, physical cha-racterization  of  the  surface  and  surface  species  are  impor-tant. Since it has been reported that well defined probe orienta-tion and  the  immobilization nature are critical, various  im-mobilization methods have been attempted [11-19].

 In this study, we controlled surface polarity by construc-ting self-assembled monolayers (SAM) of thiol molecules on the surface of  thin gold  film with  terminal groups of diffe-rent polarity. Thiolated DNA was immobilized on gold sur-face by generating mixed monolayers of thiolated oligomer and mercaptohexnol or hepthanethiol. The DNA oligomers in the resulting SAM will be in hydrophilic or hydrophobic envi-ronment, respectively. And the feasibility of molecular biologi-cal technique manipulation including nucleic acid hybridiza-tion, polymerization, restriction enzyme digestion, ligation and polymerase chain reaction (PCR) was compared. We report here that polar surface provide more favorable environment for immobilized DNA to be adaptable to solution phase mole-cular biological techniques. In addition, multiplex PCR was successfully  performed  with  solid-  phase  tethered  DNA.

Materials and Methods


 Thiolated single stranded DNA (HS-ssDNA) was purcha-sed from Research Genetics (Huntsville, AL, USA). The 5’ thiolated oligomeric DNAs and unmodified oligonucleotides were obtained from Research Genetics (USA) and Bioneer, Korea,  respectively. Au-coated slides were purchased  from EMF, USA. Mercaptohexanol  (6-mercapto-1-hexanol, MCH) hepthanethiol was purchased from Sigma-Aldrich (USA). Kle-now fragment and Taq DNA polymerase were obtained from Takara (Japan) and Perkin Elmer (USA), respectively. Rest-riction enzymes were purchased from either Promega (USA) or New England Biolabs (USA). [35S]α-dATP (1,000 Ci/mmol) was from Amersham (UK) and β-emission scintillation coc-ktail was  from Pakard  (USA). Other  chemicals were purc-hased  from  Sigma  (USA)  or  from  other  common  sources.

Preparation of self-assembled monolayers (SAM) on Au surface

 The Au  substrate used was  a glass plate of 3.0 mm×5.0 mm size on which Au was vacuum-deposited to about 1000 A thickness. The Au surface was cleaned with piranha solu-tion  [concH2SO4 :  30%  H2O2  =  2:1  (v/v)],  rinsed  with absolute ethanol and dried under nitrogen gas. Solutions of 1 mM mercaptoethanol or 1 mM hepthanthiol were prepared in  ethanol. Au-coated  glass  slides were  incubated with  the thiol solutions for 2 hrs at room temperature and rinsed with ethanol. The thiol groups were chemically adsorbed to the Au surface,  thereby creating a SAM of either mercaptoethanol or heptanthiol.

 In order  to  test  the  effect of  surface polarity  to physical adsorption of DNA molecule, we prepared radio-actively labe-led  small DNA  fragment. A  65 base  single  stranded DNA were amplified with  the KS primer and  the SK primer  (10 pmol each). The  temperature cycle was set as  follows: Hot start step: 94℃, 10 min, PCR cycle (20-45 cycles): 94℃, 30 sec; 50℃, 60 sec; 72℃, 30 sec. A part of the PCR solution was  sampled  and  analyzed  by  agarose  gel  electrophoresis. Radioactively labeled DNA (200 mL) was added to each Au glass with SAM and incubated at RT for 1 hr. After the incu-bation, Au glasses were rinsed in 1 x Tris-buffered saline with Tween 20 (TBST) at RT and the resulting radioactivity was counted in a scintillation counter (Beckman LS6500, USA).

Preparation of DNA functionalized monolayers on Au surface

 The cleaned bare Au surface was soaked in an aqueous solu-tion of a thiolated single-stranded DNA (HS-ssDNA; thiola-ted DNA 20mer-1; 5’OH(CH2)6-S-S-(CH2)6-CGA GGT CGA CGG TAT CGA TA-3’, 1 μM)  in a buffer of 1.0 M potas-sium phosphate, pH 6.7, for 10 min. The Au surface bearing pre-adsorbed HS-ssDNA was then immersed in a solution of 1 mM mercaptoethanol or 1 mM heptanthiol (100 μl) in etha-nol  for  5 min  and  then  thoroughly  rinsed with  0.5%  SDS solution. Thiol groups on the mercaptoethanol or heptanthiol was chemically adsorbed to the Au surface, thereby creating a mixed monolayer  of HS-ssDNA  and mercaptoethanol  or heptanthiol.

Hybridization and polymerization of surface tethered ssDNA

 For hybridization of  surface-immobilized  thiolated DNA 20mer-1  to  an  ssDNA  template,  gold  slides  with  an  ss DNA-functionalized monolayer on the surface were soaked in a solution of 65mer (5’-TAT AGA ACT AGT GGA TCC TTT TCT TTT CTT GAA TTC TTT CTT TTC TTT TAT CGA TAC CGT CGA CC-3’) and the mixture was heated to 65℃ for 5 min, then slowly cooled down to room tempera-ture. The surface immobilized DNA and the annealed 65-mer DNA were then polymerized with 2 U of Klenow fragment (Takara,  Japan)  for  1.5  hr  in  a  reaction  containing  4 μM dNTP, 0.2 μCi [a35S] dATP, 10 mM Tris-Cl, 7 mM MgCl2, and 0.1 mM dithiotreitol, pH 7.5. After the reaction, gold films were washed with 0.5% SDS  at  room  temperature  and  the radioactivity remaining fixed to the slides were counted in a scintillation  counter  (Beckman LS6500, USA). All  experi-ments  performed  at  least  in  triplicate. Data was  shown  as mean± standard deviation (SD).

Ligation and PCR with immobilized DNA

 To  prepare DNA  fragments  to  be  ligated  to  the  immo-bilized DNA,  pBluescriptII KS(+) was  first  enzymatically digested  with  Hind  III,  followed  digestion  with  Sca  I  to generate 1,154 bp digestion products. This DNA fragment was separated by electrophoresis on 1.2% agarose gels and puri-fied by squeezing, subsequent phenol extraction and ethanol precipitation. Concentration of the DNA fragments were ad-justed to 5 μM and then used in ligation reactions, as desc-ribed below.

 A HS-ssDNA, thiolated DNA 20mer-1 was immobilized on the gold surface, annealed  to 24-mer DNA (CH-24 mer)  to generate Hind III sticky end, and ligated to a 1,154 bp Hind III-Sca I digested DNA fragment with T4 DNA ligase (Pro-mega, USA) in a reaction buffer containing 30 mM Tris-Cl, 10 mM MgCl2, 10 mM dithiotreitol, 1 mM ATP  and 15% polyethyleneglycol  8000,  pH  7.8.  Ligation  reactions  were carried out  at 16°C  for 2 hr or overnight  at 4℃. The gold films were then washed once with a solution containing 5% SDS  in  40 mM  sodium  phosphate,  pH  7.2,  at  65℃  and  4 times with a solution of 1% SDS in 40 mM sodium phosp-hate, pH 7.2 at 65℃. The DNA immobilized on the slides was then subjected to 35 cycles of PCR, as described above. Pri-mers  used  for  the  PCR  reactions  included  20-mer-1  (5’- CGA GGT CGA CGG TAT CGA TA-3’) and Nae I primer (5’-GGC  GAA  CGT  GGC  GAG  AA-3’).  The  amplified PCR products of 431 bp were  separated by electrophoresis on 1.2% agarose gels and visualized by ethidium bromide stai-ning. All experiments performed at  least  in  triplicate. Data was shown as mean± SD.

Solid- phase mutiplex PCR

 DNA  fragments  for  ligation  to  the  immobilized  DNA, pBluescriptII KS(+) was digested with Bam HI, Eco RI or Hind III, followed digestion with Nae I to generate 359 bp, 377  bp  and  389  bp  digestion  products.  These DNA  frag-ments were separated as described above.

 HS-ssDNAs  (Thiolated  DNA  20mer-1,  Thio-T3-E  and Thio-R-B) were immobilized on a gold slide, annealed with mixture of CH-24-mer, cT3-E and c-R-B and ligated to either the 359 bp Bam HI-Nae  I, 377 bp Eco RI-Nae I or 389 bp Hind  III-Nae  I  DNA  fragments  as  described  above.  The sequence of Thio-T3-E and Thio-R-B are 5’OH-(CH2)6-S-S-(CH2)6-ATT AAC CCT CAC TAA AGC CG-3’ and 5’OH-(CH2)6-S-S-(CH2)6-AAC AGC TAT GAC CAT GCA TG-3’, respectively. The gold films were then washed as described above. The DNA  immobilized on  the  slides was  then  sub-jected to 35 cycles of PCR with 3 different pairs of primers. Forward  primers  used  were  either  20-mer  (5’-CGA  GGT CGA  CGG  TAT  CGA  TA-3’),  T3  primer  (5’-ATT  AAC CCT CAC TAA AG-3’)  or  reverse  primer  (5’-AAC AGC TAT  GAC  CAT  G-3’).  The  reverse  primer  was  Pvu  II primer  (5’- TGG CGA AAG GGG GAT GT-3’)  for  all  3 PCR reactions. The amplified PCR products were separated by electrophoresis  on  1.2%  agarose  gels  and  visualized  by ethidium  bromide  staining.  All  experiments  performed  at least in triplicate.


Comparison of hybridization and enzyme accessibility to the immobuilized DNA on gold surface with hydrop-hilic or hydrophobic matrix.

 The aim of this study is to optimize the surface conditions for solid-state molecular biological manipulation. First of all, the surface polarity of  the gold surface was modified by the well-known sulfur-gold interaction [20] to observe the relative DNA molecule  adsorption  to  a  solid  surface with different surface  polarity.  Bare  gold  surface  was  treated  with  thiol molecules with terminal group with hydroxyl group (mercap-tohexanol) or methyl group (hepthanethiol) to form self- asse-mbled monolayers  (SAM),  thus  generating  hydrophilic  and hydrophobic  surfaces,  respectively.  These  were  hybridized with  radioactively  labeled DNA  and  the  remaining  radioac-tivity was measured. As  expected  polar,  hydrophilic merca ptohexanol-treated surface revealed higher radioactivity than non-polar  hydrophobic  hepthanethiol-treated  surface  (Fig. 1). Almost  twice more DNA molecule was adsorbed  to  the polar  surface  than  the  non-polar  surface  that  suggests  that polar hydrophilic surface has higher affinity  to DNA mole-cules than non-polar hydrophobic surface.

Fig. 1. DNA molecule adsorption to hydrophilic or hydrop-hobic surfaces created on gold slide. Radioactively labeled DNA was incubated with gold slides which were functio-nalized with thiol molecules to generate hydrophilic or hydrop-hobic characteristics of surface with either mercaptoethaol or hepthanethiol. Hydrophilic surface showed higher affinity to DNA molecules.

 Regulating the surface coverage of DNA is a critical fac-tor in manipulating molecular biological techniques in solid- phase [21]. Therefore, we tried to directly visualize the amo-unt of DNA immobilized on the gold surface. The DNA mo-lecule to be immobilized was functionalized at the 5’ termi-nal with  a  thiol group. A gold-coated  glass  slide was  then treated with Hs-ssDNA and subsequently  treated with mer-captohexanol to generate a mixed SAM on the gold surface. After  immobilization,  immobilized  DNA  was  hybridization with complimentary fragment of DNA, stained with a fluores-cent dye and the result was observed under fluorescent mic-roscope. We  observed  an  increase  in  the  optical  density  of microscopically  observed  fluorescent  image  of  stained  hyb-ridized immobilized DNA as the immobilized DNA concen-tration  increased  (data  not  shown). However,  the  detection of fluorescent intensity of immobilized DNA and quantitative analysis  were  rather  limited,  thus  we  tried  to  monitor surface concentration of immobilized DNA indirectly. Vari-ous concentration of thiolated DNA on gold surface was im-mobilized and the surface was treated with either mercapto-hexanol or hapthanethiol to construct hydrophilic or hydrop-hobic  matrix  of  the  surface.  The  immobilized  DNA washybridized  with  65-mer  ssDNA.  The  resulting  hybrid was used as a template for the Klenow fragment reaction, as  described  in  Materials  and  Methods.  The  polymerization reaction  was  carried  out  in  the  presence  of  [a35S]  dATP. The gold slides were washed  thoroughly and b-emission of the  slides  was  quantified  (Fig.  2).  As  expected  the  hy-bridization  and  polymerization  efficiency  was  higher  in hydrophilic surfaces than hydrophobic surface in the range of 0 to 3 μM of thiolated DNA immobilized on gold surface. The highest efficiency was observed at 0.5 μM of  thiolated DNA was  immobilized.  And  at  the  same  concentration  of thiolated  DNA,  approximately  3.7  times  higher  efficiency was observed  in hydrophilic SAM  than hydrophobic SAM.

Fig. 2. DNA hybridization and polymerization on solid surface as a function of concentration of DNA immobilized. Thiolated single stranded DNA (HS-ssDNA) was immobilized on the gold surface derivatized with hydrophilic (●) or hydrophobic (○) molecules. partially complimentary ssDNA was hybridized to the immobilized DNA and polymerization was performed in the presence of radioisotope-labeled nucleotides.

 When  the  immobilization  reaction  time of  thiolated  time was varied from 0 to 90 min and the hybridization and poly-merization with Klenow fragment was performed in the pre-sence of radioisotope as described above. As shown in Fig. 3, higher radioactivity was measured with hydrophilic SAM of mercaptoethanol and than with hydrophobic SAM of heptha-nethiol  in  the  range  of  time  varied  in  this  experiment. Highest activity was acquired at 5-10 min of immobilization reaction time of thiolated DNA. At 5 min of immobilization reaction  time,  hybridization  and  polymerization  of  the  im-mobilized  DNA  on  hydrophilic  SAM  of  mercaptoethanol was approximately 5.9 times higher activity than hydropho-bic SAM of hepthanethiol.

Fig. 3. The DNA immobilization time dependent DNA hybri-dization and polymerization on solid surface. The reaction time was varied for 0 to 90 min and the radioactivity was measured and plotted as a function of time on the gold surface de-rivatized with hydrophilic (●) or hydrophobic (○) molecules.

It was reported  that when a  thiolated DNA molecules of 166  was immobilized on a surface the actual thickness mea-sured was about 33 ± 2  [12]. The change of surface thic-kness was far less than the actual size DNA molecule immo-bilized. This result suggests that most of immobilized DNA is not fully extended to its full length or oriented perpendicular to the surface. In order to avoid the non-specific binding of DNA molecules  to  the  surface  and, matrix molecules  such as mercaptohexanol  or  heptanthiol were  used  to  fill  in  the surface  between  DNA  molecules.  Both  of  these  matrix molecules  contained  6  carbon  backbones  which  are  the same  length  of  carbon  backbone  in  thiolated  DNA.  DNA molecules  immobilized  would  protrude  out  on  the  matrix molecules, thus could be available for subsequent reactions.

 Thiolated DNA was immobilized and subsequently incu-bated with either mercaptohexanol or heptanethiol for 0-90 min  time  period.  As  shown  in  Fig.  4,  DNA  hybridization  andpolymerization was maximized at 10 min of  incubation time with  hydrophilic  or  hydrophobic matrix molecules.  It seem that once matrix molecules occupies the space between immobilized  thiolated DNA molecules,  they  prevent  direct contact between DNA molecules and gold surface, thus they secure enough room for other complementary DNA or enzy-mes be functional. When DNA hybridization, polymerization efficiency was compared at 10 min of incubation, it was ob-vious that hydrophilic surface offered better environment for DNA and enzyme accessibility; approximately 7.5 times higher activity with  hydrophilic  surface  than  that  of  hydrophobic surface.

Fig. 4. The matrix molecule immobilization time dependent DNA hybridization and polymerization on solid surface. The rea-ction time was varied for 0 to 90 min and the radioactivity was measured and plotted as a function of time on the gold surface derivatized with hydrophilic (●) or hydrophobic (○) molecules.

 Again, the time period of polymerase reaction was varied from 0  to 3 hrs. Polymerization efficiency was higher with hydrophilic matrix molecules  than hydrophobic matrix mo-lecules (Fig. 5). Highest radioactive emission reached at 150 min  of  polymerase  reaction  with  hydrophilic  matrix  and became saturated after 150 min of reaction (solid circles  in Fig 5). The maximum was reached at 90 min of polymerase  reaction  hybridization  and  polymerization  was  performed with hydrophobic matrix (empty circles in Fig. 5).

Fig. 5. The polymerase reaction time dependent DNA hybri-dization and polymerization on solid surface. Polymerase was incubated for 0 to 180 min and the radioactivity was measured and plotted as a function of time on the gold surface deri-vatized with hydrophilic (●) or hydrophobic (○) molecules.

Solid-phase ligation and PCR with DNA immobilized on gold surface.

 As shown above we optimized the condition of solid-phase hybridization, polymerization with immobilized DNA on gold surface. For  the  next  step, we  tried  to  ligate  a DNA  frag-ments with immobilized DNA to gold surface. Thiolated DNA 20mer-1 was  immobilized on  the gold surface and  the ma-trix  was  filled  with  mercaptohexanol.  Partially  comple mentary  single  stranded  DNA  (CH-24mer,  5’-AGC  TTA TCG ATA CCG TCG ACC TCG-3’) to immobilized DNA was  hybridized  to  generate  sticky  ended  which  is  com-patible with  Hind  III  restriction  digested  DNA  for  subse-quent  ligation. A DNA  fragment of 1154 bp was prepared  to contain Hind III sticky end and Sca I blunt end by digestion of  a  plasmid with  respective  restriction  enzymes. Then  the prepared  DNA  fragment  was  ligated  with  immobilized DNA.  The  resulting  ligation was  confirmed  by  PCR with primers  20mer-1  primer  and  Nae  I  primer  where  the annealing sites of each primers reside 5’ and 3’ sides of the ligation  site.  The  target  amplicon  size  was  413  bps.  As shown in Fig. 6, the positive control solution phase ligation and PCR  resulted  in 413 bps amplicon as expected  (lane 1 in Fig 6). Solid-phase ligation and PCR generated the same size amplicon with slightly lower intensity (lane 2 in Fig. 6). However, no PCR product was detected with negative controls of no ligation addition or no thiolated DNA (lanes 3 and 4 in Fig. 6).

Fig. 6. Solid-state DNA manipulation on the gold surface. Thio-lated DNA was immobilized, hybridized to single stranded DNA and hybridized to ssDNA to generate a compatible end for restriction enzyme (Hind III)-digested DNA fragment. After DNA ligation in the presence or absence of ligase, PCR was performed with primers positioning either side of the ligation site. The amplicon generated was 413 bp. Positive solution phase control (lane 1) and solid- phase DNA manipulation (lane2) show DNA amplicons amplified. No amplicon was generated from no ligase negative control (lane 3) or no immobilized DNA control (lane 4).

Multiplex PCR with DNA immobilized on gold surface

 For  the  next  attempt,  we  tried  to  ligate  three  different 20mer-1, thio-T3-E and thio-R-B) were immobilized on the gold  surface  and  the matrix was  again  filled with SAM of mercaptohexanol.  Partially  complementary  single  stranded DNAs (CH-24mer, c-T3-E, and c-R-B) to each immobilized DNA were hybridized all together to generate 3 different sticky ended (Hind III, Eco RI, and Bam HI) DNA for ligation. Three  different size of DNA  fragments were prepared with either  Hind III/Nae I (389 bp), or Eco RI/Nae I (377 bp), or Bam HI/ Nae I (359 bp) double digestions, respectively. Then these DNA fragments were mixed and incubated all together with gold  slide  which  contained  3  different  immobilized  DNA inthe  presence  of  ligase.  The  ligation  was  confirmed  by PCR  reaction  with  primers  described  in  Materials  and methods  to generate amplicons of 212 bp, 200 bp and 182 bp,  respectively  (Fig.  7). As  a  control,  the  same  experiments were  performed  except  that  non-thiolated  single  stranded DNA with  the  same  sequence  of Thiolated DNA  20mer-1, Thio-T3-E  Thio-  R-B. And  no  detectable DNA  band was detected in the negative controls of no ligase addition (data not shown).

Fig. 7. Solid- phase multiplex PCR. Three different thiolated DNA were immobilized and ligated to a bigger size DNA frag-ments as described in Materials and methods. PRC was perfor-med each set of primers specific to each DNA fragment was used. Amplicons size of 212 bp (lane 1), 200 bp (lane 2) and 182 bp (lane 3) were detected from thiolated DNAs; Thiolated DNA 20mer-1, Thio-T3-E and Thio-R-B.


 Recently, technological progress in DNA microarrays has been extremely rapid and have been used widely in research areas  including gene discovery, detection of mutations and medical diagnostics [5, 22-24]. In order to have good DNA chips,  the  immobilization  of  DNA  molecules  is  a  critical factor. The  immobilized DNA  onto  a  solid  surface  should permit  their  accessibility  for  subsequent  steps  including binding with the complementary strands. Numerous attempts have  been  tried  to  immobilizing  DNA  molecules  on  solid surfaces  [11-19,  25-27].  A  general  approach  for  im-mobilization methodology  is  to modify  the biomolecules of interest with  a  functional  group  that  allows  covalent  attach-ment to a reactive group on the surface. These include using silanized DNA [25], amine-modified oligo nucleotides [16], poly(dT)-modified DNA by UV irradiation [26] and hetero-bifunctional DNA [19, 27].

 Thiolated DNA was used in this study. It was immobilized on  the  surface  of  a  thin  gold  film  by  generating  a mixed monolayer of thiolated oligomer and thiol molecules. It was because  shown  that mercaptoalkyl  (thiol)-modified oligonucleotide  showed  the  highest  thermal  stability with  compa-rable accessibility and specificity of the surface-bound probes [28]. Another advantage of using 5’ thio-functionalized DNA mo-lecule in immobilization is that the orientation of DNA mo-lecule  can  be  controlled.  The  orientation  of  the  immobilized DNA  is critical for  the subsequent steps  including hybridiza-tion  to a complementary DNA which  is broadly  applied  in DNA chip technology and the interaction with proteins as em-ployed  in  this  study,  and  for  the  reduction  of  background noise which is directly related to device sensitivity and sele-ctivity [29,30].

 Unlike DNA microarrays in which the subsequent step is just  hybridization  to  complementary DNA,  the DNA  chip developed  in  this  study  is  for  both  interactions with DNA and proteins, specifically enzymes. To find an optimal con-dition for solid-state DNA-Protein interaction, the surface pola-rity was controlled by changing matrix molecules which cover the  gold  thin  film.  In  addition,  we  revealed  that  various multiple  solid-state molecular biological  techniques wereas applicable  with  reasonable  efficiency  using  the  DNA  chip developed in this study.


 This research was supported by a grant from Ministry of Health and Welfare, Korea (02-PJ1-PG11-VN01-SV02-0036).


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