The Length Effect of Probe DNA for Hybridization using DNA Self-Assembled Monolayer∗

To establish a DNA sensor, DNA self-assembled monolayer (SAM) was prepared on a gold substrate. A probe DNA SAM was fabricated using thiolated DNA composed of singleand double-stranded portions on a gold substrate. The adsorption behaviors of probe DNAs were measured by surface plasmon resonance (SPR). Hybridization efficiency was optimized by controlling the length of spacer inserted in probe portion and SAM layer. Hybridization with target DNA on the DNA array prepared using probe DNA was monitored in situ by SPR imaging. The SPR image indicates that 10-thymine bases spacer shows the most efficient hybridization on the DNA array. [DOI: 10.1380/ejssnt.2005.250]


I. INTRODUCTION
The hybridization of poly-or oligo-nucleotides, such as DNA or RNA, is generally used in gene analysis.Recently, DNA microarrays, such as a DNA chip, are being used for high-throughput analysis [1,2].At present, the sensitive detection of target DNA is strongly required in medical fields because only a small amount of target DNA can be obtained from patients for the assay.Although polymerase chain reaction (PCR) is generally utilized to amplify the target DNA, the profile of gene expression might be changed after the PCR amplification.
Many attempts about immobilization of probe DNA on a solid surface were reported previously [3,4].We reported the preparation method for DNA self-assembled monolayer (SAM) on a gold substrate using a probe DNA composed of single-stranded (ss) and double-stranded (ds) oligonucleotide portions, as shown in Fig. 1.The probe DNA forms SAM on a gold substrate by addition of divalent Mg ion into the solution [5][6][7].The unique architecture of this probe DNA allows for relatively close packing of the lower section of the DNA SAM on the surface whereas in the upper section sufficient free space exists for DNA hybridization.To establish highly sensitive DNA sensors, it is essential to optimize the activity of the probe DNA on the surface.In this study, we focused on the mobility of probe portions on the substrate.The length of spacer inserted between the probe portion and the ds portion was varied to control the mobility of the probe portion on the surface.The chemical bonding of the thiol group with gold was induced to immobilize the probe DNA on a gold substrate and the formation of the monolayer was carried out by a selfassembly technique.SAMs have attracted considerable attention for well-ordered thin-film fabrication, by which thiol or disulfide derivatives can spontaneously form a closely packed monolayer on a gold surface when the substrate is immersed in a thiol or disulfide derivative solution [8][9][10].We observed the adsorption of probe DNA onto a gold substrate surface plasmon resonance (SPR).The hybridization with target DNA on the DNA array prepared using probe DNAs was observed by SPR imaging to optimize the length of the spacer for the efficiency of the hybridization.

A. Materials
Table 1 shows the nomenclature and sequences of the probe and target DNAs used in this study, which were purchased from JBios.(Saitama, Japan).The preparation of probe DNA was described elsewhere [5].To prepare the probe DNAs, ss 40−70 oligonucleotides and thiolated 20 oligonucleotide in their equimolar solutions were annealed up to 95 • C, to form ds DNA in the complemen-

B. SPR measurements
To monitor reactions occurring at the solid-liquid interface, SPR was employed in the detection of the adsorption of the probe DNA onto gold substrates.SPR measurement (NanoSensor, Nippon Laser Electronic, Japan) was carried out on the gold substrate, which was prepared by the vacuum deposition of gold onto a glass (BK-7), and the thickness of the gold was ∼50 nm.The temperature of the SPR cuvette was controlled at 20 • C. The flow rate of sample injection and rising was controlled at 0.1 ml/min with a flange pump (P.C.S. PUMP, Model 021, Toyobo, Japan).

C. Observation by SPR imaging
SPR imaging is a promising method for highthroughput detection of binding events occurring on a surface.SPR imaging measurements were carried out by SPRImager (OWG Instruments, Wisconsin, US). Figure 2 shows the experimental setup for SPR measurements.Incoherent light over the wavelength range of 600∼1100 nm was used as a source.The details of SPR imaging observation were described previously [11][12][13].A CCD camera was used to collect the images, were examined using the software package V++ (Digital Optics, New Zealand) on a personal computer.A probe DNA array was prepared on the gold substrate as follows.DNA spots were prepared on the gold substrate by an automated spotter (MultiSPRinter, TOYOBO, Japan).The DNA array was prepared on a 50-nm-thick gold evaporated on a glass slide (SF-10) that were covered first with a 2-nm-thick chromium layer to increase mechanical stability.After spotting the probe DNA, the array was incubated at 37 • C in an oven for 16 hours and then rinsed with excess aqueous MgCl 2 solution containing thiolated ds 20 base pairs oligonucleotides (Blocking DNA, approximately 100 µl, 3.0 µM) to prepare a ds DNA SAM in the space between the spots on the gold substrate.The Blocking DNA was prepared by annealing of the equimolar mixed solution of 20 merC and 20 merSH.This ds DNA SAM has a blocking effect, which decreases the nonspecific adsorption of target DNA to the areas between the spots, hence leading to high-contrast images.The flow rate of sample injection and rising was controlled at 0.1 ml/min with the same flange pump as mentioned above.The image collected by the SPR imaging measurement was further analyzed using the SPIP software (Image Metrology, Denmark).http://www.sssj.org/ejssnt(J-Stage: http://ejssnt.jstage.jst.go.jp)

A. Formation of probe DNA SAM on a gold substrate
The details about the formation of DNA SAM using the Probe (10) onto a gold substrate are described elsewhere [5].When the MgCl 2 solution was used for the solvent, the thickness of DNA SAM was remarkably change while only a small amount of the probe DNA adsorbed onto the gold substrate in the TE buffer.The DNA SAM prepared using Probe (10) was well characterized by SPR and XPS. Figure 3 shows adsorption behaviors of the probe DNAs onto the gold substrates by SPR.These results indicate that each probe DNA forms a stable monolayer on a gold substrate as well as Probe (10) does.Intriguingly, there is no significant difference in the adsorption signal of Probe (0), Probe (20) and Probe (30), whereas the signal of Probe (10) is lowest among them.If the molecular orientation in the every monolayer prepared from the probe DNA were uniform, the thickness of layer would be in proportion to the number of bases.These SPR results implied that the orientation of ds portions might be different in each SAM.In particular, in the case of the probe DNA containing the longer ss portion, most of them might adsorb on the substrates via non-specific adsorption because nucleic bases of ss portions have an affinity to a gold substrate.The formation of SAM might be bound up with the length of ss and ds portion in the probe DNA.

B. Observation of hybridization with target DNA using a DNA array
After the preparation of the DNA array containing the four kinds of probe DNA, SPR imaging measurements were carried out to monitored the hybridization with target DNA on the DNA array.The left illustration and the right image in Fig. 4 (a) show a layout of DNA array composed of four kinds of probe DNA prepared on a gold substrate and SPR image after hybridization with target DNA, respectively.The SPR image was taken by subtracting the SPR images before and after the hybridization.Each probe DNA has four spots on the same substrate to confirm the reproducibility of the hybridization.In this image, the SPR signal was observed on the spots prepared using Probe (0), Probe (10) and Probe (20).On the other hand, the spots prepared from Probe (30) showed little signal even after the hybridization assay.To discuss quantitatively, the hybridization efficiency estimated by the SPR imaging is summarized in Fig. 4 (b).The evaluated values of the hybridization signal corresponding to the brightness of SPR images were taken on the average.The spots prepared from the Probe (10) showed the best result among them, while the hybridization signal was hardly observed on the spots prepared from the Probe (30).In the cases of Probe (0) and Probe (20), both signals are approximately 40% in comparison with the case of Probe (10).The hybridization signal should be increasing with the increment of the spacer length at the viewpoint of mobility of the probe portion if the only mobility of the probe portion controls the hybridization efficiency.Although this unexpected result is still under discussion, there are two possibilities to explain the SPR imaging result: one is a dissociation of ds portion was accelerated by the flexibility of the long spacer, then hybridization signal was decreased on the spots prepared by Probe (30).The hybridization efficiency is governed by the equilibrium of association and dissociation of two complementary parts of nucleotides.In this case, the SAM prepared using Probe (10) showed the best condition of the mobility of probe portions and the stability of ds formation after hybridization.Another possibility is that the activity of probe portion was decreased because of denaturation of the probe portions on the substrate.In the second case, the Probe (30) did not form a stable monolayer on a gold substrate and most of probe portions of Probe (30) might be denatured on a gold substrate.The result of adsorption of probe DNAs implied that the orientation of each probe DNA was not uniform on the substrate.

IV. CONCLUSION
In this study, probe DNAs containing various length of spacer were employed to optimize the efficiency of hybridization.Adsorption behaviors of probe DNAs monitored by SPR suggested the formation of monolayer on gold substrates.The SPR image on a DNA array suggested that the probe DNA SAM prepared using Probe (10) showed the most efficient hybridization.Because the molecular design of probe DNA is indispensable for the highly sensitive DNA sensors, these results will become significant information in a development of DNA sensors.

FIG. 1 :
FIG. 1: Preparation of probe DNA containing ss and ds portions.

FIG. 2 :
FIG. 2: Detection of DNA hybridization on DNA array by SPR imaging.

FIG. 4 :
FIG. 4: Fig.4 (a) Layout of DNA array containing four kinds of probe DNAs and SPR difference image of the array after hybridization.(b) Summary of hybridization efficiency on the spots prepared using the probe DNAs.