Introduction

DNA plays an important role in biochemistry and biology. Numerous applications of this chain molecule were found not only in life science but also in nano science. DNA-modified gold nano particles (GNP) attracted tremendous interests in recent years.Various genetic diagnostics technologies were developed and numerous nano structures were fabricated with them. To immobilize DNA to gold surface, various mercapto modified DNA were employed. All the previously employed DNA-SH own an alkyl linker between DNA backbone and mercapto group, which made the DNA-Au system more complicated. The hydrophobic alkyllinker can partly change the property of DNA, for example, electrical characteristics or molecular flexibility. Moreover, the alkyl linker used for this indirect attachment often diminishes the transfer of signals from DNA to gold interface (and vice versa) and thus their removal is desirable. In this thesis, DNA-GNP conjugates and Au-DNA-Au nanostructures are fabricated as shown in Scheme 1. By using 5'-mercapto-5'- deoxythymidine, DNA is directly attached to gold particle surface to obtain a new type DNA-Au conjugate. This new type DNA-GNP conjugate showed good hybridizing specificity in Au-DNA-Au nanostructure fabrication. The obtained Au-DNA-Au nanostructure showed higher ability in electron transfer, which indicates promising applications for information transmission along DNA chains.

Experimental

Typical sequences of the oligonucleotides used in this study are as follows. DNA1: 5'-HS-TACCAGGATTACCGCTCACA, DNA2: 5'-HS-TGTGAGCGGTAATCCTGGTA, DNA3: 5'-HS-C6-TACCAGGATTACCGCTCACA (bearing a -C6H12- linker between its mercapto group and DNA backbone), DNA4: 5'-HS-C6-TGTGAGCGGTAATCCTGGTA DNA5: 5'-TACCAGGATTACCGCTCACA (a native DNA for control),

Gold particles (0.009μM, d=10 nm) were coated by BSPP (bis(p-sulfonatophenyl) phenyl phosphane) in 400μM BSPP solution for 1h. The Au-BSPP conjugates were separated by centrifuge. DNA-GNP conjugates were obtained by treating Au-BSPP conjugates (0.05μM) with 0.5 μM DNA-SH at a specific pH for 24 h. In order to form a self assembled monolayer (SAM), the probe DNA-SH of 0.1μM, was codeposited to gold substrates with octanethiol (10μM) and the solution was kept for 1 h. Au-DNA- Au nanostructure was obtained by hybridization of the immobilized probe DNA with their complementary DNA-GNP conjugates (0.01μM) for 3 h. Atomic force microscopy (AFM) was employed to observe the Au-DNA-Au nanostructure and measure their electric properties.

Results and Discussion

1.Fabrication of the new type DNA-GNP conjugate

The DNA-GNP conjugate is the key intermediate for various DNA-GNP applications. For the first time, DNA was directly attached to GNP surface without any alkyl linker. The formations of our new type DNA-GNP conjugates were studied by electrophoresis.

Attachment of DNA to GNPs

It was found that both the DNA-SH (DNA1 and DNA3) and native DNA (DNA5) attached to gold particles easily. Fig.2 shows the amount of free DNA in solution after incubation with GNPs by electrophoresis. After incubation of 1 h with GNPs, the amount of the three DNA in solution decreased (Lane 1, 2, 3) indicating all of the three types of DNA partly attached to gold particles. After 24 h incubation, most of the DNA attached to gold particles (Lane 4, 5, 6). The residual of the free DNA in the solution was almost undetectable. Such kind of nonspecific attachment should be avoided in DNA-GNP conjugate fabrication.

BSPP protected GNPs : Protect GNP from nonspecific attachment of DNA

It was reported that BSPP could attach to GNP by coordinated bonds. The BSPP protected GNPs (Au-BSPP conjugate) has many advantages. 1. The GNPs are stabilized against aggregation since many negative charges caused by BSPP. 2. The Au-BSPP has a good mobility in electrophoresis that attachment of DNA to it can be easily monitored. We suspect that the Au-BSPP can prevent the nonspecific attachment of DNA from GNPs. Only the DNA-SH can substitute the BSPP molecules by the more stable DNA-S-Au linkage. It is known that GNPs will show a lower mobility when attached by DNA. The disappearance of free DNA bands also provides good evidences for the DNA-GNP conjugates formation. As Fig. 3 shows, native DNA doesn't attach to BSPP protected GNP after 24 h incubation. So with BSPP protection nonspecific attachment of DNA to GNPs can be successfully prevented.

1.3 pH dependence in DNA-GNP fabrications

The Au-BSPP conjugates were employed for the fabrication of DNA-GNP conjugates. As Fig.4 shows, astonishingly, at pH 8, DNA1 or DNA5 doesn't attach to gold particles (lane 3 and 4), although the DNA3 (with a linker) attaches to the gold particles very well (lane 2). When the pH was 4.0 or lower, however, both the DNA1 and DNA3 are effectively bound to the gold particles (lane 5 and 6). Since DNA5 (without a mercapto group) doesn't attach to gold particles at any pH (lane 4 and 7 or even lower), the possibility of nonspecific attachment can be ruled out. The different behavior of the two types of DNA-SH (with or without an alkyl linker) is attributed to the electrostatic repulsion between the negatively charged Au-BSPP and DNA-SH. Because of the absence of a linker, DNA1 can't approach Au-BSPP to an accessible distance. Accordingly, an acidic pH is necessary to decrease the negative charges of both Au-BSPP and DNA1 to suppress the electrostatic repulsion.

The charges on DNA1 or Au-BSPP were directly monitored by electrophoresis at various pH. As Fig.5 shows, Both DNA1 and Au-BSPP hold much less negative charges at acidic pH indicated by their mobility in electrophoresis. When pH is 2, the negative charges on DNA1 or GNP almost decreased to 0.

Further study showed the base contents of DNA-SH would strongly influence its dependence to pH in DNA-GNP conjugate fabrication. Table 1 lists the pH necessary for various DNA-SH to obtain DNA-GNP conjugate. As expected, polyC-SH and polyA-SH can attach to BSPP protected GNPs much more easily than polyG-SH and polyT-SH. The result agrees with our hypothesis about DNA-GNP conjugate formation since the bases with higher pKa can be more easily protonated.

Table. 1 The pH necessary to fabricate DNA-GNP conjugates and pKa of the four bases

2.Fabrication of Au-DNA-Au nanostructure

The most important property of DNA-Au conjugate is its hybridizing specificity. The obtained DNA-GNP conjugates were employed to hybridize with their complementary DNA probes, which were immobilized on Au substrates. AFM assay shows that many DNA-GNP conjugates specifically hybridized with its complementary probes on the Au substrates (Fig. 6A). The observed GNPs have a height of 8-10 nm, which agrees with the size of gold particles employed.

(A)DNA-GNP conjugates (DNA1-Au) hybridized with its complementary DNA (DNA2).

(B)DNA-GNP conjugates (DNA1-Au) treated with noncomplementary DNA (DNA1).

In contrast, almost no GNP was immobilized when the DNA probes on the gold substrate were not complementary with the DNA-GNP conjugates (Fig. 6B). The fabrication of the Au-DNA-Au nanostructure is reliable and reproducible.

The influence of the conditions for the Au-DNA-Au nanostructure fabrication was studied. It was found that the amount of immobilized GNPs is proportional to the concentrations of probe DNA in low concentrations (Fig. 7). This result indicates the immobilization of gold particles is concretely caused by the hybridization of the probe DNA and DNA-GNP conjugate. When the concentration of probe DNA-SH exceeded 0.1 μM, the amount of the immobilized gold particles kept constant. Some other parameter seems to be more determinative when there are enough probe DNA on gold surface.

The influence of the DNA-GNP conjugate concentration to the Au-DNA-Au nanostructure is shown in Fig.8. More GNPs were immobilized as the concentration of DNA-GNP conjugates increased. The red color of the DNA-Au conjugate didn't change obviously after hybridization, suggesting that only very small part of the DNA-GNP conjugates hybridized with the probe DNA immobilized on gold substrate.

3.Electro characterization of Au-DNA-Au nanostructure

The obtained Au-DNA-Au nanostructure was subjected to conductive probe AFM (cp-AFM) measurement. As shown in Scheme 1, the C8H17SH SAM is expected to serve as an insulator layer. When a voltage was applied between the GNP and the gold substrate, the current through the DNA chain was recorded. For each sample, at least 200 randomly selected points were measured. The U-I curve of these points varied very much. Fig.9 shows the distribution of the currents under 100 mV.

The cp-AFM measurement of C8H17SH SAM for its conductivity was also measured, resulting currents below 1 nA under 100 mV, which agrees with the C8H17SH SAM conductivity measurements by literature (JACS, 123, 5549). In control experiment when the probe and target DNA are noncomplementary, the obtained DNA-C8H17SH SAM also showed the same conductivity (I < 1 nA). This result indicates that the DNA-C8H17SH SAM wasn't damaged by the DNA-GNP solution.

(B) Current of Au-DNA-Au nanostructure (with a linker) under 100 mV.

As Fig.9 (A) shows, when Au-DNA-Au nanostructure was measured, about half of the points showed currents lower than 1 nA, which can be presumed as the positions that no Au-DNA-Au nanostructure was fabricated. Neglecting the currents below 1 nA, the conductivity of the 20 mer ds-DNA (without a linker) can be calculated to 10-1000 nS, while the corresponding DNA with a -C6H12- linker showed a conductivity of 10-200 nS. The DNA without a linker showed a comparably higher conductivity than that with a linker or C8H17SH.

Conclusions

DNA was directly attached to gold surface through S-Au bonds to fabricate a new type DNA-GNP conjugate and the corresponding Au-DNA-Au nanostructure. pH was found to play an important role in DNA-GNP conjugate fabrication. The different behaviors of different DNA-SHs in their DNA-GNP conjugate fabrication were contributed to their molecular charges. The obtained Au-DNA-Au nanostructure was observed by AFM. Furthermore the electric properties of DNA were studied by cp-AFM. Duplex DNA without a linker showed a higher conductivity than that with a linker, which suggests our new system is more promising as molecular wires for charge transmission along DNA chains.

Fig. 1 Structures of two kinds of DNA-gold nano particle (GNP) conjugate

Scheme 1. The method developed to fabricate DNA-GNP conjugate and Au-DNA-Au nanostructure

Fig.2 Nonspecific attachment of three DNA to GNPs Lane1,4: 0.1μM DNA3 + 0.01μM GNPs; Lane2,5: 0.1μM DNA1 + 0.01μM GNPs; Lane3,6: 0.1μM DNA5 + 0.01μM GNPs; Lane7: 0.1μM DNA3; Lane8: 0.1μM DNA1; Lane9: 0.1μM DNA5

Fig.3 Prevent nonspecific attachment of DNA to GNPs by BSPP protection Lane1: Au-BSPP; Lane2: 0.5 μM DNA5 + 0.05 μM Au-BSPP; Lane3: 0.5 μM DNA5

Fig.4 Electrophoresis of DNA-Au conjugate fabrication Lane 1: Au-BSPP, lane 2, 5: DNA3 + Au-BSPP, lane 3, 6: DNA1 + Au-BSPP, lane 4, 7: DNA5 + Au-BSPP, lane 8: DNA3, lane 9:DNA1, lane 10: DNA5.

Fig.5 Mobility of DNA1 and GNP at various pH

Fig. 6 AFM images of Au-DNA-Au nanostructure (500×500 nm)

Fig. 7 Influence of Probe DNA concentration to Au-DNA-Au nanostructure fabrication

Fig. 8 Influence of DNA1-GNP conjugate concentration to Au-DNA-Au nanostructure fabrication

Fig.9 (A) Current of Au-DNA-Au nanostructure (without a linker) under 100 mV.

本論文は、リンカーのないチオール化DNAを固定化した金ナノ粒子(DNA-GNP)を用いて新しいDNAナノストラクチャを構築する手法、ならびに調製したDNAナノストラクチャの諸性質について研究した結果について述べている。本論文は、第一章の序論、第六章の結論を含む全6章から構成される。

第一章においては、DNAの自己組織化を利用して形成されたナノ構造に関するこれまでの研究経過全般の概説を行い、ナノサイエンスにおけるDNAの応用の現状ならびにその意義について述べている。特に、金粒子や金基板と結合するのに現在広く使われているチオール化DNAが、チオールとDNAとの間にアルキルリンカーを有することに着目した。そのために、本論文ではリンカーのないチオール化DNAを用いて新型DNAナノストラクチャを構築することの重要性、ならびにリンカーによる影響のない諸物性のデータを入手することの目的と意義を説明している。

第二章では、リンカーのないチオール化DNAを合成した。さらにこのチオール化DNAと、従来から使用されていたアルキルリンカーを含むDNAのマススペクトルの時間変化を測定し、それらのDNAの化学的安定性について検討を行った。その結果、高温で放置したり、あるいは室温でも長時間放置した場合にはチオール化DNAの変性が避けられないが、上記の条件を避けてナノストラクチャを構築することにより、チオール化DNAの変性は十分に抑制できることを確認した。

第三章では、S-Au結合の形成によってDNAをナノ金粒子上に固定する条件及びそのメカニズムについて述べている。S-Au結合の形成は、電気泳動により固定化されたDNAあるいはナノ金粒子を測定することにより明確に判定できることを明らかにした。まず、DNAとナノ金粒子の間の非特異的結合を抑制するために、金粒子表面をBSPPでコーティングした。次に、アルキルリンカーを持つチオール化DNA及びリンカーを持たないチオール化DNAを用い、それぞれをBSPPでコーティングされたナノ金粒子と作用させて、S-Au結合の形成条件を調べた。その結果、S-Au結合の形成は反応溶液のpHに大きく依存することを明らかにした。pH 4.0以下では、リンカーの有無に関わらずチオール化DNAはナノ金粒子上に迅速に固定される。しかし、pH 4.0~9.0では、リンカーのあるDNAしか金粒子上に固定されないことが明らかとなった。また、チオール基を持たない非修飾DNAは、どのpH条件下でも金粒子と結合しない。以上の結果より、DNAと金粒子の結合には表面電荷の間の静電力が重要な寄与をすることを示唆するとともに、チオール化DNAをナノ金粒子に固定するためにはpH 4.0以下で反応を行うことを明らかにした。以上の結論の妥当性を、異なる塩基配列を持つチオール化DNAを用いてさらに実証した。

第四章では、Au-DNA-Auナノストラクチャの構築方法について述べている。まず、リンカーのないチオール化DNAをS-Au結合で金基板上に固定した。次に、リンカーのないチオール化DNAをナノ金粒子上に固定した。ここで使用した二つのDNAは互いに相補的配列を持つ。それらを混和して、DNA同士のハイブリダイゼーションによってAu-DNA-Auナノストラクチャを構築した。その構造を原子間力顕微鏡AFMによって直接観察し、新たに開発した構築法の妥当性を確認した。

第五章では、第四章で構築したAu-DNA-Auナノストラクチャにcp-AFMを適用し、そこに含まれるDNAの電気伝導度を測定した。リンカーのあるDNA及びリンカーのないDNAで作られたAu-DNA-Auナノストラクチャの両端に電圧をかけ、流れる電流量よりDNAの電気伝導度を計算した。その結果、リンカーのあるDNAと比べてリンカーのないDNAはより高い電気伝導度を持つことが明らかとなり、本研究の本来の仮説の妥当性を確認した。

以上、本研究により、従来用いられていたチオールとDNAとの間のアルキルリンカーを除去し、このDNAをS-Au結合によってナノ金粒子上に迅速に固定することに成功した。新しいDNA-GNPの作成法におけるS-Au結合の形成の条件を最適化するとともに、その生成メカニズムを解明した。ここで、金粒子表面をBSPPでコーティングすることがDNAとナノ金粒子の間の非特異的結合を抑制するのに非常に有効であることを見出した。さらにこれらの知見を発展させて、新しいAu-DNA-Auナノストラクチャの構築に成功し、cp-AFMを用いてDNAの電気伝導度を測定した。今回の研究で確立された新たなDNAナノストラクチャの構築手法は、ナノサイエンス分野におけるDNAの応用に貢献するところが少なくない。

よって本論文は博士(工学)の学位請求論文として合格と認められる。