A Comparison of Electrochemical DNA Probes Possessing an Isomeric Ferrocene-Diamidopyridine Conjugate for SNPs Detection on Au(111)

Electrochemical single nucleotide polymorphisms (SNPs) detection was investigated on gold surface modiﬁed with 16-base-pair duplex DNAs. In these duplexes, probe DNAs consisted of complementary sequences for target DNAs and a series of ferrocene-diamidopyridine conjugates as an electrochemically active moiety. Regardless of isomeric structures in the conjugates, all the DNA probes showed electrochemical discrimination between fully matched DNAs and SNPs. A detailed comparison was carried out for these probes. [DOI: 10.1380/ejssnt.2005.393]


I. INTRODUCTION
Rapid and sensitive detection of biological targets on chemically modified surfaces has been widely investigated for developing high-throughput sensors [1][2][3][4]. Especially, DNA-based sensors have a potential for the application to large-scale typing of single nucleotide polymorphisms (SNPs) [5,6]. SNPs are common sequence variations among DNAs of individuals and relate to genetic basis disease and drug response (pharmacogenomics) [7,8]. Therefore, a simple and inexpensive protocol for largescale SNPs assay of individuals has been desired in postsequence genetics. The electrochemical discrimination strategy of SNPs on chip will be fascinating from the view point of rapidity, sensitivity, and availability of low-cost devices [9][10][11].
We recently reported a highly sensitive electrochemical SNPs detection methodology that relied upon charge transport through the π-stack of DNA duplexes on Au(111) electrodes [12]. In that report, the ferrocenemodified DNA probe HS-DNA-Fc(pS) was developed, in which a ferrocene-modified nucleoside analogue was connected at the 5' end of a single-stranded oligonucleotide (Fig. 1). The presence of a single-base mismatch in the duplexes from the probe and target DNAs caused a dramatic decrease in the electrochemical response. The key motif of the probes is the electrochemically active nucleoside residue that consists of a synthetic hydrogenbonding unit tethered to a ferrocene skeleton possessing a pS planer chirality with an acetylene linker. However, there was no reason to believe that the structure of the conjugate represented the optimal motif as an electrochemically active unit, and we have examined some other * This paper was presented at International Symposium on Surface Science and Nanotechnology (ISSS-4), Saitama, Japan, 14-17 November, 2005. † Corresponding author: inouye@ms.toyama-mpu.ac.jp structures. Here we show the electrochemical SNPs detection on Au(111) electrodes modified with new DNA probes, HS-DNA-Fc(pR) and HS-DNA-Fc(Achiral) that have isomeric structures in the nucleoside residue ( Fig. 1).
General. 1 H and 13 C NMR spectra were recorded on a Varian Gemini 300 spectrometer (300 MHz for 1 H and 75 MHz for 13 C). IR spectra were recorded on a JASCO-FT/IR 460 plus spectrometer. EI-HRMS and ESI-HRMS analyses were carried out on a JEOL-JMD-D300 and a JEOL-JMS-T100LC mass spectrometer, respectively. Melting points were determined with Yanako-MP-500D and not corrected. All synthetic reactions were carried out under argon atmosphere. THF was fleshly distilled from sodium benzophenone ketyl before use, and other solvents were purified with standard methods [15].

Phosphoramidite 3.
To a CH 2 Cl 2 (10 mL) solution of 7 (45 mg, 0.10 mmol) were added a CH 2 Cl 2 (4 mL) solution of 4-(dimethylamino)pyridine (6 mg, 0.050 mmol) and i-Pr 2 NH (0.12 mL, 0.70 mmol) at 0 • C. After the reaction mixture had been stirred for 5 min, to the mixture was added NC(CH 2 ) 2 OPClN(Pr-i) 2 (0.07 mL, 0.30 mmol) at that temperature. After warming to room temperature with stirring over 2 h period, the mixture was diluted with CH 2 Cl 2 (40 mL) and washed with a saturated NaHCO 3 aqueous solution. The organic layer was evaporated and purified by HPLC (ODS; eluent, MeOH) to give 3 as an orange oil (a mixture of diastereomers): yield 54% (

III. RESULTS AND DISCUSSION
In our previous report, electrochemical SNPs detection was performed with the probe DNAs possessing a ferrocene moiety of a pS planer chirality [12]. This chirality happens at a stage of synthesis for the nucleoside analogue and is originated from the configuration of a chiral reactant, 1,2,4-butanetriol (Scheme 1 (Fig. 2)). When a racemic nucleoside analogue was used, starting from commercially available racemate of 1,2,4-butanetriol, the corresponding probe DNA was synthesized as a mixture of diastereomers. Reverse-phase HPLC purification could separate these two DNA probes bearing each chirality of pS or pR in the nucleoside residue. Although a detailed study was carried out for the probe DNA containing the pS chirality [12], the corresponding probe of the pR one remains to be investigated. To clarify the influence of isomeric structures in the ferrocene unit on electrochemical responses, we examined the DNA probes possessing the pR ferrocene moiety. Furthermore, an achiral version of the ferrocene unit was also synthesized (Scheme 2 (Fig.  3)) and derivatized to the corresponding probe. Both the new probes were identified by MALDI-TOF mass measurements.
To unambiguously compare, the sequence of a 16-mer DNA probe (3'-HS(CH 2 ) 3 -AGT ACA GTC ATC GCG Fc-5') is selected on the basis of our previous report [12]. In this sequence, the residue of the ferrocene-modified nucleoside analogue is abbreviated to Fc, and probes are referred to as HS-DNA-Fc(pS), HS-DNA-Fc(pR), and HS-DNA-Fc(Achiral) corresponding to the chirality in each of the ferrocene moiety (Fig. 1). The sequences of a fully matched complement 8 and its SNP 9 examined in this study are 5'-TCA TGT CAG TAG CGC T-3' and 5'-TCA TGT CAC TAG CGC T-3' (underlined residue is a mismatched base), respectively.
In advance, UV and circular dichroism (CD) spectra for duplexes between the probe and the complements were measured in aqueous media. Therefore, a thiol-free version of HS-DNA-Fc(pR), DNA-Fc(pR) (3'-AGT ACA GTC ATC GCG Fc(pR)-5') was synthesized and investigated for the duplex formation. From the UV-melting profiles, the T m values of 62.4 and 47.5 • C were determined for the duplexes from the fully matched complement 8 and the SNP 9, respectively (Fig. 4). As expected, the duplex from 9 showed the lower T m value compared with that of the duplex from 8 because of the presence of a mismatched base pair in the middle of the sequences. However, even with 9, DNA-Fc(pR) firmly forms the duplex structure at 25 • C, which is the temperature for the measurements in electrochemical SNPs detection. The duplex formation was also confirmed on the basis of CD in 1 M NaClO 4 at 25 • C. Cotton effects in the wavelength region of 300 to 200 nm reflect the secondary structures of double-stranded DNAs. A mixture of DNA-Fc(pR) and the SNP 9 showed an almost identical CD spectrum in that region with those not only of DNA-Fc(pR)·8 but also of DNA-Fc(pS)·8 [12] and DNA-Fc(pS)·9 [12]; characteristic of a typical double stranded B-form DNA [16] (Fig. 5). Square wave voltammetry (SWV) measurements were performed at the electrodes modified with duplexes from the new DNA probes scanning from 0 to +0.6 V vs. Ag/AgCl in 1M NaClO 4 . When applying the duplex from HS-DNA-Fc(pR) and the fully matched complement 8, an intense anodic peak appeared at +0.30 V, resulted from the oxidation of the ferrocene moiety (Fig. 6A). This potential is almost the same value in the combination of HS-DNA-Fc(pS) and 8 [12]. On the other hand, no obvious SWV response was observed from the duplex with the mismatched complement 9. In the same manner, we also applied the duplexes from HS-DNA-Fc(Achiral) with 8 and 9 for the electrochemical measurements (Fig.  6B). A quite similar discrimination at +0.30 V was seen for these duplexes as in the cases for HS-DNA-Fc(pR) and HS-DNA-Fc(pS). Regardless of the isomeric struc- tures in the ferrocene unit, all the DNA probes showed the same "on-off" type digital action for the SNPs detection. These findings indicate that stereochemistry in the ferrocene unit has little influence on the electrochemical responses, encouraging us to develop further ferrocenebased electrochemical probes.
As a next stage of our research, we targeted naturally occurring SNPs for demonstrating the versatility of our probes. For this purpose, the SNPs sequences in the p53 gene were chosen. Therefore, a DNA probe possessing the Fc(pR) nucleoside residue and the complementary sequence for the target was newly synthesized, and the 16mer probe of 3'-HS(CH 2 ) 3 -ACT TGG CCT CCG GGT Fc(pR)-5' is referred to as HS-DNA2-Fc(pR). This new probe also revealed keen differentiation of the three SNPs from the fully matched complement 10 (Fig. 7). This experiment proves that the Fc(pR) probe can detect mutations within natural sequences of duplexes and will be independent of sequence context and composition.

IV. CONCLUSIONS
We demonstrated electrochemical SNPs discrimination on Au(111) modified with DNA probes possessing a series of isomeric structures in a ferrocene unit. Regardless of stereochemistry in the ferrocene moiety, all the DNA probes performed similar electrochemical responses for SNPs discrimination. This tolerance of the ferrocene unit for detecting SNPs is an important additional advantage for our "wire-like" DNA probes. Development of more simple and easy-made ferrocene units is now underway.