Supporting Information

Enhancement of SPR Signals by Gold Nanoparticles

on High Density DNA Microarrays

Masateru Ito,a Fumio Nakamura,a,b Akira Baba,c Kaoru Tamada,a,e +*

Hirobumi Ushijima,a King Hang Aaron Lau,d,f

Abhijit Mannae and Wolfgang Knolld,e, f

a National Institute of Advanced Industrial Science and Technology (AIST),

Tsukuba Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan b PRESTO, Japan Science and Technology (JST), 4-1-8 Honcho, Kawaguchi,

Saitama 332-0012, Japan c Department of Chemistry, University of Houston d Institute of Materials Research and Engineering, Singapore e Department of Materials Science, National University of Singapore f Max-Plank-Institute for Polymer-Research, Mainz, Germany

+Present adress: Department of Electronic Chemistry, Tokyo Institute of

Technology, G1-10 Nagatsuta 4259, Yokohama 226-8502, Japan

* Author to whom correspondence should be addressed

Materials

Carboranes were commercially available and purchased from KatChem. Column

chromatography was carried out employing Silica gel 60 N (spherical, neutral, 40~100 µm and

100~210 µm, KANTO Chemical Co., Inc.). All other reagents were commercially available and

were used without further purification. All oligonuleotides were purchased from ESPEC OLIGO

SERVICE CORP. (Tsukuba, Japan). 1H NMR and 13C NMR spectra were recorded on a JEOL

JNM AL-500 (500 MHz) spectrometer. ESI-TOFMS was performed on a Mariner (Applied

Biosystems) mass spectrometer. General instruments setting for ESI-TOFMS are as follows:

vaporizing temperature, 140 ˚C; infused rate, 3 µl / min; capillary voltage, 3100 V; nozzle

potential, 100 V. The signal intensities were averaged over five scans and recorded as the sum of

the corresponding isotopic peaks.

Synthetic procedure and compound data of 1 and 2

A series of carboxylic acid derivatives 1 and 2 were synthesized as shown in Scheme 2.

Compound 1 is composed of carborane-carboxylic acid derivative. In compound 1, carborane

moiety acts as a spacer group for the carboxylic acid end group improving the formation of

intermediate structure by the steric hindrance in a SAM. Compound 2 is the reference molecule

without carborane moiety. In detailed synthetic procedures of compounds 1 and 2 are described

below.

Synthesis of 1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane (3)

To a solution of p-carborane (1.44 g, 10 mmol) in 100 ml of 1,2-dimethoxyethane was added 1.57

M n-BuLi in hexane (7.0 ml, 11 mmol) at 0 ˚C under dry N2. The reaction mixture was stirred at

room temperature for 1 h and copper (I) chloride (1.1 g, 11 mmol) was added in one portion. The

reaction mixture was stirred at room temperature for 2 h and pyridine (6 ml) was added. The

reaction mixture was stirred at room temperature for 30 min and 4-iodoanisole (2.49 g, 10 mmol)

was added in one portion. The resulting mixture was refluxed until 4-iodoanisole was largely

consumed. After the solvent was evaporated, the residue was extracted with diethyl ether. The

organic layer was dried over Na2SO4. After the solvent was evaporated, the residue was purified

by column chromatography (silica gel, hexane : ethyl acetate = 15 : 1) to give 3 in 60% yield (1.50

g, 6.0 mmol) as a white solid. 3 : 1H NMR (500 MHz, CDCl3) δ 7.12 (d, J = 9.2 Hz, 2H), 6.68 (d,

J = 9.2 Hz, 2H), 3.74 (s, 3H), 2.75 (s, 1H); 13C NMR (500 MHz, CDCl3) δ 159.6, 129.3, 128.2,

113.2, 59.2, 55.2; HRMS (EI) Calcd for C9H18B10O: 250.2362. Found: 250.2449.

Synthesis of 1-(4-Methoxyphenyl)-12-fromic acid methyl ester

1,12-dicarba-closo-dodecaborane (4)

To a solution of 3 (1.50 g, 6.0 mmol) in 60 ml of THF was added 1.57 M n-BuLi in hexane (4.2

ml, 6.6 mmol) at 0 ˚C under dry N2. The reaction mixture was stirred at room temperature for 1 h

and Methylchroloformate (1.98 g, 6.6 mmol) was added. The reaction mixture was stirred at room

temperature for 5 h and after the solvent was evaporated, the residue was purified by flash column

chromatography (silica gel, hexane : ethyl acetate = 10 : 1) to give 4 in 88% yield (1.63 g, 5.3

mmol) as a white solid. 4 : 1H NMR (500 MHz, CDCl3) δ 7.09 (d, J = 9.2 Hz, 2H), 6.68 (d, J = 9.2

Hz, 2H), 3.74 (s, 3H), 3.65 (s, 3H); 13C NMR (500 MHz, CDCl3) δ 163.2, 159.8, 128.6, 128.1,

113.4, 55.3, 54.1; ESI-TOFMS Calcd for C11H20O3B10Na [M+Na]; 331.2, Found: 331.3.

Synthesis of 1-(4-Hydroxyphenyl)-12-fromic acid methyl ester

-1,12-dicarba-closo-dodecaborane (5)

To a solution of 4 (1.63 g, 5.3 mmol) in 60 ml of dichrolomethane was added borontribromide

(0.95 ml, 10 mmol) at 0 ˚C under dry N2. The reaction mixture was stirred for 6 h. After the

solvent was evaporated, the residue was extracted with diethyl ether. The organic layer was dried

over Na2SO4. After the solvent was evaporated, the residue was purified by flash column

chromatography (silica gel, hexane : ethyl acetate = 10 : 1) to give 5 in quantitative yield (1.56 g,

5.3 mmol) as a white solid. 5 : 1H NMR (500 MHz, CDCl3) δ 7.04 (d, J = 8.9 Hz, 2H), 6.61 (d, J =

8.9 Hz, 2H), 3.65 (s, 3H); 13C NMR (300 MHz, CDCl3) δ 163.3, 155.8, 128.8, 128.4, 114.8, 54.1;

ESI-TOFMS Calcd for C10H18O3B10Na [M+Na]; 317.2, Found: 317.3.

Synthesis of 1-(12-fromic acid methyl ester-

1,12-dicarba-closo-dodecaborane)-4-(1-bromo)-tetraethyleneglycoxybebzebene (6)

To a solution of 5 (1.88 g, 5.5 mmol) in 60 ml of acetone was added 1,

11-dibromotetraethyleneglycol (2.64 g, 8.25 mmol) and K2CO3 (2.28 g, 16.5 mmol). The

resulting mixture was refluxed for 1 day. After the solvent was evaporated, the residue was

extracted with diethyl ether. The organic layer was dried over Na2SO4. After the solvent was

evaporated, the residue was purified by column chromatography (silica gel, hexane : ethyl acetate

= 2 : 1) to give 6 in 55% yield (1.61 g, 3.0 mmol) as a colorless oil. 6 : 1H NMR (500 MHz,

CDCl3) δ 7.07 (d, J = 9.2 Hz, 2H), 6.69 (d, J = 9.2 Hz, 2H), 4.04-4.06 (m, 2H), 3.78-3.82 (m, 4H),

3.65 (s, 3H), 3.64-3.70 (m, 8H), 3.45 (t, J = 6.4 Hz, 2H); 13C NMR (500 MHz, CDCl3) δ 163.1,

159.0, 128.7, 128.0, 114.0, 71.1, 70.8, 70.6, 70.6, 70.5, 69.5, 67.4, 54.1, 30.3; ESI-TOFMS Calcd

for C18H33O6BrB10Na [M+Na]; 556.2, Found: 556.3.

Synthesis of 1-(12-fromic

acid-1,12-dicarba-closo-dodecaborane)-4-(1-bromo)-tetraethyleneglycoxybebzebene (7)

To a solution of 6 (1.61 g, 3.0 mmol) in 60 ml of THF and H2O (1 : 1) mixed solution was added

potassium hydroxide (0.84 g, 15 mmol). The reaction mixture was stirred for 6 h and neutralized

with 1 N HCl. After the solvent was evaporated, the residue was extracted with ethyl acetate. The

organic layer was dried over Mg2SO4. After the solvent was evaporated, the residue was purified

by flash column chromatography (silica gel, hexane : ethyl acetate = 1 : 1) to give 7 in quantitative

yield (1.56 g, 3.0 mmol) as a white solid. 7 : 1H NMR (500 MHz, CDCl3) δ 7.07 (d, J = 8.9 Hz,

2H), 6.69 (d, J = 8.9 Hz, 2H), 4.04-4.06 (m, 2H), 3.77-3.82 (m, 4H), 3.65-3.70 (m, 8H), 3.44 (t, J

= 6.4 Hz, 2H); 13C NMR (500 MHz, CDCl3) δ 166.1, 158.9, 128.7, 128.1, 114.0, 71.1, 70.7, 70.6,

70.5, 70.4, 69.5, 67.3, 30.1; ESI-TOFMS Calcd for C17H31O6BrB10Na [M+Na]; 542.2, Found:

542.2.

Synthesis of 12-(4-(12-fromic

acid-1,12-dicarba-closo-dodecaborane)-phenoxy)-tetraethyleneglycol-disulfide (1)

To a solution of 7 (1.56 g, 3.0 mmol) in 30 ml of ethanol was added thiourea (0.84 g, 15 mmol).

The reaction mixture was refluxed for 6 h and then 5 ml of 10% NaOH was added dropwise. The

reaction mixture was refluxed for 3 h. The reaction mixture was neutralized with 1 N HCl. The

solution was extracted with ethyl acetate. The organic layer was dried over Mg2SO4. After the

solvent was evaporated, the residue was purified by flash column chromatography (silica gel,

hexane : ethyl acetate = 1 : 1) to give 1 in quantitative yield (1.41 g, 1.5 mmol) as a white solid. 1 :

1H NMR (500 MHz, CD3OD) δ 7.10 (d, J = 9.2 Hz, 4H), 6.73 (d, J = 9.2 Hz, 4H), 4.04-4.06 (m,

4H), 3.77-3.79 (m, 4H), 3.56-3.70 (m, 20H), 2.85 (t, J = 6.4 Hz, 4H); 13C NMR (500 MHz,

CD3OD) δ 160.4, 130.4, 129.4, , 115.1, 71.8, 71.6, 71.6, 71.4, 70.7, 68.7, 67.3, 39.6; FAB-MS

Calcd for C34H62O12S2B20Na [M+Na]; 965.9, Found: 965.8.

Synthesis of 4-Hydroxy-benzoic acid ethyl ester (8)

To a solution of 4-Hydroxy-benzoic acid (2.76 g, 20 mmol) in 200 ml of ethanol was added

sulfuric acid (0.2 g, 2.0 mmol). The reaction mixture was refluxed for 6 h and then neutralized

with sat. NaHCO3. The solution was extracted with ether. The organic layer was dried over

Na2SO4. After the solvent was evaporated, the residue was purified by flash column

chromatography (silica gel, hexane : ethyl acetate = 1 : 1) to give 8 in quantitative yield (3.32 g,

20 mmol) as a white solid. 8 : known product.

Synthesis of 4-(1-bromo)-tetraethyleneglycoxy-benzoic acid ethyl ester (9)

To a solution of 8 (3.32 g, 20 mmol) in 200 ml of acetone was added 1,

11-dibromotetraethyleneglycol (9.61 g, 30 mmol) and K2CO3 (8.29 g, 60 mmol). The resulting

mixture was refluxed for 1 day. After the solvent was evaporated, the residue was extracted with

diethyl ether. The organic layer was dried over Na2SO4. After the solvent was evaporated, the

residue was purified by column chromatography (silica gel, hexane : ethyl acetate = 2 : 1) to give

9 in 64% yield (5.19 g, 12.8 mmol) as a colorless oil. 9 : 1H NMR (500 MHz, CDCl3) δ 7.98 (d, J

= 8.9 Hz, 2H), 6.93 (d, J = 8.9 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 4.17-4.19 (m, 2H), 3.87-3.89 (m,

2H), 3.80 (t, J = 6.4 Hz, 2H), 3.72-3.74 (m, 2H), 3.67-3.3.70 (m, 6H), 3.46 (t, J = 6.4 Hz, 2H),

1.37 (t, J = 7.0 Hz, 2H); 13C NMR (500 MHz, CDCl3) δ 166.2, 162.3, 131.3, 122.9, 114.0, 71.0,

70.7, 70.5, 70.5, 70.4, 69.4, 67.4, 60.5, 30.2, 14.2; ESI-TOFMS Calcd for C17H25O6BrNa

[M+Na]; 427.1, Found: 427.1.

Synthesis of 4-(1-bromo)-tetraethyleneglycoxy-benzoic acid (10)

To a solution of 9 (5.19 g, 12.8 mmol) in 120 ml of Ethanol and H2O (1 : 1) mixed solution was

added lithium hydroxide monohydrate (2.69 g, 64 mmol). The reaction mixture was stirred for 6 h.

After the solvent was evaporated, the residue was extracted with ethyl acetate. The organic layer

was dried over Mg2SO4. After the solvent was evaporated, the residue was purified by flash

column chromatography (silica gel, hexane : ethyl acetate = 1 : 1) to give 10 in quantitative yield

(4.83 g, 12.8 mmol) as a white solid. 10 : 1H NMR (500 MHz, CDCl3) δ 8.05 (d, J = 8.6 Hz, 2H),

6.96 (d, J = 8.6 Hz, 2H), 4.20-4.22 (m, 2H), 3.89-3.90 (m, 2H), 3.63-3.82 (m, 10H), 3.47 (t, J =

6.4 Hz, 2H); 13C NMR (500 MHz, CDCl3) δ 171.3, 163.2, 132.3, 121.8, 114.3, 71.2, 70.9, 70.7,

70.6, 70.5, 69.5, 67.6, 30.3; ESI-TOFMS Calcd for C15H21O6BrNa [M+Na]; 399.0, Found: 399.0.

Synthesis of 4-(1-Mercapto)-tetraethyleneglycoxy-benzoic acid (2)

To a solution of 10 (4.83 g, 12.8 mmol) in 120 ml of ethanol was added thiourea (3.9 g, 51 mmol).

The reaction mixture was refluxed for 6 h and then 5 ml of 10% NaOH was added dropwise. The

reaction mixture was refluxed for 3 h. The reaction mixture was neutralized with 1 N HCl. The

solution was extracted with ethyl acetate. The organic layer was dried over Mg2SO4. After the

solvent was evaporated, the residue was purified by flash column chromatography (silica gel,

hexane : ethyl acetate = 2 : 3) to give 2 in 36% yield (1.52 g, 4.6 mmol) as a white solid. 1 : 1H

NMR (500 MHz, CDCl3) δ 8.04 (d, J = 8.6 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H), 4.19-4.21 (m, 2H),

3.88-3.90 (m, 2H), 3.60-3.75 (m, 10H), 2.70 (q, J = 7.9 Hz, 2H), 1.59 (t, J = 8.3 Hz, 1H); 13C

NMR (500 MHz, CDCl3) δ 171.0, 163.2, 132.3, 121.8, 114.3, 72.9, 70.9, 70.7, 70.6, 70.2, 69.5,

67.6, 24.3; ESI-TOFMS Calcd for C15H22O6SNa [M+Na]; 353.1, Found: 353.1.; HRMS (EI)

Calcd for C15H23O6S [M+H]; 331.1216. Found: 331.1215.

Synthesis of DNA modified gold nanoparticle

(i) Synthesis of SDS capped gold nanoparticle

To an aqueous solution of HAuCl4 (20 mg, 50 µmol) in 50 ml was added sodium dodecyl sulfate

(SDS) (50 mg, 170 µmol). The reaction mixture was reflux for 10 min and then trisodium citrate

aqueous solution (2 wt%, 2.5 ml) was added quickly with stirring vigorously for 30 min. Within a

few minutes, a sharp color change (from colorless to wine red) was observed. Subsequently, the

solution was allowed to cool to room temperature. The reaction mixture was centrifuged at 6000

rpm and removed the supernatant. The characterization of SDS capped gold nanoparticle was

carried out by a UV-vis absorption spectroscopy.

(ii) Synthesis of DNA modified gold nanoparticle

To an aqueous solution of SDS capped gold nanoparticle was added 5’-thiol terminated

oligonucleotide (20 complementary, SH- 5’-CTGTGTCGATACGTTCTCCA-3’) in 50 mM NaCl

of PBS buffer. The reaction mixture was stirred for 12 h. The exchange reaction was occurred on

the gold nanoparticle surface. The reaction mixture was centrifuged at 10000 rpm and removed

the supernatant, washed with 50 mM NaCl of PBS buffer 2 times. The characterization of DNA

modified gold nanoparticle was carried out by a UV-vis absorption spectroscopy.