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Electronic Supplementary Information

for

Antibody-graphene oxide nanoribbon conjugate as a surface enhanced

laser desorption/ionization probe with high sensitivity and selectivity

Jing Wang1,2, Mengting Cheng1, Zhen Zhang3, Liangqia Guo2, Qian Liu1,*, Guibin Jiang1

1 State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-

Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

2 Department of Chemistry, Fuzhou University, Fuzhou 350002, China

3 School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China

*Correspondence to: qianliu@rcees.ac.cn

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2015

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1. Experimental section

Chemicals and reagents

Multi-walled carbon nanotubes (MWCNTs) were purchased from Chengdu Organic Chemicals

Co. of Chinese Academy of Sciences (Chengdu, China). KMnO4, H2SO4, H2O2 (30%), hydrochloric

acid (HCl), ethanol, and ethyl ether were purchased from Sinopharm Chemical Reagent Beijing Co.

(Beijing, China). Graphene and graphene oxide (GO) were from ACS Material (Medford, MA). 2-

Morpholino-ethanesulfonic acid (MES), N-hydroxysuccinimide (NHS), N-(3-

dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), α-cyano-4-hydroxycinnamic

acid (CHCA), and O-(2-aminoethyl)-O’-(2-carboxyethyl)polyethylene glycol hydrochloride (NH2-

PEG-COOH, Mp 3000) were from Sigma-Aldrich (St. Louis, MO). Chloramphenicol (CAP) was

from Alfa Aesar (Ward Hill, MA). The water used in all experiments was prepared from a Milli-Q

water purification system (Millipore, Milford, MA). All reagents were of analytical grade unless

otherwise noted.

Synthesis of graphene oxide nanoribbons (GONRs)

GONRs were synthesized from longitudinal unzipping of MWCNTs by chemical oxidation.

Typically, MWCNTs (O.D. > 50 nm) were suspended in 150 mL of concentrated H2SO4 (98%) for

12 h. Then, 750 mg of KMnO4 was added to the mixture and stirred for 1 h at room temperature.

The mixture was heated in sequence at 55 °C for 30 min, 65 °C for 1 h, and 70 °C for 20 min. After

reaction, the mixture was cooled down to room temperature and 400 mL of ice water containing 5

mL of H2O2 (30%) was poured into the mixture under stirring. The solids were collected by

filtration through a PTFE membrane (0.45 μm pore size), and then dispersed in 150 mL of water by

aid of stirring for 30 min and sonication for 15 min. The, 30 mL of HCl solution (20% v/v) was

added and the solids were filtered again. The product was finally washed with ethanol and ethyl

ether for several times and air-dried.

Preparation and purification of anti-CAP monoclonal antibody

The anti-CAP monoclonal antibodies were prepared in lab using the hybridoma technique.

Protein–CAP conjugates were synthesized using an activated ester method.1 CAP-bovine serum

albumin (BSA) conjugate was used as an immunogen and CAP-ovalbumin (OVA) conjugate was

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used as a coating antigen. The monoclonal antibody (MAb) was produced as described previously

with some modifications.2 Briefly, five female BALB/c mice of 8 weeks old were immunized

subcutaneously with CAP-BSA (100 μg) in Freund’s complete adjuvant. After three booster doses

of CAP-BSA in Freund’s incomplete adjuvant at 2-week intervals, the sera were collected and

assayed for anti-CAP antibodies by a non-competitive indirect ELISA. The mouse exhibiting the

highest titer was immunized with a final dose of CAP-BSA (100 μg) in distilled water. The spleen

was removed and splenocytes were fused with SP2/0 myeloma cells and cultured in 96-well plates.

The positive hybridomas were rescreened using a competitive indirect ELISA with CAP as the

competitor. The hybridomas that excreted antibodies specific to CAP were subcloned twice with the

limiting dilution method. The isotype of the antibodies was detected with mouse heavy and light-

chain specific antisera using a commercial ELISA kit (Southern Biotech Co., Beijing, China).

The specificity of as-prepared anti-CAP monoclonal antibody was characterized by the

equation: Cross-reactivity (%) = [(IC50 value of CAP)/(IC50 value of other analogs)] × 100. Three

structurally related chemicals were tested as analogs. The results based on three parallel

experiments are given in Table S4. It can be seen that the obtained anti-CAP monoclonal antibody

showed high specificity for CAP.

Preparation of antibody-GONR conjugates

The procedures for preparation of antibody-GONR conjugates (GONR-PEG-Ab) are depicted

in Fig. 1. PEG was used as a linker between GONRs and antibody. Briefly, GONRs (10 mg) were

dispersed in 10 mL of water and sonicated for 2 h. Then, 250 μL of MES buffer solution (500 mM,

pH 6.1), 250 μL of NHS aqueous solution (50 mg/mL), and 300 μL of fresh prepared EDC aqueous

solution (10 mg/mL) were added to 500 μL of GONR dispersion. The mixture was shaken for 30

min under room temperature. Afterward, the mixture was centrifugated at 13000 rpm for 5 min and

the supernatant was discarded. The precipitates were washed with MES buffer solution (50 mM) to

remove excess EDC and NHS. In this way, the carboxyl groups in GONRs were activated. The

activated GONRs were re-dispersed in 500 μL of MES buffer solution (50 mM, pH 6.1) with

sonication. Then, 6.2 mg of EDC, 3.6 mg of NHS, and 1.0 mg of NH2-PEG-COOH were added to

the dispersion. The mixture was shaken at room temperature for 2 h to produce PEGylated GONRs.

The product was centrifugated and washed thoroughly with MES buffer solution to remove

unreacted NH2-PEG-COOH. To functionalize with antibody, the PEGylated GONRs were dispersed

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in 500 μL of MES buffer solution and incubated with 50 μL of anti-CAP monoclonal antibody

solution (6.5 mg/mL) for 2 h. The final product was washed with water for three times and

dispersed in 500 μL of water. The concentration of the GONR-PEG-Ab in the dispersion was

determined by UV-Vis spectrometry to be 0.5 mg/mL.

Characterization of materials

AFM images were captured on an Agilent 5500 Atomic Force Microscope (Santa Clara, CA).

The AFM samples were deposited on a fresh mica wafer. Low-resolution SEM images were

captured on a Hitachi S-3000N scanning electron microscope (Tokyo, Japan). High-resolution SEM

images were captured on a Hitachi SU-8020 field-emission scanning electron microscope (Tokyo,

Japan) equipped with an IXRF Systems energy dispersive X-ray spectrometer (Austin, TX). TEM

images were captured on a Hitachi H-7500 transmission electron microscope (Tokyo, Japan). XPS

spectra were obtained on an AXIS Ultra DLD X-ray photoelectron spectrometer (Kratos,

Manchester, UK) with Al Kα X-ray radiation as the X-ray source excitation. XRD patterns were

obtained on a PANalytical X’Pert PRO X-ray diffraction system (Almelo, Netherlands). FT-IR

spectra were obtained on a JASCO FT/IR Fourier transform infrared spectrometer (Victoria, B. C.,

Canada). The samples were mixed and ground with KBr and then pressed into transparent disks for

measurement. The solid-state UV-visible absorption spectra were obtained on a Shimadzu UV-3600

UV-VIS-NIR spectrophotometer (Kyoto, Japan). The film of the sample was prepared by placing a

drop of its dispersion (0.5 mg/mL) on the surface of a quartz cuvette followed by air-drying.

SELDI-TOF MS analysis

For SELDI-TOF MS analysis, GONR-PEG-Ab was used as a SELDI probe. First, 50 μL of the

probe dispersion (0.5 mg/mL) was added to 5 mL of sample solution and incubated for 2 h. The

probe was isolated from the solution by using a centrifugal ultra-filter device (Amicon Ultra,

MWCO 10 kD, Millipore, MA). The filter residue was further centrifugated at 12000 rpm for 15

min at 4 °C. Then, the precipitates were re-dispersed in 10 μL of water and 2 μL of the dispersion

was dropped onto a stainless steel MTP target frame III (Bruker Daltonics) for measurement.

MALDI-TOF MS measurements were performed on a Bruker Daltonics Autoflex III Smartbean

MALDI-TOF mass spectrometer in reflector mode controlled by FlexControl software. A 337 nm

nitrogen laser with the frequency of 100 Hz was used. The spectra were recorded by summing 200

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laser shots. The negative ion mode was used throughout the experiments. The data processing was

performed with FlexAnalysis 3.0 software.

Determination of enrichment factor

The enrichment factor is defined as the concentration ratio of analyte before and after

enrichment in the sample. To measure the enrichment factor, 0.025 μg of GONR-PEG-Ab probe

was added to 5 mL of CAP solution (1 ng/mL). After enrichment, the probe was isolated by

ultracentrifugation, and the concentration of CAP in the supernatant was measured by HPLC-

MS/MS on a Waters 2695 separation module coupled to a Micromass Quattro Premier XE mass

spectrometry. Then, the CAP concentration in the final extract was calculated. In this way, the true

enrichment factor was obtained. The HPLC-MS/MS conditions are as follows: Dionex Acclaim

RSLC 120 C18 column (100 mm × 2.1 mm, 2.2 μm) at 40 °C; Isocratic elution with a mobile phase

consisting of 75% ACN/25% H2O; Multiple reaction monitoring (MRM) detection: 321.3

→151.3;256.8.

River water and human serum sampling

River water samples were collected from Xiaoling River (Beijing, China). The collected water

samples were filtered through a 0.45 μm Millipore PTFE membrane immediately after sampling

and stored in amber glass bottles at 4 °C. Human serum samples from healthy male athletes were

kindly provided by Dr. Jing Shao from National Institute of Sports Medicine of China. The serum

samples were stored at -21 °C.

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2. Supporting tables

Table S1. Intensities and S/N values of the feature peak of CAP in the Fig. 3.

River water Human serumProbe

Intensity S/N Intensity S/N

GONR-PEG-Ab 116 27 122 35

GONR-Ab 28 7 24 7

GONR-PEG 43 7 25 8

GONR 0 0 45 13

No probe 0 0 0 0

Table S2. Intensities and S/N of the feature peak of CAP in the Fig. S7.

Matrix Intensity S/N

GONR 1045 293

G 782 176

MWCNT 352 99

GO 160 43

CHCA 143 38

Table S3. Intensities and S/N of the feature peak of CAP in the Fig. S9 (left column).

Matrix Intensity S/N

GONR-PEG-Ab 235 53

GONR-Ab 63 20

GONR-PEG 98 23

GONR 51 14

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Table S4. The cross-reactivity (CR, %) of the anti-CAP monoclonal antibody to CAP and its

analogs (n = 3).Compound Chemical structure Cross-reactivity (%)

CAP

O2N C C CH2OH

OH

H NH

H

C CHCl2

O

100

CAP succinateO2N C C CH2O

OH

H NH

H

C CHCl2

O

C CH2CH2

O

C OH

O330

ThiamphenicolSO2 C C CH2OH

OH

H NH

H

C CHCl2

O

H3C< 0.1

FlorfenicolSO2 C C CH2F

OH

H NH

H

C CHCl2

O

H3C< 0.1

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3. Supporting figures

Figure S1. FE-SEM images of MWCNTs (A), GONRs (B), and GONR-PEG-Ab (C). As shown in

B, the MWCNTs can be completely unravelled to form GONRs by chemical oxidation. No

aggregation was observed in GONRs. From C, GONR-PEG-Ab can maintain the geometry of

nanoribbon after functionalization with Ab.

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Figure S2. EDX spectra of MWCNTs (A) and GONRs (B). The spectra were obtained using silicon

plate as a substrate, so a strong Si peak was observed for all the samples. From the EDX results, the

C/O ratios of MWCNTs and GONRs were measured to be 26 and 2.4, respectively, indicating that

GONRs were successfully oxidized.

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Figure S3. TEM images of MWCNTs (A), GONRs (B), GONRs at high magnification (C), and

GONR-PEG-Ab (D). The TEM results are consistent with those of SEM (Figure S1). The B shows

that MWCNTs were cut into short segments and completely unravelled to form GONRs. From D,

GONR-PEG-Ab keeps the morphology of GONR with no aggregation observed. Some amorphous

substances were observed probably due to the attached polymers and proteins.

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Figure S4. UV-Vis absorption spectra of GONRs, GONR-PEG, and GONR-PEG-Ab films. The

films were formed on the surface of a quartz cuvette with a density of 4.65 × 10-3 g/m2. The GONRs

have a strong absorption over a wide wavelength range and the maximum absorption band is at

~264 nm. After modified with PEG and Ab, the absorbance decreases slightly due to the low optical

absorption of PEG. The absorbance at 337 nm (i.e., the wavelength used in MALDI) of GONRs,

GONR-PEG, and GONR-PEG-Ab is 0.58, 0.55, and 0.50, respectively, indicating that GONR-

PEG-Ab can keep good optical absorption capability in MALDI.

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Figure S5. C1s XPS spectra of MWCNTs (a) and GONRs (b). MWCNTs show a dominant C-C

peak at 284.8 eV. Peak fitting of the C1s bands of GONRs yields three main components at 284.8,

287.0, and 288.9 eV assigned to C-C, C-O, and O-C=O bonds, respectively,3 demonstrating the

successful unravelling of MWCNTs by chemical oxidation. These results also indicate that GONRs

possess many carboxyl groups for further modification with NH2-PEG-COOH. The XPS spectrum

of GONR-PEG-Ab was not measured because the amount of the prepared material was limited.

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Figure S6. XRD patterns of MWCNTs and GONRs. MWCNTs show a predominant peak at 26.0°,

corresponding to a d spacing of 3.4 Å. The graphite (002) spacing increases upon the oxidation.3a In

GONRs, the peak at 26.0° is diminished, and a new peak appears at 10.5°, corresponding to a d

spacing of 8.4 Å. The broadening of the peak indicates the disruption of the well-ordered

multilayered structure in MWCNTs. The XRD spectrum of GONR-PEG-Ab was not measured due

to insufficient sample amount.

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Figure S7. Comparison of different materials as MALDI matrices for MALDI-TOF MS

measurement of CAP. The CAP solution (50 μg/mL) was mixed with the matrix, and 2 μL of the

mixture was deposited on the MALDI target for measurement. The molar ratio of analyte to matrix

was 7.7 × 10-3 mol/g. The dual peaks of [M-H]- of CAP were detected at m/Z 320.4 and 322.4

(marked with asterisk). The intensities and S/N of the feature peak of CAP are given in Table S2.

The strongest peak intensity was obtained with GONR, suggesting that GONR is a more efficient

matrix than other materials. The peak intensity decreases in the following order: GONR > G >

MWCNT > GO ≈ CHCA.

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Figure S8. SEM images of different films (GONR-PEG-Ab (A), GONRs (B), G (C), and GO (D))

deposited on the MALDI target. GONR-PEG-Ab, GONRs and GO can form flat and homogenous

films. On the contrary, G sheets aggregate or re-stack to discrete clusters during the film formation

process, and thereby a considerable part of the MALDI target surface cannot be covered.

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Figure S9. Detection of CAP in pure water samples by SELDI-TOF MS using different materials

as probes. The left column was samples spiked with CAP at 1 ng/mL. The right column was

unspiked blank samples. The feature peak of CAP was marked with asterisk. The intensities and

S/N of the feature peak of CAP are given in Table S3. No peaks of CAP could be detected in blank

samples (right column). After spike at 1 ng/mL, CAP could be detected with all the probes (left

column). It should be noted that there is no matrix inference in pure water, so all the MS spectra are

relatively clean. Even so, GONR-PEG-Ab shows the strongest peak intensity among the tested

probes, indicating that GONR-PEG-Ab can effectively capture the target analyte. In fact, the

superiority of GONR-PEG-Ab is more prominent in analysis of complex samples (see Fig. 3 in the

paper).

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