Supplementary Materials

Investigate electrochemical immunosensor of cortisol based on

gold nanoparticles/magnetic functionalized reduced graphene

oxideBolu Suna, Yuqiang Goub, Xiaoping Zhenga, Yuling Maa, Ruibin Baia,

Ahmed Attia Ahmed Abdelmoatya, Fangdi Hua*

a School of Pharmacy, Lanzhou University, Lanzhou 730000, Chinab Lanzhou Military Command Center for Disease Prevention and Control, Lanzhou 730000, China* Corresponding authors. Tel./fax: +86 931 8915686.

Contents1. Reagents and apparatus

2. Preparation of magnetic functionalized reduced graphene oxide (Fe3O4-rGO,

MrGO)

3. SEM characterization of electrode

4. Optimize conditions of depositing AuNPs

5. Optimization of incubation time

6. Cross-reactivity

7. Application of the immunosensor in human serum

1. Reagents and apparatus

Graphite powder (KS-10), concentrated sulfuric acid (H2SO4), potassium

persulfate (K2S2O8), phosphorus pentoxide (P2O5), potassium permanganate (KMnO4),

sodium hydroxide (NaOH), sodium chlorate (NaClO3), Nafion, branched

polyethylenimine 25 K (PEI), Poly(diallyldimethylammonium chloride) (PDDA),

absolute ethanol, hexane, dichloromethane, iron(III) acetylacetonate (Fe(acac)3), oleic

acid, oleylamine, meso-2,3-dimercaptosuccinic acid (DMSA), dimethylsulfoxide

(DMSO), 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide Hydrochloride (EDAC,

>99%), and N-hydroxysuccinnimide (NHS) were purchased from Sigma-Aldrich. o-

Phenylenediamine (o-PD, 99%), H2O2 (30%), chloroauric acid and trisodium citrate

were from Shanghai reagent Co. Inc. (Shanghai, China). Cortisol (Cor) (Ag),

biotinylated cortisol monoclonal antibody (Biotin-Ab), HRP-labeled streptavidin

(HRP–Strept), Cortisol enzyme-linked immunosorbent assay (ELISA) kits, bovine

serum albumin (BSA) and Tween-20 were purchased from Elabscience Biotechnology

Co., Ltd. (Lanzhou, China). The clinical serum samples were kindly provided by the

First Hospital of Lanzhou University (Lanzhou, China). For the cross-reactivity

assays, corticosterone, adrenocorticotropic hormone (ACTH), prolactin, β-estradiol,

testosterone, progesterone, human growth Hormone (hgH), all from Sigma, were

tested. The AuNPs solution used in all experiments were prepared according to the

reported method by boiling HAuC14 aqueous solution with trisodium citrate (Enustun

et al., 1963). The average diameter of the prepared AuNPs was about 20 nm.

Electrochemical measurements were performed on a CHI 6041E workstation

(Shanghai Chenhua, China) with a conventional three electrode system comprised of a

platinum wire auxiliary, a saturated calomel reference and the modified glass carbon

(GCE) working electrode. The process of prepared MrGO was characterized by

atomic force microscope (AFM, Veeco Dimension 3100), X-ray diffraction (XRD),

Fourier transformed infrared (FT-IR, Spectrum One, Pekin-Elmer, USA) and vibrating

sample magnetometer (VSM, Lakeshore 7307, USA). The morphology and structure

of the modified electrode were studied by scanning electron microscopy (SEM, JEOL

JSM-6380LV, Japan). Transmission electron microscopy (TEM) was performed on a

JEOL JEM-1230 (Japan). Ultrasonication was performed using a KQ2200 ultrasonic

cleaner (40 kHz, 100 W, China). The cortisol ELISA reaction was recorded at 450 nm

using Bio-Rad 680 microplate reader.

2. Preparation of magnetic functionalized reduced graphene oxide (Fe3O4-

rGO,

2.1 Preparation of rGO-PEI

Water soluble GO was prepared from graphite according to the modified

Hummers method and its concentration was estimated by calibration curve from the

absorbance at 231 nm in the UV–Vis spectra (Marcano et al., 2010). Then the

carboxyl graphene oxide was prepared by 5 g NaOH and 5 g NaClO3 in 100 mL

aqueous suspension of GO (1 mg/mL) with the aid of ultrasonic agitation for 2 h, and

dialyzed. Subsequently, the 20 mL PEI (0.1 mg/mL) was mixed with 20 mL carboxyl

graphene oxide (1 mg/mL) and 20 mg EDAC, and heated to 60 ºC for 12 h. The color

had changed gradually from brown to black during the heating process, indicating

reduction of GO to reduced GO (rGO) during heating with PEI and obtaining the

reduced graphene oxide modified with PEI (Yu et al., 2009).

2.2 Preparation of DMSA-Fe3O4

The Fe3O4 NPs modified with meso-2, 3-dimercaptosuccinic acid (DMSA-Fe3O4)

was prepared according to the following steps. Initially, 20 mL of octylamine and 10

mL of phenyl ether were added to a three-necked flask, then the 3 mM Fe(acac)3 was

injected into mixture at 110 ºC for 2 h, and heated to reflux (280 ºC) for 1 h. This

solution was cooled to room temperature. The resulting solution was washed using

excess ethanol and separated by centrifugation. The black product was redissolved in

cyclohexane with 0.05 mL oleic acid and 0.05 mL oleylamine. The undissolved

residue of the solution was removed by centrifugation (8000 rpm, 30 min) obtaining

the Fe3O4 nanoparticle (Zhang et al., 2011). Subsequently, 20 mg of the prepared

Fe3O4 NPs was dissolved in 2 mL toluene, then 20 mg DMSA and 2 mL DMSO were

added to the solution and stirred at 25 ºC for 12 h. This precipitant was washed with

ethyl acetate for three times and separated using a permanent magnet bar. Finally, the

DMSA-Fe3O4 NPs were collected and transferred into 2 mL water.

2.3 Synthesis of Fe3O4-rGO composites.

The 10 mL aqueous solution of DMSA-Fe3O4 NPs (~0.8 mg/mL) blended with

30 mg of EDAC, 30 mg of NHS, and 15 mL of PEI-rGO (~0.3 mg/mL) and stirred at

room temperature for 48 h. Then, the raw product was centrifuged and washed 3 times

with milli Q water. Meso-2,3-dimercaptosuccinnic acid (DMSA)-modified Fe3O4 NPs

and PEI-rGO can be combined via formation of amide bonds between COOH groups

of DMSA molecules bound on the surface of the Fe3O4 NPs and amine groups of PEI.

The Fe3O4-GO composites were obtained after centrifugation and through washing

with ultrapure water.

3. SEM characterization of electrode

The morphologies of Nafion@GCE (A), PDDA-G/Nafion@GCE (B) and

MrGO/Nafion@GCE (C) were investigated by using scanning electron microscopy

(SEM). Compared with Nafion@GCE, the surface of MrGO/Nafion@GCE was

scattered a great number of small particles, while PDDA-G/Nafion@GCE showed the

undulating morphology of PDDA-G. As shown in Fig. S1C, the MrGO was obviously

modified on the electrode. The rugged morphology of MrGO was beneficial to

increase the specific surface area of electrode, which not only provided a good

condition for large electrochemical deposition of AuNPs, but also increased the

capacity of cortisol on the surface of modified electrode.

Fig. S1 SEM images of Nafion@GCE (A), PDDA-G/Nafion@GCE (B) and

MrGO/Nafion@GCE (C) with the magnifying power of 20000× (a) and 10000× (b).

4. Optimize conditions of depositing AuNPs

The conditions of depositing AuNPs were optimized by cyclic voltammetry

(CV). The AuNPs were deposited on the MrGO/Nafion@GCE at -0.2 V for different

time in the 5 mL HAuCl4 solution with different concentration. As shown in Fig. S2,

when the MrGO/Nafion@GCE electrode was incubated for 30 s in 5 mL HAuCl4

solution (0.05 g/L) at -0.2 V by electrochemical deposition, the △DP presented the

maximum. Therefore, the AuNPs were deposited on the MrGO/Nafion@GCE at -0.2

V for 30 s in the 5 mL HAuCl4 solution (0.05 g/L) by electrochemical deposition.

△DP was calculated according to the following equation:

(△DP=（Ip0－Ip′）/Ip0×100%)

Where Ip0 is the CV reduction peak current of MrGO/Nafion@GCE before modified

AuNPs, and Ip′ is the CV reduction peak current of MrGO/Nafion@GCE after

modified AuNPs.

Fig. S2 Dependence of electric deposition time and concentration of HAuCl4 on △P (a. 0.02

g/L HAuCl4; b. 0.05 g/L HAuCl4; c. 0.1 g/L HAuCl4; d. 0.2 g/L HAuCl4; e. 0.5 g/L HAuCl4; f.

1.0 g/L HAuCl4).

5. Optimization of incubation time

In this work, the method of incubation was used to modify Cor on the surface of

AuNPs/MrGO/Nafion@GCE, and the effect of incubation time on immunoreaction

has been studied by DPV. The AuNPs/MrGO/Nafion@GCE were initially dipped into

50 μL cortisol solution (0.1 mg/mL at pH 7.4) for different time at 4 ºC. Next, they

were rinsed with PBST to remove un-immobilized cortisol and blocked with 50μL 2%

BSA solution containing 0.05% Tween for 50 min at 37 ◦C to block possible

remaining active sites against non-specific adsorption. Subsequently, 50 μL of a

mixture of the appropriate standard cortisol solution (or the sample) and 500 ng/mL

Biotin-Ag were placed onto the basic electrode surface and incubated for 50 min at 37 ◦C. 50 μL of 500 ng/mL HRP-Strept was added and reacted for 50 min, then washed

thoroughly with PBST to remove non-specifically bounded Biotin-Ab and HRP-

Strept, resulting the electrochemical immunosensor successfully fabricated. After that,

the immunosensor was deposited into 5.0 mL of pH 7.0 PBS involving 2.0 mM o-PD

and 4.0 mM H2O2 solution and allowed to incubate for 8 min. As shown in Fig. S3,

the peak current increased rapidly for the first 10 h (overnight) with the increasing

incubation time of AuNPs/MrGO/Nafion@GCE, then tended to level off. It indicated

that the amount of cortisol (Cor) adsorbed on the electrode surface to be maximized.

To ensure the promotion of work efficiency, 12 h (overnight) was determined as the

optimum incubation time to be used in the experiments.

Fig. S3 The effect of incubation time on immunoreaction.

6. Cross-reactivity

To evaluate the selectivity of the proposed immunosensor for cortisol, various

potential interfering species were examined in connection with their effect on the

determination of cortisol. Some species commonly existing in biological samples

were chosen for a fixed cortisol concentration of 10 ng/mL, and the results are shown

in Table. S1.

Table. S1 Interference of different species to the DPV determination of cortisol with the

proposed immunosensor.

Interferents Interferents concentration/ng mL-1 Recovery (%)

corticosterone 100 104.68

Adrenocorticotropic

Hormone (ACTH)100 102.52

Prolactin 100 96.73

β-Estradiol 100 97.38

Testosterone 100 98.70

Progesterone 100 99.36

human growth hormone

(hgH)100 99.73

8. Application of the immunosensor in human serum

Table. S2 shows the correlation results obtained using the proposed

immunosensor and the ELISA method. The relative standard deviations of the

proposed immunosensor for determination ranged from 3.89% to 5.41% and the

recovery rates of the samples ranged between 97.89% and 103.36%.

Table. S2 Comparison of serum cortisol levels determined using two methods.

SampleMeasuerd

(ng mL-1)a

Add/

ng mL-1

ELISA This work

Found

(ng mL-1)aRSD(%)

Recover

y（%）Found

(ng mL-1)aRSD(%)

Recover

y（%）1 76.3 10 91.3 6.78 105.79 89.2 5.41 103.36

2 77.8 50 131.6 5.31 102.97 128.6 4.57 100.16

3 75.6 100 163.9 4.92 93.33 171.9 3.89 97.89

a The average value of three successive determinations.

As shown in Fig. S4, the proposed method showed good precision, acceptable

stability and reproducibility, when it was compared with the commercially available

enzyme-linked immunoadaordent assay. Therefore, the developed design exhibits an

excellent analytical performance in terms of sensitivity, selectivity, wide range of

quantifiable antigen concentrations, indicating the immunosensor could be used for

the sensitive, efficient and real-time detection of cortisol in real samples.

Fig. S4. The comparison of ELISA and electrochemical immunosensor.

References

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