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Electrophoretic approach for the simultaneous deposition and functionalization of reduced graphene oxide nanosheets with diazonium compounds: application for sensing in serum Qian Wang, Alina Mihaela Vasilescu, Qi WANG, Yannick Coffinier, Musen Li, Rabah Boukherroub, and Sabine Szunerits ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15955 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 24, 2017

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ACS Applied Materials & Interfaces

Electrophoretic Approach for the Simultaneous Deposition and Functionalization of Reduced Graphene Oxide Nanosheets with Diazonium Compounds: Application for Sensing in Serum Qian Wang,1,2 Alina Vasilescu,3 Qi Wang,2 Yannick Coffinier,1 Musen Li,2 Rabah Boukherroub1 and Sabine Szunerits1* 1

Univ. Lille, CNRS, Centrale Lille, ISEN, Univ. Valenciennes, UMR 8520 - IEMN, F-59000 Lille, France 2

Key Laboratory for Liquid-Solid Structural Evolution and Processing of Materials (Ministry of Education), Shandong University, Jinan 250061, China

3

International Center of Biodynamics, 1B Intrarea Portocalelor, Sector 6, 060101, Bucharest, Romania

Abstract Electrophoretic deposition (EPD) of reduced graphene oxide nanosheets (rGO) offers several advantages over other surface coating approaches, including process simplicity, uniformity of the deposited films and good control of the film thickness. The EPD conditions might also be of interest for the reduction of diazonium salts, which upon the release of N2 molecules and generation of radicals, can form covalent bonds with the sp2 hybridized carbon lattice atoms of rGO films. In this work, we report on the coating of gold electrodes in one–step with rGO/polyethyleneimine (PEI) thin films and their simultaneous modification using different phenyl (Ph) diazonium salt precursors bearing various functionalities such as –B(OH)2, COOH and –C≡CH. We show further the interest of such interfaces for designing highly sensitive sensing platforms. Azide-terminated lysozyme aptamers were clicked onto the rGO/PEI/Ph-alkynyl matrix and used for the sensing of lysozyme levels in patients suffering *

To whom correspondence should be addressed. Sabine Szunerits ([email protected]; Tel: +33 3 62 53 17 25; Fax: +33 3 62 53 17 01)

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from Inflammatory Bowel Disease (IBD), where lysozyme levels are up-regulated. The approach attained the required demand for the determination of lysozyme level in patients suffering from IBD with a 200 femtomolar detection limit and a linear range up to 20 picomolar without signal amplification.

Keywords: reduced graphene oxide, electrophoretic deposition, diazonium salts, lysozyme sensing, serum samples


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1. Introduction Reduced graphene oxide (rGO) modified interfaces have found widespread interest in recent years in electrochemical sensors and biosensors.1-8 The conductive nature of rGO together with its large surface area and presence of oxygen containing groups have been postulated to be responsible for enhanced heterogeneous electron transfer rates and excellent electrochemical properties of rGO coated electrodes.9-10 The abundant oxygen containing groups, especially carboxylic acid functions, provide furthermore a suitable platform for the immobilization of biomolecules. To take full advantage of the properties of rGO for sensing, the controlled and reproducible deposition of rGO onto electrical interfaces is required. The fabrication of rGO coated electrodes employs mainly solution-based deposition methods such as dip coating, layer-by-layer assembly, spin coating, drop casting or spray coating.11 However, the electrochemical behavior of these electrodes is representative of the electrochemical behavior from numerous graphene fragments with ill-defined coverage, layer numbers and orientation. Such interfaces are also often graphite-like, as the number of graphene sheets often exceeds 100 sheets. Reducing the thickness of rGO coating in a controlled manner through electrophoretic deposition (EPD) was shown to be highly beneficial for the construction of electrochemical sensors.4, 12-13 EPD of rGO offers several advantages over other surface coating approaches, such as process simplicity, uniformity of the deposited films and good control of the film thickness.4, 14-21 Wu et al. pioneered the EPD process of rGO films showing that Mg2+ charged GO can be deposited on a cathode under applied voltage at 100-160 V.22 Ruoff and coworkers demonstrated that uniform, thickness adjustable graphene films can be obtained by controlled EPD of hydrazine or KOH modified GO.15 We have developed more recently a sensitive and stable non-enzymatic platform for D-glucose sensing based on a electrophoretically deposited reduced graphene oxide (rGO) matrix modified with Ni(OH)2 or Cu nanostructures.4,

12-13

Indeed, metal ions are commonly used as additives to produce

positively charged GO suspensions allowing cathodic EPD with simultaneous reduction of GO. One of the materials having received increased attention is polyethyleneimine (PEI) modified graphene.17, 23-25 Its high amine density of primary amines situated at the chain ends and their accessibility allow PEI to be covalently linked to GO and rGO using carbodiimide cross-linking reaction between the -COOH groups of GO and the -NH2 groups of PEI. PEI



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polymers can also interact with the oxygenated groups of GO nanosheets via electrostatic interactions, resulting in positively charged nanosheets.

Here we present a one-step production of thin-film electrodes that can be suitably modified with various chemical functionalities based on cathodic electrophoretic deposition (EPD) (Figure 1A). It is shown that the EPD process of GO/PEI in the presence of diazonium salt precursors results in the coating of electrodes with functional rGO/PEI/Ph-R (R=carboxylic acid, boronic acid or alkynyl functions) films (Figure 1B). To further underline the interest of this surface modification procedure for the construction of a biosensing platform, rGO/PEI/Ph-alkynyl modified electrodes were post-functionalized by “clicking” azide-terminated aptamers to rGO/PEI/Ph-alkynyl modified electrodes. We opted for showing the potential of rGO/PEI/Ph-aptamer electrodes for analyzing the serum lysozyme of inflammatory bowel disease (IBD) infected people, where the level of lysozyme is up-regulated. Indeed, lysozyme has emerged as one of the preferred analytical targets, very often investigated to prove new aptasensing concepts, particularly with electrochemical detection.26 In aptasensors, as the one described here, efficient immobilization of the aptamer is paramount to achieve sensitive and selective detection. Various immobilization strategies have been explored in lysozyme aptasensors to ensure appropriate stability, surface coverage, while preserving the affinity of immobilized aptamer: adsorption or π-π stacking interactions between the DNA bases of the lysozyme aptamer and graphene oxide-modified interfaces, covalent coupling of amine-ended aptamer to carboxylated surfaces, chemisorption of thiolated aptamers on Au, click chemistry between azide-modified gold particles and alkyneterminated aptamers, affinity binding of biotynilated aptamers on avidin-modified surfaces, electrostatic interactions between the negatively-charged phosphate backbone of the aptamer and positively-charged interfaces or hybridization of the aptamer with a partially complementary DNA strand, immobilized on the electrode surface.26 Among these approaches, the usefulness of generic supports such as a “clickable” interface has been underlined and demonstrated for an aptasensor array.27 Nevertheless, highly sensitive biosensing schemes for electrochemical detection of lysozyme in serum were described so far only in a few studies and are mostly connected with signal amplification strategies to achieve the required sensitivity.25, 28-33 Human lysozyme is found in tissues and body fluids and the level is increased in ill patients. Serum lysozyme 4 ACS Paragon Plus Environment

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levels of healthy people are in the range of 0.462–2.958 mg/L,34-35 while increased concentrations, associated with the expression of monocyte/macrophage activity were found in a number of diseases, including AIDS36, pulmonary tuberculosis37 and Crohn’s disease.38 The heightened levels of lysozyme in IBD (Crohn’s disease and ulcerative colitis) add to changes of other biomarkers (e.g. C-reactive protein, serological antibodies such as atypical perinuclear antineutrophil cytoplasmic antibodies (pANCAs), anti-Saccharomyces cerevisiae antibodies (ASCA), erythrocyte sedimentation rate and fecal calprotectin, to improve the diagnostic of this disease.39 We will show that electrophoretically formed rGO/PEI/Ph-aptamer electrodes allow direct lysozyme detection using differential pulse voltammetry in the femtomolar range with a linear range up to picomolar concentrations. The simplified electrode construction, especially when compared to the recently reported lysozyme sensor using nitrogen-doped vertically aligned carbon nanotube electrodes,30 makes the proposed approach highly appealing.

2. Experimental 2.1. Materials Graphite powder (< 20 micron), potassium permanganate (KMnO4), sulphuric acid (H2SO4), phosphoric acid (H3PO4), hydrogen peroxide (H2O2), sodium nitrite (NaNO2), 4ethynylaniline,

4-aminobenzoic

polyethyleneimine

(PEI),

acid,

copper

4-aminophenylboronic

sulphate

(CuSO4),

sodium

acid

hydrochloride,

ascorbate,

tris(3-

hydroxypropyltriazolylmethyl)amine (THPTA), phosphate buffer (PBS, 0.01 M, pH=7.4), potassium ferrocyanide ([K4Fe(CN)6]), bovine serum albumin (BSA) and lysozyme were purchased from Sigma-Aldrich and used as received. The aqueous solutions used in the experiments were prepared using Milli-Q water. Azide-terminated lysozyme aptamers were purchased from Eurogentec (France): 5’-N3-TTT TTT TTT TTT GGG AAT GCA TCC ACA TCT ACG AAT TCA TCA GGG CTA AAG AG-3’. Human samples were provided by Dr. Ionescu Andra.30

2.2. Electrophoretic deposition (EPD)



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Electrophoretic deposition (EPD) was performed on Au coated glass substrates prepared by thermal evaporation of 5 nm of titanium and 50 nm of gold onto cleaned glass slides (76×26×1 mm3, CML France). The EPD was carried out in a two-electrode cell, where the two electrodes are placed parallel to each other and are separated by a distance of 1 cm. A platinum foil (1x2 cm2) acts as the anode and the glass/Ti/Au substrate as the cathode. The diazonium precursors used here were 4-ethynylaniline, 4-aminobenzoic acid and 4aminophenylboronic acid hydrochloride. For the formation of rGO/PEI/Ph-R, first the diazonium salts were formed by stirring the mixture of diazonium reagents (15 mM, 0.5 mL), NaNO2 (15 mM, 0.5 mL) and HCl (0.75 M, 0.5 mL) at 0 °C for 5 min. To this solution GO (0.5 mg mL-1, 2 mL) and PEI (2 mg mL-1, 0.5 mL) were added and a DC voltage of 15 V was applied for 2 min. After deposition, the interfaces were rinsed with deionized water (three times) followed by blow drying with nitrogen. Similarly, a rGO/PEI interface and a gold/PEI/4-ethynylaniline was formed by EPD under similar conditions. For the modification of gold thin films electrodes with 4ethynylaniline, after the diazonium salts formation, the gold electrode was biased at -0.2 V for 5 min, rinsed with water (3x) followed by blow drying with nitrogen.

2.3. Formation of lysozyme sensor (rGO/PEI/Ph-aptamer) Immobilization of the azide-terminated aptamer probe is achieved by “click” chemistry.27 The rGO/PEI/Ph-alkynyl matrix was immersed into a solution containing the azide-terminated aptamer (1 µM in 0.02 M Tris buffer), CuSO4 (1 mM), sodium ascorbate (1 mM) and THPTA (2 mM) at 4 °C overnight. After washing with PBS, the electrode was incubated with BSA (5 wt.%) for 1 h to block non-specific adsorption. After removing excess BSA by rinsing in Tris buffer, the interface was kept in Tris buffer at 4°C for further sensing application.

2.4. Instrumentation Scanning electron microscopic images were obtained on a Nova NanoSEM (FEI, USA) 450 SEM.30 An optical profilometer (Zygo NewView 6000 Optical Profilometer with MetroPro software) with 1 nm height resolution as used for thickness measurements.


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The zeta potential was measured using the electrophoretic mode with the Zetasizer® Nano ZS (Malvern Instruments S.A., Worcestershire, UK). X-ray photoelectron spectroscopy (XPS) experiments were performed in a PHl 5000 VersaProbe - Scanning ESCA Microprobe (ULVAC-PHI, Japan/USA) instrument. 30 Micro-Raman spectroscopy measurements were performed on a LabRam HR Micro-Raman system (Horiba Jobin Yvon, France) combined with a 473-nm laser diode as excitation source. 30 An Autolab potentiostat 30 (Eco-Chemie) was used for cyclic voltammetry (CV) measurements. A platinum mesh and Ag/AgCl electrode were used as a counter and a reference electrode, respectively. The scan rate was 50 mV s-1 and the active surface area was 0.04 cm2. Lysozyme detection was achieved by immersion of the rGO/PEI/Ph-aptamer in lysozyme solutions in PBS (0.01 M, pH=7.4) for half an hour as described previously.30

Spectrophotpmetric measurements of lysozyme’s enzymatic activity were performed on a Evolution 600 UV −vis spectrophotometer (Thermo Scientiﬁc, 220France).

2.5. Determination of lysozyme concentration in human serum based on its enzymatic activity The determination of lysozyme in the human serum sample was based on its enzymatic activity using a classic turbidimetric assay40 with small modifications. In short, Micrococcus lysodeykticus (0.1 mg/mL; 480 µL) in PBS (50 mM, pH=7.0) was incubated with either 20 µL of undiluted serum or lysozyme standard solution and the decrease in absorbance at 450 nm was monitored over time. The rate of absorbance decrease at 450 nm was plotted against the concentration of lysozyme to build a calibration curve. The concentration of lysozyme in the human serum samples was deduced from the calibration curve and expressed based on hen egg lysozyme.

3. Results and discussion 3.1. Electrophoretic deposition of rGO/PEI thin films on gold electrodes Aqueous suspensions of GO have a negative zeta potential of ζ = -41.3±0.8 mV, and the 7 ACS Paragon Plus Environment


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GO platelets migrate towards the anode upon application of a DC voltage.15 For the reduction of the different diazonium salts under cathodic EPD, the formation of positively charged GO is necessary. This can be achieved by charging GO with metallic ions such as Ni2+ or Cu2+ ions4,

13

or through the interaction with polyethyleneimine (PEI), a cationic polymer with

repeated amine groups. A solution with equal weight amounts of chemically derived GO and PEI, GO/PEI=1, results in GO/PEI nanosheets with ζ = +20.3±1.7 mV (Table 1).

Table 1: Zeta potential of different GO/PEI solutions. Matrix GO GO/PEI GO/PEI/Ph-COOH GO/PEI/Ph-B(OH)2 GO/PEI/Ph-alkynyl

Weight ratio GO/PEI (mg mL-1) 0.5 /0.0 0.5/0.5 0.5/5 -1 0.25 mg mL /0.25 mg mL-1/1.875 mM -1 0.25 mg mL /0.25 mg mL-1/1.875 mM -1 0.25 mg mL /0.25 mg mL-1/1.875 mM

Zeta potential / mV -41.3±0.8 +20.3±1.7 +36.4±1.3 +9.9±0.3 +13.1±1.1 +11.3±0.6

A ten times higher PEI amount (GO/PEI=0.1) results in ζ = +36.4±1.3 mV and was used in the following for EPD. Figure 1A shows the change in coating density of a gold electodes with time. Upon the application of +15 VDC for 30 s, the gold electrode is coated with only some rGO/PEI nanoplatelets. The coating is more dense after 1 min and a homogenous film of about 1 nm, as determined by profilometry measurements, is formed after 2 min EPD. Longer deposition times did not result in a further significant increase of the film thickness in contrast to EPD of rGO.4 Figure 1B displays the EDX mapping spectra of the rGO/PEI film deposited at 15 V for 2 min. It becomes clear that rGO and PEI are homogeneously distributed in the deposited matrix.

(A)


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(B)

Figure 1: (A) SEM images of Au electrodes coated with rGO/PEI nanoflakes upon EPD at 15 VDC for different times; (B) EDX mapping of C (green), O (blue) and N (red) of a Au electrode coated with rGO/PEI by EPD at 15 VDC for 2 min. The Raman spectrum of the rGO/PEI matrix formed at 15 VDC for 2 min (Figure S1A) exhibits the characteristic D-band (1352 cm-1) and G-band (1592 cm-1) corresponding to sp3 carbon atoms of the defect structures and sp2 hybridized carbon from the aromatic structure. The ID/IG ratio was determined to be 0.63, smaller than that of the initial used GO (ID/IG = 1.19). It is also smaller than that of electrophoretically deposited rGO on gold with a ID/IG = 0.83.16 The smaller ID/IG peak intensity ratio of rGO/PEI corresponds to lower defect/disorder in the graphitized structure due to an improved reduction of rGO/PEI. XPS was in addition used to analyse the chemical composition of electrophoretically deposited rGO/PEI films (Table 2). The XPS survey spectrum shows peaks at binding energies of 285, 532 and 400 eV corresponding to the C1s (77.9 at.%), O1s (14.4 at.%) and N1s (5.7 at.%) respectively. The amount of PEI integrated into the rGO nanosheets is lower when compared to other PEI-loaded GO or rGO nanoformulations in solution with up to 17 at.% of N1s.41-42 The high resolution N1s spectrum reveals the presence of the PEI amine groups at 399.5 eV (Figure S1B). The conversion of GO to rGO is indicated in the C1s core level XPS spectrum of rGO-PEI, which displays the characteristic bands of rGO at 284.6 eV (sp2hybridized carbon), together with bands at 285.0 eV (sp3-hybridized carbon/C-H), 286.2 eV (C-O/C-N), 288.2 eV (C=O) and 291.2 eV (O-C=O) (Figure S1C).



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3.2. Formation of rGO/PEI functionalized with diazonium salts (rGO/PEI/Ph-R) via electrophoretic conditions Diazonium-based chemistry is a widely accepted surface modification approach for graphene based materials.43 Under reductive conditions, release of N2 from the diazonium salt results in the formation of radicals, which add to the sp2 hybridized carbon lattice atoms on the surface of rGO through covalent bond formation. We were interested if cathodic EPD from a mixture of GO/PEI in the presence of diazonium salts would result in the coating of gold electrodes with rGO/PEI thin films where rGO was covalently modified with the respective diazonium salt derivative (Figure 2). The grafting behavior of three p-substitured phenyl compounds, 4-aminobenzoic acid (I), 4-aminophenylboronic acid (II) and 4ethynylaniline (III) on rGO/PEI films was investigated by mixing GO/PEI (1/1 ratio, 0.5 mg mL-1) with the corresponding aryl diazonium salt precursor (1.8 mM). The zeta potential values of the GO/PEI/diazonium salt mixtures dropped but remained positive (Table 1). Application of a DC potential of 15 V for 2 min in the presence of NaNO2/HCl results in the in situ formation of diazonium salts, elimination of nitrogen followed by electrophoretic reduction of the diazonium salt to aryl radicals and formation of a covalent bond with rGO

Figure 2:Schematic illustration of the one-step production of thin-film electodes that can be suitably modified with various chemical functionalities.

The successful covalent grafting was validated by XPS analysis (Figure 3). The atomic percentages of each element are seen in the Table S1. In the case of 4-aminobenzoic acid (I) as the diazonium precursor, the C1s high resolution spectrum was deconvoluted into five 10 ACS Paragon Plus Environment

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bands at 284.6 eV (sp2-hybridized carbon), 285.0 eV (sp3-hybridized carbon/C-H), 286.2 eV (C-O/C-N), 288.2 eV (C=O) and 291.2 eV (O-C=O) (Figure 3A). When compared to rGO/PEI (Figure S1C), the band at 291.2 eV (O-C=O) presents 5.1 at.%, while in the case of rGO/PEI only 1.2 at.% were detected, indicating the integration of phenylcarboxylic acid functions onto the rGO/PEI matrix. The N1s high resolution spectrum of rGO/PEI/Ph-COOH shows bands at 399.5 eV (NH2 groups of PEI) and 401.1 eV, attributed to the formation of azobenzoic acid multilayers (-N=N-) in the film, advocated as one of the possible side reactions in diazonium based coupling reactions (Figure 3A).44-45 The absence of a band at 402.0 eV due to the -N2 group of the diazonium salt is a further indication of the sucessful incorporation of the ligand onto the rGO nanosheets. Most indicative that covalent linking took place is that the overall nitrogen content in the presence and absence of 4-aminobenzoic acid (I) stayed unaltered at 5.7 at.%, pointing towards covalent coupling rather than π−π stacking of 4-aminobenzoic acid onto rGO/PEI. Comparable results are obtained using the other two diazonium salt precursors, 4-aminophenylboronic acid (II) and 4-ethynylaniline (III) (Figures 3B, C). EPD of a mixture of GO/PEI/4-aminophenylboronic acid (II) resulted in a matrix containing 4.3 at.% of B1s (Table 2). Figure 3B depicts the B1s high resolution XPS spectrum with a band at 191.1 eV corresponding to -C-B(OH)2 bond in accordance with the chemical composition of 4-aminophenylboronic acid. Interestingly, in the case of 4aminophenylboronic acid (II), no additional band at 401.1 eV, is observed in the N1s XPS spectrum, suggesting the absence of

-N=N- functions in the film.The C1s high resolution

spectrum of rGO/PEI modified using 4-ethynylaniline (III) as diazonium salt precursor was deconvoluted into bands at 284.6 eV (sp2-hybridized carbon), 285.0 eV (sp3-hybridized carbon/C-H), 286.2 eV (C-O/C-N), 288.2 eV(C=O) and 291.2 eV (O-C=O) (Figure 3A). The band at 285.0 eV (C-C/C-H) is more dominant as expected for the integration of alkynyl units (Figure 3C). (A)



rGO/PEI/Ph-COOH

rGO/PEI/Ph-COOH

C

N

1s

Intensity / a.u.

Intensity / a.u.

1s

294 292 290 288 286 284 282 Binding energy / eV

404 400 396 Binding energy / eV

(B) 2

B

rGO/PEI/Ph-B(OH)

2

1s

N

1s

Intensity / a.u.

Intensity / a. u.

rGO/PEI/Ph-B(OH)

193


192 191 190 Binding energy / eV

(C) rGO/PEI/Ph-alkynyl

C

rGO/PEI/Ph-alkynyl

1s

N

1s

Intensity / a.u.

Intensity / a.u.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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406

404 402 400 398 Binding energy / eV

396

Figure 3: Characterization of Au electrodes coated electrophoretically with rGO/PEI/Ph-R nanosheets: (A) C1s and N1s high resolution core level spectra of rGO/PEI/Ph-COOH films formed in the presence of 4-aminobenzoic acid; (B) B1s and N1s high resolution core level spectra of rGO/PEI/Ph-B(OH)2 films formed in the presence of 4- aminophenylboronic acid


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(C) C1s and N1s high resolution core level spectra of rGO/PEI/Ph-alkynyl films formed in the presence of 4-ethynylaniline. The above presented results for three types of diazonium salt precursors, largely used as sensor functionalization units, support the versatility of EPD for one step surface functionalization of electrical interfaces with rGO/PEI/Ph-R. Considering the advantages of “click” chemistry for the integration of surface ligands, the rGO/PEI/Ph- alkynyl interface was selected in future studies.

3.3. Formation of lysozyme sensor using rGO/PEI/Ph-alkynyl modified electrodes The presence of surface functions on electrodes is essential for the construction of sensors allowing for the immobilization of analyte specific ligand. Motivated by the wide interest in protein analysis, we opted for lysozyme sensing as model system.26 The majority of electrochemical aptasensors for lysozyme used thiolated aptamers on gold electrodes as linkers.26 The rGO/PEI/Ph-alkynyl surface allows for “click” chemistry to be used

for

27

aptamer attachment (Figure 4A) as shown recently by Xie et al. The success of the surface modification was confirmed by an increase in the N1s component in the XPS survey spectrum (Table 2). The high resolution N1s XPS spectrum of rGO/PEI/Ph-aptamer surface (Figure 4B) reveals in addition the presence of nitrogen components related to triazole ring at 402.2 eV (-C-N-) and 401.2 eV (-N=N- of triazole and of film itself) with an additional band at 399.5 eV (PEI component). The electrochemical behavior of the rGO/PEI/Ph-aptamer electrode was determined by cyclic voltammetry using [Fe(CN)6]4-/3- as redox couple (Figure 4C). Fast heterogeneous electron transfer is observed on rGO/PEI coated electrodes due to possible electrostatic attraction between the negatively charged redox probe and the positively charged rGO/PEI interface. The rGO/PEI/Ph-alkynyl modified interface shows an almost complete blocking of the electron transfer linked probably to the less permeable phenyl layer as reported by Belanger and co-workers.45 Clicking of the aptamer carrying negatively charged phosphate backbones does not alter the electrochemical behavior of the rGO/PEI/Ph-aptamer interface.

(A)



N N N HOOC

HOOC

COOH

COOH N3

N3-lysozyme aptamer Cu(I) rGO/PEI/Ph-alkynyl

rGO/PEI/Ph-aptamer

(B)

(C) N

rGO/PEI/Ph-aptamer

300

1s

200

Intensity / a.u.

rGO/PEI rGO/PEI/Ph-alkynyl rGO/PEI/Ph-aptamer

100 i / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 -100 -200

406

404 402 400 398 Binding energy / eV

396

-300 -0,2

0

0,2

0,4

0,6

E /V vs. Ag/AgCl

Figure 4: (A) Schematic illustration of the lysozyme sensor; (B) N1s high resolution core level spectrum of rGO/PEI/Ph-aptamer; (C) CV of rGO/PEI (black), rGO/PEI/Ph-alkynyl (green) and rGO/PEI/Ph-aptamer (blue) in [Fe(CN)6]4-/3- (5 mM)/ PBS (10 mM); scan rate: 50 mVs-1.

3.4. Lysozyme sensing Figure 5A shows the change in differential pulse voltammetry (DPV) signals upon increase in lysozyme concentrations. Higher lysozyme concentrations result in a recovery of the redox current as upon lysozyme binding, the aptamer experiences conformational changes, screening the negative charges of the phosphate backbone in DNA.46,47 The negatively charged [Fe(CN)6]4-/3- might interact now more freely with the positive charges of rGO/PEI matrix, resulting in an overall increase of the redox current due to increased electrostatic interactions upon lysozyme addition. A linear relationship up to 30 pM according to i (µA)=0.160+0.092×[lysozyme] (pM) is obtained (Figure 5B) with a detection limit of ≈200 14 ACS Paragon Plus Environment

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fM from five blank noise signals (95% confidence level). Healthy people have a serum lysozyme level between 27-301 nM. IBD patients can have lysozyme levels of up 1.4 µM.35. The performance of the sensor also compares favorably to other electrochemical based lysozyme sensing platforms (Table 3). While low fM detection limits were reported on other electrical interfaces, these approaches need sophisticated amplification strategies. The one step formulation of the rGO/PEI/Ph-aptamer sensor is in addition an advantage for large scale production. For impedance based lysozyme aptasensor,

32

with a detection limit of 6 fM, the

construction of the sensing interface is lengthy. It furthermore shows a very short linear range of up to 500 fM only.32 The simple fabrication step which can be performed without any special equipment in any laboratory settings makes this sensor also competitive to the recently reported VA-NCNTs-aptamer with a 100 fM detection limit.

30

Compared to the previously

reported direct lysozyme sensor using vertically aligned N-doped carbon nanotubes (VANCNTs), 30 where response saturation occurred at 20 pM, the wider linear range is likely due to the presence of more binding sites in rGO-PEI/Ph-aptamer interface. We finally compared the performance of the rGO/PEI/Ph-aptamer sensor with that of a gold electrode modified with PEI/Ph-aptamer without rGO present (Figure S2A). A linear relationship up to 15 pM (R=0.9995) according to i(µA)=0.056+0.065×[lysozyme] (pM) is obtained (Figure S1B). The decreased linear range might be due to the smaller amount of lysozyme linked to the surface (Table S2) as indicated in the lower atomic percentage of N1s detected. The detection limit of lysozyme was determined to be ≈1 pM from five blank noise signals (95% confidence level).

Table 3: Characteristics of different lysozyme sensors Approach Electroluminescence DPV

Electrode Au electrode-aptamer rGO/PEI/Ph-aptamer

LOD 120 pM 200 fM

Linear range 64 pM–0.64 µM 0.2-30 pM

Square wave voltammetry DPV

Au NCs/GCE-aptamer

100 fM

100 fM–10 nM

1 pM

1-15 pM

100 fM

1 pM–1 nM

this work 33

100 fM 6 fM 4.3 fM

100 fM-7 pM 10 fM–500 fM 5 fM–5 nM

30 32 49

Cyclic voltammetry DPV Impedance DPV

Au/Ph-aptamer Au with thiocyanuric acid /AuNPs-aptamer VA-NCNTs-aptamer GCE/rGO-aptamer Carbon electrode-

Ref. 48 this work 29



aptamer-antibody sandwich GCE: glassy carbon electrode, Au NCs: tetrahexahedral Au nanocrystals; rGO: reduced graphene oxide; VA-NCNTs: vertically aligned nitrogen-doped carbon nanotubes

To illustrate the selectivity of the sensor for lysozyme, the electrochemical response of other proteins such as bovin serum albumin (BSA), cytochrome C and casein at ten times higher concentrations than that of lysozyme was tested. The change in currents were insignificant when compared to lysozyme (Figure 5C).

The reproducibility of the rGO-PEI/Ph-aptamer electrode, was 3.3% (lysozyme=10 pM; n=5). The long-term stability of the rGO-PEI/Ph-aptamer sensor when stored in PBS showed a loss of 2.1% after storage at 4 °C for 2 weeks. Storage in water shows however a loss of 4.3% in the anodic peak current after a week as well as storage at room temperature. Increasing the solution PBS pH to 8 as well as lowering it to 6 but keeping storage at 4 °C, showed comparable stability to storage at pH 7 indicating that the system is rather pH robust. Furthermore, we determined for how many cycles a single rGO-PEI/Ph-aptamer sensor can be used for accurate lysozyme detection. As can be seen from Figure 5D, using a 10 pM lysozyme solution more than 50 cycles could be run with one single electrode. (A)

(B)

5

2 1

4 3

0,3 0,25

2

i / µA

3

5

i / µA

100 pM 50 pM 30 pM 20 pM 10 pM 5 pM 1 pM 0.1 pM 0 pM

4 i / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0,2 0,15

1

0,1

0

0

0,1

0,2

0,3

0,4

0

0

E / V vs Ag / AgCl

20

40

0

0,2

0,4 0,6 0,8 [Lysozyme] / pM

60

80

1

1,2

100

[Lysozyme] / pM

(C)

(D)


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3 1,5

2

1 BSA

cyt C

casein

i / µA

Lysozyme

1

i / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0,5

0

1

0

2

3

different proteins

4

5

6 10 20 40 60

cycles

Figure 5: (A) DPVs of a rGO/PEI/Ph-aptamer electrode upon lysozyme addition solution: Fe(CN)6]4-/3- (5 mM) / PBS (10 mM); (B) Calibration curve; (C) Comparison of signal of the rGO/PEI/Ph-aptamer sensor upon addition of other proteins (5 nM) and lysozyme (30 pM); (D) Determination of number of sensing cycles of a single rGO-PEI/Ph-aptamer sensor using a 10 pM lysozyme solution.

3.5. Serum analysis Human serum samples were investigated in the following (Figure 6). Using the calibrativen curve in Figure 5B, lysozyme levels of 238 ± 20 nM were measured in the sample of a healthy human, 792 ±70 nM for an IBD infected person and 243 ± 22 nM after treatment. When comparable to lysozyme concentrations determined by a classic turbidimetric assay

30

analogous results were obtained (Table 4). By this later method, for

healthy individuals, the human serum sample contains 240 ±30 nM lysozyme, while patients with IBD showed levels of 823 ± 73nM. (Table 4). The lysoyme level was again decreased for IBD patients after treament. The results indicate that the sensor is adequate for the analysis of clinical samples.



1,2 healthy patient 1 IBD patient IBD treated patient 0,8 i / µA

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0,6 0,4 0,2 0

0

0,15 0,3 E / V vs. Ag/AgCl

0,45

Figure 6: DPVs of a rGO/PEI/Ph-aptamer electrode in [Fe(CN)6]4-/3- (5 mM) / PBS (10 mM) after incubation with diluted serum samples from a person (black), a IBD diagnosed patient (red) and a IBD patient after treatment and diagnosed as cured (green).

Table 4: Lysozyme levels in serum of a healthy patient, and IBD infected person and an IBD infected person after treatment method

healthy patient

IBD patient

IBD patient after treatment

DPV with our sensor Turbidometric assay

238 ± 20 nM 240± 23 nM

792 ±70 nM 823±73 nM

240 ±30 nM 278± 60 nM

4. Conclusion The interest of electrophoretic deposition for the formation of rGO/PEI modified gold electrodes with simultaneous surface modification of rGO with carboxylic acid, phenyl boronic acid and alkynyl functions is presented. It is based on the simultaneous reduction of GO to rGO and reduction of different diazonium salts, which upon the release of N2 molecules and generation of radicals can form covalent bonds with the rGO part of the film. The one step process together with high control over film thickness make this approach of great interest for large scale production. To show the possibility of further modification of rGO/PEI/Ph-alkynyl films, azide-terminated lysozyme aptamers were clicked onto the matrix and used for lysozyme sensing in serum samples of patients suffering from IBD. Our data 18 ACS Paragon Plus Environment

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emphasizes that Au interfaces modified with rGO/PEI/Ph-alkynyl films by one-step EPD can be used to achieve efficient aptamer immobilisation and that the electrochemical label-free aptasensors obtained are both sensitive and selective. The analytical characteristics of the sensor with detection level down to 200 fM and linearity up to 20 pM together with good stability and reproducibility are appropriate for its use in other biological fluids. Moreover the presented strategy for obtaining “generic” rGOPEI/Ph-alkynyl interfaces allows for developing aptasensors for other interesting analytes by clicking the specific aptamers. Such generic interface will be very interesting for developing aptasensor arrays. Not in the least, functionalization of thin gold interfaces with nanometer films of rGO/PEI/Ph-alkynyl of controlable thickness demonstrated in this report could be extended for “clickable” SPR and QCM interfaces and can be an useful approach when designing sensors for dual electrochemical –SPR or QCM investigations.

Acknowledgments R.B. and S.S. gratefully acknowledge financial support from the Centre National de la Recherche Scientifique (CNRS), the Lille1 University, the Hauts-de-France region, the CPER “Photonics for Society” and the ANR through FLAG-ERA JTC 2015-Graphtivity. Q.W. thanks Chinese government for the China Scholarship Council (CSC) Award. Qi WANG thanks the National Natural Science Foundation of China (Grant No.51502159) and Natural Science Foundation for Outstanding Young and Middle-aged Scientists of Shandong Province (Grant No. BS2015CL001).

ASSOCIATED CONTENT Supporting Information Available: [XPS anamysis of all interfaces (Table S1, S2); Raman and XPS spectra of different materials (Figure S1); DPV and calibration curves of gold electrodes modified with phenyl-alkynyl ligands (Figure S2)]. This material is available free of charge via the Internet at http://pubs.acs.org.

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