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Vertically aligned nitrogen-doped carbon nanotube carpet electrodes: highly sensitive interfaces for the analysis of serum from patients with inflammatory bowel disease Qian Wang, Palaniappan Subramanian, Alex Schechter, Eti Teblum, Reut Yemini, Gilbert Daniel Nessim, Alina Mihaela Vasilescu, Musen Li, Rabah Boukherroub, and Sabine Szunerits ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00663 • Publication Date (Web): 25 Mar 2016 Downloaded from http://pubs.acs.org on March 26, 2016

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ACS Applied Materials & Interfaces

Vertically aligned nitrogen-doped carbon nanotube carpet electrodes: highly sensitive interfaces for the analysis of serum from patients with inflammatory bowel disease

Qian Wang,1,2 Palaniappan Subramanian,3 Alex Schechter,3* Eti Teblum,4 Reut Yemini,4 Gilbert Daniel Nessim,4 Alina Vasilescu,5 Musen Li,2 Rabah Boukherroub1 and Sabine Szunerits1* 1

Institute of Electronics, Microelectronics and Nanotechnology (IEMN), UMR CNRS 8520, Lille1 University, Avenue Poincaré-BP60069, 59652 Villeneuve d’Ascq, France 2

Key Laboratory for Liquid-Solid Structural Evolution and Processing of Materials (Ministry of Education), Shandong University, Jinan 250061, China 3

4

Department of Biological Chemistry, Ariel University, Ariel 40700, Israel

Department of Chemistry and Bar Ilan Institute of Nanotechnology and Advanced Materials (BINA), Bar-Ilan University, Ramat Gan, 52900, Israel 5

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

Abstract The number of patients suffering from Inflammatory Bowel Disease (IBD) is increasing worldwide. The development of non-invasive tests that are rapid, sensitive, specific and simple would allow preventing patient discomfort, delay in diagnosis, and the follow-up of the status of the disease. Herein, we show the interest of vertically aligned nitrogen-doped carbon nanotube (VA-NCNT) electrodes for the required sensitive electrochemical detection of lysozyme in serum, a protein that is up-regulated in IBD. To achieve selective lysozyme detection, biotinylated lysozyme aptamers were covalently integrated onto the VA-NCNTs. Detection of lysozyme concentrations in serum was achieved by measuring the decrease in the peak current of the Fe(CN)63-/4- redox couple by differential pulse voltammetry upon addition of the analyte. We achieved a detection limit as low as 100 fM with

*

To whom correspondence should be addressed. Sabine Szunerits ([email protected];Tel: +33 3 62 53 17 25; Fax: +33 3 62 53 17 01) and Alex Schlechter ([email protected]; Tel : 972-3-9371470 ; Fax : 972-54-7740254

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a linear range up to 7 pM, in line with the required demands for the determination of lysozyme level in patients suffering from IBD. We attained the sensitive detection of biomarkers in clinical samples of healthy patients and individuals suffering from IBD and compared the results to a classical turbidimetric assay. The results clearly indicate that the newly developed sensor allows for a reliable and efficient analysis of lysozyme in serum.

Keywords: Inflammatory Bowel Disease, nitrogen-doped vertically-aligned carbon nanotubes; aptamers; lysozyme; differential pulse voltammetry; sensing


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1. Introduction Biosensors based on electrochemical detection principles have become routine analytical tools for the sensitive and selective detection of a variety of biological analytes in an inexpensive way.1 The good electron transfer kinetics and biocompatibility of carbon-based electrodes, together with well established surface functionalization strategies has put them to the forefront of interest as transducer materials in electrochemical biosensors.2 Carbon nanotubes (CNTs) have in particular attracted enormous interest in electrochemistry thanks to their small size and good electrochemical properties.3-6 CNT electrodes can be fabricated by adsorption of CNTs onto electrodes or by embedding CNTs in polymer networks and binders and linking the matrix to the surface of the chosen transducer.4 The direct growth of CNTs on different substrates is a particular appealing approach as it allows controlling morphological parameters such a CNT length and density.7-10 Dense arrays of vertically aligned carbon nanotubes, often called CNT forests or CNT carpets, which can be directly synthesized on bulk metals using chemical vapor deposition (CVD),9 are promising materials for the construction of reproducible interfaces required for reliable sensing applications.8,10-13 Some of us showed recently that nitrogen plasma-treated vertically aligned carbon nanotube interfaces are promising electrocatalytic interfaces towards oxygen reduction in alkaline media.8 The inclusion of heteroatoms14 such as nitrogen creates turbostratic disorder, thus increasing the electrocatalytic behavior of CNTs.15-16 Additionally, nitrogen-doped CNTs are characterized by faster electron kinetics compared to undoped CNTs, showing even better performance as electrochemical transducers. Herein, we show the potential of nitrogen-doped VA-NCNTs for the extreme sensitive and selective detection of lysozyme concentrations in serum of patients suffering from inflammatory bowel disease (IBD). This disease has been found to result from an aberrant innate and acquired immune response to commensal microorganisms in genetically susceptible individuals.17 The diagnosis of IBD relies on clinical findings after radiological, endoscopic and histological examinations. The development of noninvasive alternative tests that are rapid, sensitive, specific and simple are thus of high importance to prevent patient discomfort, delay in diagnosis, and follow-up of the status of the disease.18-27 It is now well established that, human lysozyme is up-regulated in a number of gastrointestinal inflammatory conditions, including inflammatory bowel disease.28-29 However, only some reports achieved the required picomolar sensitivity for real time sensing of biological samples.25-27,30 In all these sensing platforms, they achieve high sensitivities using different signal amplification strategies. We demonstrate here a femtomolar (fM) level detection limit of lysozyme in serum using nitrogendoped VA-NCNTs modified with lysozyme aptamers without any additional amplification step.

2. Experimental part 3 ACS Paragon Plus Environment


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2.1. Materials 4-Aminobenzoic acid, sodium nitrite (NaNO2), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), 2-(N-morpholino)ethanesulfonic acid (MES), phosphatebuffered saline (PBS, 0.1M), hydrochloric acid (HCl), lysozyme, Micrococcus lysodeykticus and potassium ferrocyanide ([K4Fe(CN)6]) were purchased from Aldrich and used as received. Neutravidin was purchased from Pierce. The biotinylated aptamer was purchased from Integrated DNA Technologies (Belgium) and had the following sequence: 5’-biotin-TTT TTT TTT TTT TTT TTT TTT TTT ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG-3’.31 The oligonucleotide was dissolved in Tris-EDTA buffer solution (pH=8.0) and kept frozen. Human samples from healthy patients and patients suffering of IBD were kindly provided by Dr. Ionescu Andra (Fundeni Clinical Institute, Department of Gastroenterology and Hepatology, Bucharest, Romania) in accordance with the local ethical committee.

2.2. Preparation of nitrogen-doped vertically aligned carbon nanotube carpet (VA-NCNTs) electrodes We performed CNT synthesis in a three-zone atmospheric-pressure tube furnace (Carbolite model HZS-E), using a single fused-silica tube with an internal diameter of 22 mm.7,10 The two-first zones of the furnace preheated the precursor gases at 770 °C, decomposing the hydrocarbon gases and forming water vapor from O2 and H2.32 The sample was positioned in the last zone of the three-zone furnace (G3) for the annealing and growth steps at 755 °C. Flows of He (99.9999%), ArO2 (a mixture of 99.9999% argon with 1% oxygen), C2H4 (99.999%), and H2 (99.9999%) were maintained using electronic mass flow controllers (MKS P4B) with digital mass flows control unit (MKS model 247D). All experiments were performed by using the “fast-heat” technique, that we describe in detail in a previous paper,10 in which the samples are initially positioned outside the heated zone of the furnace with a fan blowing on the exposed quartz tube to keep the sample at room temperature. Using this technique, the heating is applied to the sample only during annealing and growth (and not during the initial purging and ramping of the furnace to the set temperature). We also employed our technique to form controlled part per million (ppm) amounts of water vapor inside the reactor, from controlled flows of ArO2 and H2, which is described in detail in a previous publication.32 Purging of the system was performed by 100 standard cubic centimeters per minute (sccm) and 400 sccm H2 for 15 minutes while the furnace was ramped to the desired temperature. Once the set temperatures of three-zone furnace were reached, the quartz tube was shifted, positioning the sample in the growth zone (G3) to start the VACNT synthesis which includes three steps. First the stainless steel coin surface was 4 ACS Paragon Plus Environment

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oxidized at 755 °C for 45 min by flowing 1000 sccm ArO2 and 100 sccm Ar. Then the surface was reduced by flowing 400 sccm hydrogen and 100 sccm Ar at 755 °C for another 40 min. The third step comprises CNTs growth. The flows of Ar, H2, ArO2 and C2H4 were respectively set at 100, 400, 250 and 200 sccm for 15 min. After the growth was completed, the quartz tube was shifted out of the furnace to slowly cool down to room temperature under a flow of helium before the sample was removed from the furnace Nitrogen plasma treatment of VA-CNTS was performed in a Zepto Low pressure plasma system (Diner electronic Gmbh, Ebenhausen, Germany) equipped with a 40 kHz HF-generator operating between 0 and 100 W. The VA-CNTs coated steel coins were introduced into the plasma chamber for 2 min. The chamber pressure and the plasma power were maintained at 1.2 mbar and 130 W, respectively.

2.3. Formation of VA-NCNTs-aptamer The fabrication of the lysozyme sensor includes three steps. The diazonium cations were prepared in situ by mixing NaNO2 (10 mM) with 4-aminobenzoic acid (10 mM) in HCl (0.5 M) .33-34 The mixture was left stirring for 5 min at 0 ℃ before being transferred to the electrochemical cell. Electrochemical modification was then performed y the reduction of the in situ generate 4-carboxyphenyl diazonium salt using five cyclic voltammtry scans from +0.4V to -0.6 V at a scan rate of 100 mV s-1.. The VANCNTs-COOH electrode was then thoroughly washed with distilled water to generate The terminal carboxylic acid groups of VA-CNTs-COOH electrode were activated by immersion in MES buffer (pH=5.0, 100 mM) containing EDC (100 mM) and NHS (20 mM) for 2 h at room temperature. The electrode was washed with MES buffer to remove excess reagents. The electrode was subsequently functionalized with neutravidin (100µg mL-1) in PBS buffer (pH=7.4, 100 mM) for 1 h at room temperature, and rinsed copiously with PBS. Immobilization of the biotinylated aptamer probe is based on the high affinity between avidin and biotin.35 The neutravidin modified VA-CNTs electrode was immersed in PBS buffer (pH=7.4, 100 mM) containing the biotinylated aptamer probe (1 µM) for 2 h at room temperature. After washing with phosphate-buffered saline (PBS, 0.1M), the electrode was rinsed with 0.1 % SDS to remove unbound aptamer.

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X-ray photoelectron spectroscopy (XPS) X-ray photoelectron spectroscopy (XPS) experiments were performed in a PHl 5000 VersaProbe Scanning ESCA Microprobe (ULVAC-PHI, Japan/USA) instrument at a base pressure below 5×10-9 mbar. Core-level spectra were acquired at pass energy of 23.5 eV with a 0.1 eV energy step. All spectra were acquired with 90° between X-ray source and analyzer and with the use of low energy electrons and low energy argon ions for charge neutralization. After subtraction of the Shirley-type background, the core-level spectra were decomposed into their components with mixed GaussianLorentzian (30:70) shape lines using the CasaXPS software. Quantification calculations were conducted using sensitivity factors supplied by PHI. In the case of VA-NCNTs a short oxygen plasma treatment was performed to exclude the presence of surface absorbed oxygen species in the C1s spectrum. Raman spectroscopy 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. Visible light is focused by a 100x objective. The scattered light is collected by the same objective in backscattering configuration, dispersed by a 1800 mm focal length monochromator and detected by a CCD. Scanning electron microscopy (SEM) Scanning electron microscopic images of the films were obtained using Nova NanoSEM (FEI, USA) 450 scanning electron microscope with FEG (field emission gun, Schottky type) systems equipped with an energy dispersive X-ray analyzer at an accelerating voltage of 20 kV. High resolution transmission electron microscopy (HRTEM) The CNTs were characterized by high resolution transmission electron microscopy (HRTEM) using a JEOL-2100 (JEOL, USA) operating at 200 keV. HRTEM samples were prepared by dispersing a section of the CNT carpet in 2-propanol with gentle sonication for 30 min and then placing 1 drop of the solution on a 300 mesh Cu holey carbon grid (from SPI).

Electrochemical measurements Cyclic voltammetry (CV) measurements were performed with an Autolab potentiostat 30 (EcoChemie, Utrecht, The Netherlands). A platinum mesh and a Ag/AgCl electrode were used as a counter and a reference electrode, respectively. The scan rate was 100 mV s-1 and the active surface area was 0.04 cm2.


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Electrochemical detection of lysozyme was achieved by incubating the VA-NCNT-aptamer electrode in lysozyme solution in PBS for 30 min at room temperature, followed by rinsing with PBS buffer and transfer to a 5 mM [Fe(CN)6]4- solution in 0.1 M PBS pH 7.4 for the detection by differential pulse voltammetry (DPV). Electrochemical measurements were carried out in the following optimized conditions: modulation time 0.05 s; interval time 0.5 s; initial potential -0.1 V; end potential 0.5 V; step potential 5 mV; modulation amplitude 25 mV. The data recorded in this manuscript are collected from five different experiments. UV/Vis measurements Absorption spectra were recorded using a Evolution 600 UV-VIS spectrophotometer (Thermo Scientific, France).

2.5. Determination of lysozyme concentration in human serum based on its lytic activity The determination of lysozyme in the human serum sample was based on its enzymatic activity using a classic turbidimetric assay36 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 deducted from the calibration curve and expressed based on hen egg lysozyme.

3. Results and discussion 3.1. Construction of vertically aligned nitrogen-doped carbon nanotubes (VA-NCNTs) electrodes for lysozyme sensing Some of us showed recently that nitrogen plasma-treated VA-NCNTs are promising electrocatalytic interfaces towards oxygen reduction in alkaline media.8 We were thus intrigued to investigate whether VA-NCNTs modified with specific ligands could also be highly sensitive interfaces for electrochemical sensing applications. The synthesis of dense carpets of VA-NCNTs is based on chemical vapor deposition using C2H4 as hydrocarbon source, followed by N2-plasma treatment for 2 min.8 The morphology of the resulting VA-NCNT carpet is depicted in Figure 1A. The SEM image shows a dense VA-NCNTs carpet with a height of 7 ± 1 µm. HRTEM image of the dispersed CNTs (Figure 1B) indicates that the CNTs have an average diameter of 9 ± 1 nm with 4-5 walls and exhibit a high degree of crystallinity. A further analysis of the CNT structure was inferred by the intensity of 7 ACS Paragon Plus Environment


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the G/D bands in the Raman spectrum, which displays the characteristic D and G bands at 1340 cm-1 and 1578 cm-1, respectively (Figure 1C). The chemical composition of the VA-NCNTs was further examined by X-ray photoelectron spectroscopy (XPS). The XPS survey spectrum shows next to C1s and O1s the presence of N1s with 2.1 at. % (Table 1). The high resolution N1s spectrum can be deconvoluted into two bands at 400.1 eV and 401.9 eV assigned to pyrrolic and charged quaternary type nitrogen groups (Figure 1D).37 In the case of the C1s band, the main peak from sp2-hybridized carbon at 284.5 eV is characterized by an asymmetric profile, with the broad band ranging from 287-292 eV due to shake-up satellites. The additional contribution at 285.4 eV is due to surface functional groups C-O/C-N and organic species that are not fully carbonized (Figure 1E).37 The electrochemical behavior of the VA-NCNT carpet electrodes was determined by cyclic voltammetry using ferri/ferrocyanide as redox couple. The VA-NCNT electrode shows a quasireversible electron transfer behavior with a ∆E=116 mV (Figure 2). The electrochemical behavior depends strongly on the length of the VA-NCNTs, with a decrease in electron transfer rate for longer wires. For the use in electrochemical sensing, VA-NCNTs with fast heterogeneous electron transfer are advantageous38-40 and the VA-NCNT carpet electrodes of 7 µm in height were further investigated for the construction of a lysozyme biosensor. Compared to the electrochemical characteristics of vertically aligned CNTs without nitrogen doping (VA-CNT) (see supporting information, Figure S1), the VA-NCNT carpet electrodes showed excellent electrochemical properties. This motivated us to pursue using these interfaces for sensing applications The immobilization of the analyte specific ligand on the sensing interface is a vital step to achieve selective detection. In our case, we achieved selectivity towards lysozyme by linking a lysozymespecific aptamer to VA-NCNTs (Figure 3). We grafted 4-carboxyphenyl radicals to the VA-NCNTs using a standard procedure involving the reduction of the 4-carboxybenzenediazonium cations to generate aryl radicals. 33-34,41 Electro-reduction of 4-carboxyphenyl diazonium salts results in carboxylterminated interfaces, followed by covalent linkage between the NH2 groups present on neutravidin and the carboxyl-terminated VA-NCNTs via amide bond formation. The 4-carboxyphenyl diazonium salt is formed in situ upon addition of NaNO2/HCl to a solution of 4-aminobenzoic acid, where nitrogen is spontaneously eliminated, forming the 4-carboxyphenyl diazonium salts. Electrochemical reduction of the diazonium salt to its aryl radical results in the formation of a robust covalent bond with the VA-NCNTs (Figure 3A). Five voltammetric reduction cycles are sufficient to generate a satisfying organic layer (Figure 3B).


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The XPS analysis validated the introduction of carboxylic acid groups onto the VA-NCNTs after electrochemical reduction (Figure 3C). In addition to bands at 284.5 eV (sp2 C-C), 285.0 eV (C-C/CH), 286.5 eV (C-O/C-N), and 288.9 eV (C=O), the presence of a band at 291.3 eV (O-C=O), absent in the C1s core level spectrum of VA-NCNTs, indicates the successful incorporation of COOH groups onto VA-NCNTs (Figure 1E). Additionally, the atomic percentage of oxygen increased (Table 1). The introduction of carboxylic groups onto VA-NCNTs had an important effect on the electron transfer (Figure 4). We observed inhibition of the electron transfer due to electrostatic repulsion between the negatively charged COOH groups and negatively charged ferro/ferri cyanide redox probe, consistent with observations of Chung et al.42. Neutravidin was covalently linked to carboxylterminated VA-NCNTs via amine coupling between the NH2 groups of neutravidin and the carboxylic acid groups present on the wires. XPS analysis of the VA-CNTS-neutravidin electrode showed a significant increase in the N1s signal (Table 1). Furthermore, we observed a partial restoration of the electron transfer (Figure 4A). Finally, a biotinylated lysozyme aptamer was coupled to the neutravidin ligands present on the VA-CNTS-neutravidin electrode. The presence of P2p and S2p in the XPS survey spectrum (Table 1) is a first indication of the successful immobilization of the biotinylated aptamer onto VA-NCNTs. We observe a further decrease of the voltammetric peak current response of the ferro/ferri cyanide redox probe, which might be due to electrostatic repulsion with the negatively charged phosphate backbone of the aptamer. Figure 3D displays the HRTEM image collected from the functionalized VA-NCNTs. The images clearly indicate the presence of a functionalized layer both on the lateral sides and tip (see inset image) of the carbon nanotubes, suggesting that VA-NCNTs are uniformly modified throughout their length. The electrochemical responses of five different VANCNT electrodes before and after aptamer modification are shown in Figure 4B. The currents recorded are fairly comparable in all cases, indicating a good reproducibility in the fabrication as well as in the modification of the VA-NCNT electrodes.

3.2. Lysozyme detection on VA-CNTs-aptamer electrodes: sensitivity, selectivity, and reproducibility To evaluate the performance of the developed sensor for the quantitative analysis of lysozyme, we recorded the change of the oxidative current values of [Fe(CN)6]4- upon addition of increasing concentrations of lysozyme using differential pulse voltammetry (DPV) (Figure 5A). Upon binding its target, the surface linked aptamer experiences conformational changes, and the negative charges of the phosphate backbone in DNA are screened in part by the positively charged protein, as indicated by other authors.43-44 At the same time, the formation of a bulkier lysozyme-aptamer complex at the surface of the VA-NCNTs results in partial blocking of electron transfer from the redox probe to the electrode. The overall effect when using [Fe(CN)6]4- probe is the decrease in peak current density upon



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increasing lysozyme concentrations. From Figure 5B, it can be seen that the peak current decreases with increasing lysozyme concentration as a consequence of the efficient capture of lysozyme by the aptasensor. A similar behavior has been recently reported by Elissa et al. for electrochemical sensing of ovalbumin on a reduced graphene oxide modified interface.23 We observed a good linear relationship with a correlation coefficient of r=0.999 between the anodic peak current density of [Fe(CN)6]4- redox probe and lysozyme concentration according to j(mA cm-2)=0.56-0.04×[lysozyme] (pM) (Figure 5C). The response curve saturates at ≈7 pM, most likely to the saturation in the number of binding sites. The detection limit of lysozyme was determined to be ≈100 fM from five blank noise signals (95 % confidence level) a concentration range that is appropriate for analysis of biological samples. Concentrations of lysozyme in serum of healthy people range from 27-301 nM, while for IBD patients significantly higher levels have been reported (1.4 µM).45-46 Consequently, our method allows accurate detection of lysozyme both in healthy and IBD patients. Moreover, the analytical characteristics are appropriate for detection in other biological fluids such as urine or cerebrospinal fluid where lysozyme levels as low as 5.8 nM are recorded.46 Therefore, further applications of the sensor described here can be envisaged, that require high sensitivity. The analytical performance was compared to that of VA-CNTs-aptamer sensors without nitrogen doping (Figure 5C). While a linear response upon lysozyme addition was found for a similar concentration range, the sensitivity of the interfaces decreased more than 2 times to j(mA cm-2)=0.560.017×[lysozyme] (pM). We also tested a glassy carbon electrode using the same surface modification strategy, and measured sensitivity comparable to that of undoped VA-CNTs. To ensure that anions or species present in biological fluids do not interfere with the measurement strategy, a calibration curve in Fe(CN)6]4- solutions spiked with bovine serum albumin (10 mM) and increased salt concentration (100 mM NaCl) was recorded (Figure 5C). No influence of proteins and salt concentration was observed. The same was true when ascorbic or uric acid (0.1 mM) were present (see supporting information, Figure S2). The performance of the VA-NCNTs-aptamer interface for lysozyme sensing is considerably improved when compared to most reported electrochemical interfaces as well as optical detection approaches (Table 2). The possibility to obtain low detection limits without the need of sophisticated amplification strategies or additional modification steps makes the developed approach a viable alternative to the highly sensitive impedimetric lysozyme aptasensor based on a glassy carbon electrode modified with graphene,43 or the recently developed aptamer-antibody sandwich assay using chemical amplification.30 In the case of the impedimetric lysozyme aptasensor, the construction of the sensing interface necessitates several lengthy steps such as manual deposition of chitosan-graphene oxide mixture, chemical reduction of graphene oxide, activation of carboxyl groups and covalent attachment of amine-ended aptamer, totaling 52 h as compared to around 5.5 h required for functionalizing the VA-NCNT electrodes used here. However, one drawback is the limited linear 10 ACS Paragon Plus Environment

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range of our sensor, comparable with the impedimetric lysozyme aptasensor, but much smaller than the one reported on screen printed carbon electrodes using chemical amplification.30 The reproducibility of the VA-NCNTs-aptamer electrodes was expressed in terms of the relative standard deviation, which was determined to be 2.3 % at a lysozyme concentration of 4 pM (n=5). The long-term stability of the VA-NCNTs sensor when stored in PBS was also evaluated showing a loss of 2.5 % in the anodic peak current when testing 2 pM lysozyme solution after the electrode has been stored at 4 °C for 2 weeks. This indicates that the stainless steel substrate electrode used for the growth of the carbon carpets are not passivating or corroding under these conditions.

To illustrate the selectivity of the sensor, VA-CNTs-aptamer interfaces were incubated with four nonspecific proteins. As can be seen in Figure 6A, the change in current was much lower with the other proteins when compared to lysozyme, indicating the good selectivity of the sensor with negligible non-specific interaction with other proteins. In addition, the current response of lysozyme addition (5 pM) to VA-NCNTs without linked aptamer was examined to ensure the lysozyme’s specific binding to the aptamer. The observed current change was significantly smaller than in the presence of the aptamer. A major concern inherent in protein detection assays is the possibility of background interference. It is thus important to assess the impact of serum proteins. Human serum protein solutions (diluted 10 times with PBS) were spiked with lysozyme (5 pM final concentration) and the current density signal was recorded by DPV. When compared to the current density observed for the same lysozyme concentration in PBS, no change in signal was observed. Together with the good analytical performances, this demonstrated the feasibility of detecting lysozyme in real serum samples and the potential utility of VA-NCNTs-aptamer electrodes to monitor the lysozyme level in patients affected by inflammatory gastrointestinal diseases.

3.3. Measurements of clinical samples To evaluate the reliability and potential application of the proposed sensor, the lysozyme level in human serum samples from healthy patients and patients suffering of inflammatory bowel disease was determined using our VA-NCNTs-aptamer sensor (Table 3). Figure 6B, shows the electrochemical signals determined from diluated serum samples of a healthy and IBB patient. In the case of healthy individuals, the human serum sample was found to contain 0.23 ±0.05 µM lysozyme. IBD infected patients showed on the other hand lysozyme levels as high 0.85 ±0.07 µM. To confirm the results, the lysozyme concentration was in parallel determined by a classic turbidimetric assay.36 In this assay a suspension of killed Micrococcus lysodeykticus is made up in PBS, test serums are added and the decrease in optical density is recorded at 450 nm. The activity obtained was compared to that using 11 ACS Paragon Plus Environment


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standard (0-5 µg mL-1) concentrations of egg white lysozyme (Figure 6C). For healthy individuals, the human serum sample was found to contain 3.36 ±0.45 µg/mL (0.24 ±0.03 µM) lysozyme. IBD infected patients showed 11.20 ±0.98 µg/mL (0.80 ±0.07 µM). These levels are consistent with lysozyme concentrations found using the newly developed VT-NCNTs-aptamer based assay (Table 3) as shown by statistical analysis using the Analysis ToolPak add-in in Microsoft Excel. While the F-test showed that the variances of the results obtained by the two methods are equal (p=0.416, p>0.05), the T-test confirmed that the results provided by the electrochemical sensor do not differ significantly from those obtained by the turbidimetric method (p>0.05).The results indicate that the newly developed biosensor is highly reliable for the analysis of clinical samples and that potential interferences by other electroactive serum components are low.

4. Conclusion We demonstrated the great potential of chemically modified vertically aligned nitrogen-doped carbon nanotubes electrodes for the electrochemical sensing of lysozyme. Specific sensing was achieved through covalent integration of a biotinylated lysozyme aptamers on the carbon nanostructures. The decrease in the differential pulse voltammetry current using [Fe(CN)6]3- as redox probe was used as indicator for the presence of lysozyme. The described sensor exhibited a detection limit of ≈100 fM without any need for amplification, appropriate for detection of lysozyme levels in serum and urine. We demonstrated the feasibility and reliable use of the lysozyme sensing assay in clinical samples through the analysis of serum samples from healthy and IBD infected patients. We believe that the assay presented here can also surve as an interesting alternative for the analysis and diagnosis of patients suffering from leukemia and other diseases associated with higher or lower lysozyme levels.

Acknowledgments R.B. and S.S. gratefully acknowledge financial support from the Centre National de la Recherche Scientifique (CNRS), the University Lille 1 and Nord Pas de Calais region. S.S thanks the Institut Universitaire de France (IUF) for financial support. A.S. and G.D.N. acknowledge the Israel Science Foundation (ISF) for funding the research work through the Israel National Research Center for Electrochemical Propulsion (INREP) and I-CORE Program (number 2797/11). P.S. acknowledges the support of INREP and Ariel University postdoctoral fellowship program for providing the research scholarship.


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Table 1: Atomic percentage (at. %) of elements as determined by XPS analysis Sample

C1s

O1s

N1s

P2p

S2p

VA-NCNTs (7 µm height)

86±1

12±1

2±0.5

-

-

VA-NCNTs (37 µm height)

85±1

13±1

2±0.5

-

-

VA-NCNTs-COOH

82±1

16±1

2±0.5

-

-

VA-NCNTs-neutravidin

76±1.1

18±1

6±1

-

-

VA-NCNTs-aptamer

69±1

19±1

9±1

1.5±0.5

1.5±0.5


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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


Table 2: Characteristics of other lysozyme sensing platforms Method

Interface

LOD

Linear range

Ref.

Fluorescence

GO/aptamer + amplification using exonuclease III

5.6 nM

8.7-70 nM

47

Electroluminescence

Au electrode, aptamer, Ru(bpy)32+

120 pM

64 pM–0.64 µM

48

Square wave voltammetry

THH Au NCs/GCE-aptamer

0.1 pM

0.1 pM–10 nM

44

Cyclic voltammetry

Au with thiocyanuric acid /AuNPs-aptamer

0.1 pM

5 pM–1 nM

49

SPR

rGO-Au, aptamer

0.5 nM

0.5-200 nM

19

Impedance

GR–GCE-aptamer

6 fM

0.01–0.5 pM

43

DPV

SPCE-aptamer-antibody sandwich

4.3 fM

5 fM–5 nM

30

DPV

VA-NCNTs-aptamer

100 fM

0.1-7 pM

this work

SPCE: screen-printed carbon electrode; GCE: glassy carbon electrode, THH Au NCs: tetrahexahedral Au nanocrystals; GR: electrochemically reduced graphene oxide; rGO: reduced graphene oxide



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Page 18 of 27

Table 3: Results of lysozyme concentrations in serum of healthy patients (sample 1) and IBD infected patients (sample 2) sample

Analysis method

detected

1 (healthy patient)

Turbidometric assay

3.36 ±0.45 µg/mL (0.24 ±0.03 µM)

2 (IBD patient) 1 (healthy patient) 2 (IBD patient)

Turbidometric assay

11.2 ±0.98 µg/mL (0.80 ±0.07 µM) 3.22 ±0.89 µg/mL (0.23 ±0.05 µM) 11.9 ±0.48 µg/mL (0.85 ±0.03 µM)

DPV on VA-NCNTs-aptamer electrodes DPV on VA-NCNTs-aptamer electrodes


Page 19 of 27

(A)

(B)

(C)

D

G

Raman 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


2D

1000

1500

2000

2500

Raman shift / cm

3000

3500

-1



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

Page 20 of 27

(D)

N 1s

404

402

400

398

396

Binding energy / eV

(E)

C1s

296

294

2

sp C-C C-N/C-O satellites

292

290

288

286

284

282

Binding energy / eV

Figure 1: Morphology and chemical composition of VA-NCNTs: (A) SEM and (B) HRTEM images;

(C) Raman spectrum; (D) N1s and (E) C1s high-resolution XPS spectra.


Page 21 of 27

300 VA-NCNTs (7 µm) 200 VA-NCNTs (37 µm) 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


0 -100 -200 -300 -0,4

-0,2

0

0,2

0,4

0,6

0,8

E / V vs. Ag / AgCl Figure 2: Cyclic voltammograms of VA-NCNTs (7 µm in length) (black) and VA-NCNTs VA-

NCNTs (37 µm in length) (blue) in 5 mM [Fe(CN)6]4-/3- in 0.1 M PBS scan rate=100 mV s-1.



(A)

(B)

(C)

100

VA-NCNTs-COOH 2

sp C-C Intensity / a.u.

0 -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

Page 22 of 27

-200

1. scan 2. scan 3. scan 4. scan 5. scan

-300 -400

-0,6

-0,4

-0,2

0

0,2

0,4

3

sp C-C C-N/C-O C=O O-C=O

294

292

290

288

286

284

282

Binding energy / eV

E / V vs. Ag / AgCl

(D)

2 nm


Page 23 of 27

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


Figure 3: (A) Surface modification of VA-NCNTs electrode through electroreduction of 4-

carboxyphenyl diazonium salt formed in situ followed by covalent immobilization of neutravidin and interaction with biotinylated lysozyme aptamer; (B) Cyclic voltammograms of VA-NCNTs electrode obtained in a solution of 4-aminobenzoic acid (10 mM), NaNO2 (10 mM)/HCl (0.5 M); scan rate: 100 mVs-1; (C) C1s high resolution spectrum of VA-NCNTs-COOH.; (D) HRTEM image of modified VANCNTs



(A)

(B) 300

300

100

VA-NCNTs 250 VA-NCNTs-aptamer

VA-NCNTs VA-NCNTs-COOH VA-NCNTs-avidin VA-NCNTs-aptamer

200 i / µA

200

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

Page 24 of 27

0

150

-100

100

-200

50

-300 -0,2 -0,1

0

0,1

0,2

0,3

0,4

0,5

0,6

0

E /V vs. Ag / AgCl

1 2 3 4 number of VA-NCNTs electrodes

Figure 4: (A) Cyclic voltammograms of VA-NCNTs (black), VA-NCNTs–COOH (orange), VA-

NCNTs-neutravidin (grey), and VA-NCNTs-aptamer (blue) in 5 mM [Fe(CN)6]4-/3- in 0.1 M PBS ; scan rate: 100 mVs-1; (B) Bar diagram of peak currents recorded at 0.28 V for 5 different VA-NCNTs interfaces before (black) and after aptamer modification (blue).


Page 25 of 27

(A)

(B)

(C)

0,6 0 100 fM 500 fM 1 pM 2 pM 5 pM 10 pM 20 pM

0,3 0,2

0,5

-2

0,4

0,6

j /mA cm

-2

0,5

j /mA cm

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


0,4 0,3 0,2

0,1

VA-NCNTs (PBS 0.1M) VA-CNTs (PBS 0.1M) VA-NCNTs (PBS 0.1M/10 mM BSA/100 mM NaCl) GCE

0,1

0

0

0,1

0,2

0,3

E / V s. Ag / AgCl

0,4

0

0

5

10 [Lysozyme] / pM

15

20

Figure 5: (A) Principle of lysozyme sensing with the VA-NCNT-aptasensor. Upon binding lysozyme, the bulky complex aptamer–lysozyme at the tip of VA-NCNTs blocks the electron transfer from the redox probe [Fe(CN)6]4- added in solution to the electrode. This effect is observed as a decrease in the anodic peak of [Fe(CN)6]4- recorded by DPV; (B) DPVs of the VA-NCNTs-aptamer electrode in 5 mM [Fe(CN)6]4- in 0.1 M PBS after incubation with different concentrations of lysozyme; (C) Calibration curve for lysozyme based on peak current intensity recorded by DPV for VA-NCNTaptasensor (black circles), undoped VA-CNT-aptasensor (squares, blue), glassy carbon electrode (GCE, square, green) in a solution of 5 mM [Fe(CN)6]4- / 0.1 M PBS and for VA-NCNT-aptasensor (black circles) in 5 mM [Fe(CN)6]4- / BSA (10 mM)/NaCl (100 mM)/ 0.1 M PBS. 25 ACS Paragon Plus Environment


(A)

(B) 0,6 healthy patient IBD patient

0,25

0,5

-2

j /mA cm

-2

0,2 ∆j / mA cm

0,15

0,4 0,3

0,1

0,2

0,05

0,1 0

0 BSA

cyt C

Lysozyme

casein

0

0,05

0,1

Lysozyme on VA-NCNTs

0,15

0,2

0,25

0,3

0,35

0,4

E / V vs. Ag/AgCl

(C)

0,05 turbidometric assay

450 nm

-1

(UA min )

0,04 0,03 sample 1 (healthy patient)

0,02

∆A

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

Page 26 of 27

0,01 0

sample 1 (IBD patient) 0

5

10

15

20

-1

[lysozyme] / µg mL

Figure 6: (A) Comparison of the analytical signal of the VA-NCNTs-aptamer sensor upon addition of different proteins (5 nM) and lysozyme (5 pM); also shown is the response of unmodified VA-NCNTs to 5 pM of lysozyme; (B) DPVs of the VA-NCNTs-aptamer electrode in 5 mM [Fe(CN)6]4- in 0.1 M PBS after incubation with serum samples (10.000 times diluted) from healthy (black) and IBD patients (blue); (C) Calibration curve of turbidometric assay for the determination of lysozyme concentrations in serum from healthy and IBD infected patients.


Page 27 of 27

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