Modified Floating Electrode-based Sensors for the Quantitative

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Biological and Medical Applications of Materials and Interfaces

Modified Floating Electrode-based Sensors for the Quantitative Monitoring of Drug Effects on Cytokine Levels Related with Inflammatory Bowel Diseases Viet Anh Pham Ba, Yoo Min Han, Youngtak Cho, Taewan Kim, Byung Yang Lee, Joo Sung Kim, and Seunghun Hong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04287 • Publication Date (Web): 27 Apr 2018 Downloaded from http://pubs.acs.org on April 28, 2018

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ACS Applied Materials & Interfaces

Modified Floating Electrode-based Sensors for the Quantitative Monitoring of Drug Effects on Cytokine Levels Related with Inflammatory Bowel Diseases Viet Anh Pham Ba†, Yoo Min Han‡, Youngtak Cho†, Taewan Kim§, Byung Yang Lee§,*, Joo Sung Kim‡,┴,*, Seunghun Hong†,║,* †

Department of Physics and Astronomy and Institute of Applied Physics, Seoul National

University, Seoul 08826, Korea ‡

Department of Internal Medicine and Healthcare Research Institute, Seoul National

University Hospital Healthcare System Gangnam Center, Seoul 06236, Korea §

Department of Mechanical Engineering, Korea University, Seoul 02841, Korea

┴

Department of Internal Medicine and Liver Research Institute, Seoul National University

College of Medicine, Seoul 03080, Korea ║

Department of Biophysics and Chemical Biology, Seoul National University, Seoul 08826,

Korea KEYWORDS: carbon nanotube field effect transistors, lupeol, lipopolysaccharides, tumor necrosis factor alpha, Raw 264.7 cells

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ABSTRACT: Modified floating electrode-based sensors were developed to quantitatively monitor the levels of tumor necrosis factor α (TNF-α), a pro-inflammatory cytokine related with inflammatory bowel disease (IBD), and to evaluate the effect of drugs on the cytokine levels. Here, antibodies (anti-TNF-α) were immobilized on the floating electrodes of carbon nanotube devices, enabling selective and real-time detection of TNF-α among various cytokines linked to IBD. This sensor was able to measure the concentrations of TNF-α with a detection limit of 1 pg/L, allowing the quantitative estimation of TNF-α secretion from mouse macrophage Raw 264.7 cells stimulated by lipopolysaccharides (LPS). Notably, this method also allowed us to monitor the anti-inflammatory effect of a drug, lupeol, on the activation of LPS-induced nuclear factor κB (NF-κB) signaling in Raw 264.7 cells. These results indicate that our novel TNF sensor can be a versatile tool for biomedical research and clinical applications such as screening drug effects and monitoring inflammation levels.

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1. INTRODUCTION Inflammatory bowel disease (IBD) is a serious chronic gastrointestinal disorder which may be caused by the abnormal activation of nuclear factor κB (NF-κB) pathway.1,2 This abnormal activation increases the production and secretion of pro-inflammatory cytokines including tumor necrosis factor α (TNF-α). Previous studies have shown that TNF-α plays a crucial role in the pathogenesis of IBD, therefore the estimation of TNF-α levels is important in the diagnosis and therapy of IBD.3,4 For example, the average TNF-α levels in the blood of healthy donors and patients with Crohn's disease - a type of IBD - were about 3 ng/L and 6 ng/L, respectively.5 Moreover, anti-TNF agents have been widely used in the treatment of IBD.6 Labeling methods, such as enzyme-linked immunosorbent assay (ELISA), flow cytometry, polymerase chain reaction (PCR), and fluorescence-based immunoassays, have been used for the detection of TNF-α and other markers with a high sensitivity.7-12 However, previous methods often require complex equipment and time-consuming preparatory procedures. Various label-free techniques have been developed to overcome these drawbacks while still maintaining a high sensitivity and selectivity.13-18 For example, immunosensors, which are usually disposable devices due to the formation of stable immune complexes, have exhibited specific, rapid and real-time responses by the small volume of samples.15,19,20 Recently, field effect transistor (FET)-based immunosensors have been considered as a potential candidate for the detection and quantification of inflammation-related cytokines.15-17 However, their applications for the monitoring of drug effects related with clinical diseases have been still limited. Herein, we developed a modified floating electrode-based CNT-FET sensor for the quantitative monitoring of drug effects on cytokines related with IBD. In this work, antibody (anti-TNF-α) molecules were immobilized on the floating electrodes of a CNT-FET sensor so 3



that the TNF sensor can detect TNF-α at various concentrations via a specific interaction between TNF-α and anti-TNF-α molecules. In this sensor structure, floating electrodes enhanced the sensor sensitivity, and, also, their gold surface could be easily functionalized using well-known chemical processes to build selective sensors.21 Our TNF sensor recognized TNF-α from other IBD-related cytokines such as interleukin-1β (IL-1β) and interleukin-6 (IL6) with a quick response at a low concentration down to 1 pg/L. Using this method, we could quantitatively measure the levels of TNF-α secreted from mouse macrophage Raw 264.7 cells pretreated with various concentrations of LPS at different time periods. Significantly, we monitored the effect of an anti-inflammatory agent, lupeol, on the production and secretion of the pro-inflammatory cytokine, TNF-α, from Raw 264.7 cells. The results indicate that lupeol inhibits the TNF-α production of Raw 264.7 cells by the stimulation of LPS as previously reported.7 This method exploits the advantages of floating electrode-based sensor structures such as high sensitivity and label-free simple detection, and it has the potential for monitoring other biomaterials of clinical interests simply by using different antibodies. Thus, our novel strategy can be a powerful tool for various basic biomedical research and clinical applications.

2. RESULTS AND DISCUSSION 2.1. Characteristics of a TNF Sensor Figure 1a illustrates the hybridization of a floating electrode-based CNT-FET sensor with anti-TNF-α molecules. Firstly, a CNT-FET sensor including five floating electrodes was fabricated by a photolithography method described in previous works (Figure 1a-i, Materials and Method).21-23 Secondly, the gold floating electrodes were modified by N-acetyl-Lcysteine (Figure 1a-ii). After that, the CNT-FET sensor was immersed in the mixture of 1ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). As a result, COOH terminal groups were activated as succinimide ester groups on the floating 4


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electrodes of the CNT-FET sensor (Figure 1a-iii).24 Finally, an anti-TNF-α solution was applied on the sensor. The NH2 terminals of anti-TNF-α molecules interacted with the succinimide ester terminals, resulting in the covalent immobilization of anti-TNF-α molecules on the floating electrodes (Figure 1a-iv). Since anti-TNF-α can specifically bind with TNF-α, this method allows us to obtain selective sensors for the detection of TNF-α secreted from cells (Figure 1c). To confirm the formation of anti-TNF-α molecules on gold floating electrodes, the topography images of a sensor device were taken using an atomic force microscopy (AFM) system. Figure 2a shows the AFM images of a floating electrode surface before and after the modification with anti-TNF-α. First, the bare surface of a gold floating electrode was imaged, and then, the surface was functionalized with anti-TNF-α. Successively, the topography image of the gold electrode surface was taken at the same position and condition. The images show the spots on the gold electrode after the modification with the size at a nanometer level which is consistent with the size of a single protein molecule.25 The comparison of height profiles shows a height increase by the presence of anti-TNF-α molecules, indicating the successful immobilization of the anti-TNF-α molecules on the floating electrodes. In our sensors, the floating electrodes play the role as substrates for the immobilization of anti-TNF-α. Furthermore, floating electrode structures improve the sensitivity of biosensors, resulting from the increased number of Schottky barriers.21 We also performed a fluorescent assay to confirm the bioactivity of anti-TNF-α on floating electrodes. Figure 2b shows the fluorescent image of a sensor stained with fluorescent dye-conjugated anti-rabbit IgG. Note that, the fluorescent signals appeared only on the regions of floating electrodes. Since anti-rabbit IgG was specific for rabbit clonal anti-TNF-α, this result indicates that anti-TNF-α molecules were successfully immobilized only on the

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floating electrodes. Furthermore, it also implies that the bioactivity of anti-TNF-α was not affected by the immobilizing procedure. Figure 2c shows the liquid gating effect curve of a sensor before and after the sensing of TNF-α. The electrical characteristics of our sensor were investigated by applying a liquid gate bias (Vlg) using a gold electrode to deionized water on the CNT channel of the sensor. Here, a source-drain bias was maintained at 0.1 V while a liquid gate bias was swept from 0.5 V to 0.5 V. The curve shows the drastic decrease of electric currents through the sensor in the small range of a gate bias voltage, indicating the typical p-type characteristics and sensitivity of our sensor. Moreover, the electric currents through the sensor increased after a TNF-α sensing measurement, which suggests that the binding between TNF-α and anti-TNF-α on the sensor gave a field effect on the underlying CNTs as reported previously.16,17,21

2.2. Detection of TNF-α by Using TNF Sensors Figure 3a illustrates the sensing mechanism of our sensor for the detection of TNF−α. When TNF−α was introduced on the surface of a sensor, TNF−α specifically interacted with anti-TNF−α immobilized on the floating electrodes. Previous reports have shown that this interaction could generate a negative field effect on the underlying CNTs, which should increase the electric currents in p-type semiconducting channels like the p-type channels in our TNF sensor (Figure 2c).15-17 By measuring the electrical current changes of TNF sensors, the levels of TNF−α could be quantitatively evaluated. Sensors immobilized with anti-TNF−α were utilized to detect various concentrations of TNF−α solutions. Figure 3b shows the real-time electrical current changes of a sensor during the addition of TNF−α solutions with a concentration range of 100 fg/L to 100 pg/L. During the sensing measurements, a source-drain bias voltage of 0.1 V was applied on the sensors. The currents in the sensor increased immediately when a TNF-α solution was 6


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introduced. Such quick and real-time responses are one of the main advantages of FET-based sensors, which have been reported by many other groups.15,20-23,26,27 Note that when the sensor was exposed to a TNF-α solution with 1 pg/L or higher concentrations, it exhibited current changes much larger than noise amplitudes. For example, electrical currents in the sensor increased 32, 61 and 83 nA during the addition of TNF-α solutions at concentrations of 1, 10 and 100 pg/L, respectively. It implies that our sensor can be utilized as a highly-sensitive sensor for the TNF-α detection. Here, similar signals were obtained even when TNF-α solutions with various concentrations were added to different sensors separately. Since TNF-α levels in the human blood were reported to be about 3 ng/L, these results show that our TNF sensors have a high sensitivity enough for the monitoring of TNF-α levels in clinical samples.5 On the other hand, there were no significant changes in the electrical currents of an unmodified floating electrode-based CNT-FET sensor by the addition of TNF-α solutions (Figure S1). Moreover, electrical currents in a TNF sensor were monitored for 10 min to test the long-term stability of the sensor signal (Figure S2). The currents in the sensor increased and gradually stabilized after the addition of a TNF-α solution. These results clearly show that the signals of our sensors were caused by the interaction between TNF-α and anti-TNF-α molecules on the floating electrodes of our sensors. A dose dependence curve presented in Figure 3c was obtained by converting the current changes of sensors to normalized conductance changes (∆G/∆Gmax) which is defined as the conductance change ratio over the maximum conductance change of the sensor. We performed the sensing experiments with different sensor chips for statistical analysis. The relative conductance change increased as the concentration of TNF-α increased, and it saturated at around 1 µg/L of TNF-α. Note that the error bars were much smaller than the sensor signals ∆G/∆Gmax from 1 pg/L of TNF-α, indicating the high sensitivity and reliability of our sensors. Moreover, the dose dependent curve was also analyzed by the Hill equation 7



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model for the quantification of TNF-α levels. Previous works show that the responses of label-free sensors can be written like,26,27

∆G /∆Gmax = γ

Cβ KDβ + C β

(1)

where C and KD are the concentration of TNF-α in a solution and the dissociation constant of the target-receptor pair like the TNF-α and anti-TNF-α, respectively. γ is a conversion parameter, and β is Hill’s coefficient. By fitting the dose dependent response data using equation (1), we could estimate the dissociation constant KD of the TNF-α - antibody pair as 36.1 fM which is similar to the previously-reported values for similar TNF- α proteins.17,28 Figure 3d shows the real-time responses of a TNF sensor to various pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and TNF-α which are largely produced in IBD.3,29 During the addition of IL-1β and IL-6, there were no significant changes in electrical currents in the TNF sensor. Note that, IL-1β and IL-6 were added at rather high concentration of 100 µg/L, whereas, the addition of relatively low 1 µg/L concentration of TNF-α caused the sharp increase of the electrical currents. Moreover, the size of IL-1β is quite equivalent with that of TNF-α. It clearly shows that the TNF sensor responded to TNF-α without being disturbed by other cytokines. This result also implies that our TNF sensors could be applied as selective biosensors for the detection of cytokines related with IBD.

2.3. Monitoring the TNF-α Secretion of Raw 264.7 Cells We utilized TNF sensors to monitor the TNF-α secretion of Raw 264.7 cells induced by LPS stimulation. Figure 4a illustrates the stimulation mechanism of a Raw 264.7 cell to secrete TNF-α. Here, the binding of LPS molecules to toll-like receptor 4 (TLR4) on a cell membrane induces the activation of NF-κB signaling pathway, resulting in the production and secretion of pro-inflammatory cytokines including TNF-α.7,30,31 In our experiments, Raw 8


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264.7 cells were cultured in six-well plates (Figure 1b-i) for 2 days and then stimulated by medium containing LPS with different concentrations and treatment times (Figure 1b-ii). The medium solutions obtained after the treatment process were introduced on TNF sensors to quantify the level of TNF-α (Figure 1c). Figure 4b presents the dependence of the TNF-α levels in the medium solutions harvested after the LPS stimulation on the concentrations of LPS. We prepared various concentrations of LPS in the range of 0.01 – 10 µg/mL to stimulate Raw 264.7 cells for 2 h. The TNF sensor exhibited significant responses to the samples, indicating that the TNF-α secretion from Raw 264.7 cells was stimulated by LPS. By fitting the conductance change of TNF sensors via the equation (1), we could quantify the levels of TNF-α in the harvested medium solutions. These results indicate that this cell culture model with our sensor devices could be used to study the efficient dose of stimulating drugs. Figure 4c shows the dependence of the TNF-α levels in harvested medium solutions on LPS stimulation time. For time-dependent experiments, Raw 264.7 cells were stimulated by 10 µg/mL LPS. The results show that the electrical current in the sensor clearly increased by the addition of medium solutions harvested after 1 h. From the conductance changes of TNF sensors, TNF-α levels could be estimated via the equation (1). This indicates that a large amount of TNF-α was produced once Raw 264.7 cells were stimulated by 10 µg/mL LPS for 2 h, which is consistent with reported results.11

2.4. Monitoring the Effect of Anti-inflammatory on NF-κB Signaling Pathway NF-κB signaling pathway has been found to be a key regulator in IBD, and, thus the therapeutic method of IBD has been recently suggested using inhibitors blocking this pathway.7,32 Figure 5a illustrates the inhibition mechanism for NF-κB signaling pathway by lupeol which may be a strong anti-inflammatory and anti-cancer agent. A previous report 9



showed that lupeol inhibited the DNA binding via the suppression of the phosphorylation and degradation of IκBα which was required for the activation of NF-κB signaling pathway. Consequently, lupeol could strongly suppress the production and secretion of LPS-induced pro-inflammatory cytokines including TNF-α in mouse macrophages.7,30,31 Here, our TNF sensors were utilized to monitor the effect of lupeol on the activation of NF-κB signaling pathway in RAW 264.7 cell line via the estimation of TNF-α levels. Figure 5b shows the dependence of LPS-induced TNF-α levels on the different concentrations of lupeol. In this experiment, we pretreated Raw 264.7 cells with lupeol at concentrations in the range of 1 – 1000 µM for 24 h, followed by the stimulation of 10 µg/mL LPS for 2 h. Medium solutions were then harvested and introduced onto a TNF sensor to quantify the level of TNF-α. To quantitatively estimate the inhibition of lupeol on NF-κB pathway, electric current changes were converted to the concentrations of TNF-α using equation (1). When Raw 264.7 cells were pretreated with lupeol, the TNF-α production significantly decreased. This clearly indicates that lupeol acted as an inhibitor on Raw 264.7 cells, blocking the production and secretion of TNF-α. Moreover, the results are consistent with the previous works performed by our group via the ELISA method.7 Furthermore, this result clearly shows that the TNF sensor could be used as a highly-sensitive tool to monitor the effect of versatile drugs related with IBD and other diseases.

3. CONCLUSIONS We report a modified floating electrode based sensor as a simple but effective method for the quantitative evaluation of drugs related with IBD. In this method, floating electrodebased CNT-FET sensors were modified by anti-TNF-α, enabling the selective and quantitative monitoring of TNF-α among other inflammatory cytokines related with IBD. Our TNF sensors exhibited a high sensitivity to TNF-α with the detection limit of 1 pg/L. Importantly, 10


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the TNF sensors could be utilized to monitor the secretion of TNF-α from Raw 264.7 cells under LPS stimulation. Also, the inhibiting effects of lupeol, an anti-inflammatory agent, on the NF-κB pathway signaling were estimated via TNF-α levels by using TNF sensors. Since our method can be applied to rapidly and accurately monitor biomaterials of clinical interests, it should open versatile biomedical applications such as drug screening and inflammation monitoring. 4. EXPERIMENTAL SECTION 4.1. Materials TNF-α (D2D4) XP rabbit mAb (mouse specific), mouse tumor necrosis factor-α (mTNF-α), mouse interleukin-1β (mIL-1β), mouse interleukin-6 (mIL-6), anti-rabbit IgG (alexa fluor 488 conjugate) were purchased from Cell Signaling Technology (Danvers, MA, USA). Semiconducting single-walled carbon nanotubes (CNTs) were purchased from NanoIntegris, Inc. Lipopolysaccharides (LPS), lupeol and other chemical reagents were purchased from Sigma-Aldrich and used as received.

4.2. Fabrication of CNT-FET Sensors First, a CNT film was dispersed in 1,2-dichlorobenzene by applying sonication for 2 h. To form a CNT channel pattern, an octadecyltrichlorosilane (OTS) self-assembled monolayer (SAM) with nonpolar terminal groups was patterned on a SiO2 substrate using the standard photolithography method. Then, the substrate was placed in the CNT solution (0.05 mg/mL) for 1 min and rinsed with 1,2-dichlorobenzene.21,22 Since the OTS SAM prevented the adsorption of CNTs, CNTs were directly assembled on a bare SiO2 region to form the CNT network channel with 3 µm in width and 300 µm in length. Afterward, metal electrodes (Pd/Au 10 nm/30 nm) were fabricated via photolithography, thermal evaporation, and lift-off 11



processes.21,23 Our sensors have five floating electrodes, and the dimension of each floating electrode was 15 µm × 300 µm. Lastly, source and drain electrodes were insulated by a photoresist layer (DNR) to eliminate leakage currents during electrical measurements in aqueous environments.

4.3. Preparation of TNF sensors A CNT-FET sensor was immersed in the solution of N-acetyl-L-cysteine (0.5 M) for 15 min to form linkers with COOH terminals on the gold surface of floating electrodes. After that, the device was immersed in a mixture containing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 0.5 M) and N-hydroxysuccinimide (NHS, 0.1 M) for 1 h to activate the COOH terminals as succinimide ester groups. Subsequently, the CNT-FET sensor was incubated in 100 µg/mL TNF-α (D2D4) XP rabbit mAb (mouse specific) solution for 30 min. As a result, anti-TNF-α molecules were immobilized on the floating electrodes. Finally, 0.1 g/mL bovine serum albumin (BSA) solution was added and incubated for another 30 min to block excess COOH terminals. After each step, the device was gently rinsed in DI water and dried under a N2 flow. The antibody-based kits such as ELISA kits used to quantify TNF-α levels in our previous works could be stored up to one month at 2 – 8 °C.7,32 Therefore, we think our TNF-α sensors based on the same antibody also can be stored for about one month at the same conditions because the shelf-life of antibody-based sensors usually depend on that of antibody. However, since the preparation of our TNF-α sensors was rather simple, TNF-α sensors used in our experiments were freshly prepared prior to sensing measurements.

4.4. Fluorescence Imaging A TNF sensor was incubated in the solution containing 200 µg/mL anti-rabbit IgG conjugated with alexa fluor 488 for 1 h. Then, the sensor was lightly washed with a PBS 12


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buffer to remove excess fluorescent dyes. The fluorescence imaging of the TNF sensor was performed using a fluorescence microscope (Nikon, Eclipse TE2000-U) equipped with an EMCCD (Nikon, DQC-FS) and a fluorescence excitation system (CoolLED, pE) at the excitation wavelength of 490 nm.

4.5. Measurement for the Liquid Gating Effect of Transistors A gold electrode was inserted into deionized water on a TNF sensor to apply a liquid gate bias (Vlg). For liquid gating effect measurements, a gate bias was swept from -0.5 to 0.5 V while a source-drain bias was maintained with 0.1 V. The source-drain current of the transistor was measured by a semiconductor characterization system (Keithley, 4200, USA).

4.6. Preparation of RAW 264.7 Cells The mouse macrophage cell-line RAW 264.7 was provided by the Korean Cell Line Bank (KCLB No. 40071). The RAW 264.7 cells were seeded in six-well plates and cultured for 2 days to facilitate attachment before drug treatments. To stimulate the secretion of TNF-α, the RAW 264.7 cells were cultured for 2 h in the complete growth medium containing various concentrations of LPS. To test the dependence of the TNF-α secretion on LPS-stimulated time, the RAW 264.7 cells were cultured in the complete growth medium containing 10 µg/mL of LPS. The culture media were harvested after defined periods. TNF-α levels in the culture media were evaluated by using TNF sensors. For the NF-κB pathway inhibition experiments, Raw 264.7 cells were pretreated with various concentrations of lupeol for 24 h and subsequently stimulated by 10 µg/mL LPS for 2 h.7,32 After the treatment processes, the culture media were harvested and introduced onto TNF sensors to evaluate TNF-α levels.

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4.7. Electrical Measurements As test samples for sensor responses, a series of TNF-α solutions with well-defined concentrations was prepared by diluting a stock TNF-α solution (100 mg/L). It has been reported that the pH value of a solution could influence FET sensor responses and antibody activity.33,34 Therefore, target solutions were prepared in a physiological buffer solution with a fixed pH value of ~ 7.4 as reported previously.14,16,18 For the detection of sensing signals, TNF sensors were connected to a Keithley 4200 semiconductor analyzer, and a source-drain bias voltage of 0.1 V was maintained during electrical measurements. A droplet of complete growth medium was introduced on the floating electrode region of the sensor, and a sourcedrain current was monitored in response to the addition of TNF-α solutions at various concentrations. For experiments on RAW 264.7 cells, complete growth medium was introduced on TNF sensors and, then the culture media harvested after treatment processes was injected on the sensors. Note that, in the mentioned cell culture condition, the pH values of samples were maintained as a physiological pH (∼ 7.4). During the addition of the culture media, electrical currents in the sensors were measured by the Keithley 4200 semiconductor analyzer in the same conditions as above.

ASSOCIATED CONTENT Supporting Information Additional data for control experiments about the selectivity and stability of TNF-α sensors.

AUTHOR INFORMATION Corresponding Authors *Email: [email protected].

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*Email: [email protected]. *Email: [email protected]. ORCID Viet Anh Pham Ba: 0000-0002-4096-4328 Byung Yang Lee: 0000-0003-0125-2501 Author Contributions V.A.P.B. and Y.M.H. contributed equally to this work. Notes The authors declare no competing financial interest

ACKNOWLEDMENTS This work was supported by the Ministry of Science and ICT (MSIT) of Korea (Nos. 2015R1A2A2A04002733, H-GUARD_ 2013M3A6B2078961). SH acknowledges the support from (2014M3A7B4051591, 2015M3C1A3002152 and 2017R1A2B2006808). This study was also supported by a National Research Foundation of Korea (NRF) grant by the Korean government (The Ministry of Science and ICT; NRF-2015R1A2A2A04002733)

REFERENCES (1)

Abraham, C.; Cho, J. H. Inflammatory bowel disease. N. Engl. J. Med. 2009, 361 (21),

2066-2078. (2)

Barnes, P. J.; Karin, M. Nuclear factor-kappaB: a pivotal transcription factor in

chronic inflammatory diseases. N. Engl. J. Med. 1997, 336 (15), 1066-1071. (3)

Neurath, M. F. Cytokines in inflammatory bowel disease. Nat. Rev. Immunol. 2014, 14

(5), 329-342.

15



(4)

Page 16 of 31

Schreiber, S.; Nikolaus, S.; Hampe, J. Activation of nuclear factor kappa B

inflammatory bowel disease. Gut 1998, 42 (4), 477-484. (5)

Gutierrez, A.; Frances, R.; Amoros, A.; Zapater, P.; Garmendia, M.; Ndongo, M.;

Cano, R.; Jover, R.; Such, J.; Perez-Mateo, M. Cytokine association with bacterial DNA in serum of patients with inflammatory bowel disease. Inflamm. Bowel. Dis. 2009, 15 (4), 508514. (6)

Nielsen, O. H.; Ainsworth, M. A. Tumor necrosis factor inhibitors for inflammatory

bowel disease. N. Engl. J. Med. 2013, 369 (8), 754-762. (7)

Lee, C.; Lee, J. W.; Seo, J. Y.; Hwang, S. W.; Im, J. P.; Kim, J. S. Lupeol inhibits

LPS-induced NF-kappa B signaling in intestinal epithelial cells and macrophages, and attenuates acute and chronic murine colitis. Life Sci. 2016, 146, 100-108. (8)

Tricot, S.; Meyrand, M.; Sammicheli, C.; Elhmouzi-Younes, J.; Corneau, A.; Bertholet,

S.; Malissen, M.; Le Grand, R.; Nuti, S.; Luche, H.; Cosma, A. Evaluating the efficiency of isotope transmission for improved panel design and a comparison of the detection sensitivities of mass cytometer instruments. Cytometry A 2015, 87 (4), 357-368. (9) Orfao,

Bueno, C.; Rodriguez-Caballero, A.; Garcı́a-Montero, A.; Pandiella, A.; Almeida, J.; A.

A

new

method

for

detecting

TNF-α-secreting

cells

using

direct-

immunofluorescence surface membrane stainings. J. Immunol. Methods 2002, 264 (1-2), 7787. (10)

Komatsu, M.; Kobayashi, D.; Saito, K.; Furuya, D.; Yagihashi, A.; Araake, H.; Tsuji,

N.; Sakamaki, S.; Niitsu, Y.; Watanabe, N. Tumor necrosis factor-alpha in serum of patients with inflammatory bowel disease as measured by a highly sensitive immuno-PCR. Clin. Chem. 2001, 47 (7), 1297-1301. (11)

Schildberger, A.; Rossmanith, E.; Eichhorn, T.; Strassl, K.; Weber, V. Monocytes,

peripheral blood mononuclear cells, and THP-1 cells exhibit different cytokine expression 16


Page 17 of 31 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


patterns following stimulation with lipopolysaccharide. Mediators Inflamm. 2013, 2013, 697972. (12)

Szmacinski, H.; Toshchakov, V.; Piao, W.; Lakowicz, J. R. Imaging of Protein

Secretion from a Single Cell Using Plasmonic Substrates. Bionanoscience 2013, 3 (1), 30-36. (13)

Liu, Y.; Yan, J.; Howland, M. C.; Kwa, T.; Revzin, A. Micropatterned aptasensors for

continuous monitoring of cytokine release from human leukocytes. Anal. Chem. 2011, 83 (21), 8286-8292. (14)

Liu, Y.; Kwa, T.; Revzin, A. Simultaneous detection of cell-secreted TNF-alpha and

IFN-gamma using micropatterned aptamer-modified electrodes. Biomaterials 2012, 33 (30), 7347-7355. (15)

Pui, T. S.; Agarwal, A.; Ye, F.; Huang, Y.; Chen, P. Nanoelectronic detection of

triggered secretion of pro-inflammatory cytokines using CMOS compatible silicon nanowires. Biosens. Bioelectron. 2011, 26 (5), 2746-2750. (16)

Zhang, Y.; Chen, R.; Xu, L.; Ning, Y.; Xie, S.; Zhang, G. J. Silicon nanowire

biosensor for highly sensitive and multiplexed detection of oral squamous cell carcinoma biomarkers in saliva. Anal. Sci. 2015, 31 (2), 73-78. (17)

Nam, H.; Oh, B. R.; Chen, P.; Chen, M.; Wi, S.; Wan, W.; Kurabayashi, K.; Liang, X.

Multiple MoS2 Transistors for Sensing Molecule Interaction Kinetics. Sci. Rep. 2015, 5, 10546. (18)

Mazloum-Ardakani,

M.;

Hosseinzadeh,

L.;

Khoshroo,

A.

Ultrasensitive

Electrochemical Immunosensor for Detection of Tumor Necrosis Factor-α Based on Functionalized MWCNT-Gold Nanoparticle/Ionic Liquid Nanocomposite. Electroanalysis 2015, 27 (11), 2518-2526. (19)

Moina, C.; Ybarr, G., Fundamentals and Applications of Immunosensors. In Advances

in Immunoassay Technology, Chiu, N., Ed. InTechOpen: 2012; pp 65-80. 17



(20)

de Moraes, A.; Kubota, L. Recent Trends in Field-Effect Transistors-Based

Immunosensors. Chemosensors 2016, 4 (4), 20. (21)

Kim, B.; Lee, J.; Namgung, S.; Kim, J.; Park, J. Y.; Lee, M. S.; Hong, S. DNA sensors

based on CNT-FET with floating electrodes. Sens. Actuators B: Chem. 2012, 169, 182-187. (22)

Lee, M.; Lee, J.; Kim, T. H.; Lee, H.; Lee, B. Y.; Park, J.; Jhon, Y. M.; Seong, M. J.;

Hong, S. 100 nm scale low-noise sensors based on aligned carbon nanotube networks: overcoming the fundamental limitation of network-based sensors. Nanotechnology 2010, 21 (5), 055504. (23)

Pham Ba, V. A.; Cho, D. G.; Kim, D.; Yoo, H.; Ta, V. T.; Hong, S. Quantitative

electrophysiological monitoring of anti-histamine drug effects on live cells via reusable sensor platforms. Biosens. Bioelectron. 2017, 94, 707-713. (24)

Fujiwara, K.; Watarai, H.; Itoh, H.; Nakahama, E.; Ogawa, N. Measurement of

antibody binding to protein immobilized on gold nanoparticles by localized surface plasmon spectroscopy. Anal. Bioanal. Chem. 2006, 386 (3), 639-644. (25)

Erickson, H. P. Size and shape of protein molecules at the nanometer level determined

by sedimentation, gel filtration, and electron microscopy. Biol. Proced. Online 2009, 11, 3251. (26)

Jin, H. J.; An, J. M.; Park, J.; Moon, S. J.; Hong, S. "Chemical-pain sensor" based on

nanovesicle-carbon nanotube hybrid structures. Biosens. Bioelectron. 2013, 49, 86-91. (27)

Kim, T. H.; Lee, B. Y.; Jaworski, J.; Yokoyama, K.; Chung, W. J.; Wang, E.; Hong,

S.; Majumdar, A.; Lee, S. W. Selective and sensitive TNT sensors using biomimetic polydiacetylene-coated CNT-FETs. ACS Nano 2011, 5 (4), 2824-2830. (28)

Bettazzi, F.; Enayati, L.; Sanchez, I. C.; Motaghed, R.; Mascini, M.; Palchetti, I.

Electrochemical bioassay for the detection of TNF-alpha using magnetic beads and disposable screen-printed array of electrodes. Bioanalysis 2013, 5 (1), 11-19. 18


Page 18 of 31

Page 19 of 31 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


(29)

Atreya, I.; Atreya, R.; Neurath, M. F. NF-kappaB in inflammatory bowel disease. J.

Intern. Med. 2008, 263 (6), 591-596. (30)

Sharif, O.; Bolshakov, V. N.; Raines, S.; Newham, P.; Perkins, N. D. Transcriptional

profiling of the LPS induced NF-kappaB response in macrophages. BMC Immunol. 2007, 8, 1. (31)

Kang, C. C.; Chuang, Y. J.; Tung, K. C.; Chao, C. C.; Tang, C. Y.; Peng, S. C.; Wong,

D. S. A genetic algorithm-based Boolean delay model of intracellular signal transduction in inflammation. BMC Bioinformatics 2011, 12 Suppl 1, S17. (32)

Seo, J. Y.; Lee, C.; Hwang, S. W.; Chun, J.; Im, J. P.; Kim, J. S. Nimbolide Inhibits

Nuclear Factor-capital KA, CyrillicB Pathway in Intestinal Epithelial Cells and Macrophages and Alleviates Experimental Colitis in Mice. Phytother. Res. 2016, 30 (10), 1605-1614. (33)

Kaisti, M. Detection principles of biological and chemical FET sensors. Biosens.

Bioelectron. 2017, 98, 437-448. (34)

Reverberi, R.; Reverberi, L. Factors affecting the antigen-antibody reaction. Blood

Transfus 2007, 5 (4), 227-240.

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Figures

Figure 1. Schematic diagram depicting the preparatory procedure to the TNF-α detection by using a TNF sensor. (a) Schematic diagram showing the fabrication of a TNF sensor: (i) fabrication of a CNT-FET sensor with five floating electrodes; (ii) modification of the floating electrodes by N-acetyl-L-cysteine; (iii) activation of COOH terminal groups as succinimide ester groups; (iv) immobilization of anti-TNF-α on the floating electrodes via linkers. (b) Stimulation of Raw 264.7 cells secreting TNF-α by LPS: (i) culturing Raw 264.7 cells on a six-well plate; (ii) treatment of the cells with LPS for TNF-α secretions. (c) Detection of TNFα in the harvested medium solutions using a TNF sensor. The drawing is not to scale.

20


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Figure 2. Characterization of a TNF sensor based on a CNT-FET sensor with floating electrodes. (a) AFM images (top) of the surface of a floating electrode before and after antiTNF-α immobilization and height profile comparison (bottom) of that surface before (blue) and after (red) the immobilization. (b) Fluorescence image of a TNF sensor obtained after the incubation of the sensor with alexa-fluor-488-conjugated anti-rabbit IgG. The scale bar represents 50 µm. (c) Liquid gating effect of a TNF sensor before (blue) and after (red) binding TNF-α. Here, a source-drain bias of 0.1 V was maintained during the sweep of gate bias from -0.5 V to 0.5 V.

21



Figure 3. Response of TNF sensors to the addition of cytokines. (a) Schematic diagram showing the sensing mechanism of a TNF sensor. Once TNF-α binds to anti-TNF-α on floating electrodes, a field effect is induced on the underlying CNTs. (b) Real-time electrical current changes of a TNF sensor during the addition of TNF-α solutions. A source-drain voltage bias was maintained at 0.1 V. The addition of TNF-α solutions at concentrations of 1, 10 and 100 pg/L caused the increase of electrical currents by 32, 61 and 83 nA, respectively. (c) Dose-dependent relative conductance changes of TNF sensors in response to the concentration changes of TNF-α. Data are expressed as means ± SEM (standard error of the mean), (n=3). (d) Real-time response of a TNF sensor to various cytokines. The addition of IL-1β and IL-6 resulted in the negligible electrical current changes of the TNF sensor, while TNF-α caused a sharp increase for the currents in the sensor. 22


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Figure 4. Monitoring of the TNF-α secretion of Raw 264.7 cells under the stimulation by LPS. (a) Schematic drawing showing the mechanism of the cytokines secretion of Raw 264.7 cells stimulated by LPS (IKK = IκB kinase). (b) Dose response curve for the stimulation of different LPS concentrations on the TNF-α secretion of Raw 264.7 cells. The cells were cultured on six-well plates for 2 days before LPS stimulation. After that, the cells were stimulated by various concentrations of LPS for 2 h. (c) Effect of LPS-stimulated time on the TNF-α secretion of Raw 264.7 cells. Medium solutions harvested at different time after the stimulation of 10 µg/mL LPS. The concentrations of TNF-α were calculated from the relative conductance change of the TNF sensor by using equation (1) and given as means ± SEM, (n=3).

23



Figure 5. Evaluation of the inhibiting effect of lupeol on LPS-induced NF-κB signaling pathway. (a) Schematic drawing depicting the inhibition mechanism of lupeol on the LPSinduced TNF-α secretion in mouse macrophages. (b) Concentration of TNF-α secreted from lupeol-pretreated Raw 264.7 cells. The cells were pretreated with various concentrations of lupeol for 24h, followed by the stimulation of 10 µg/mL LPS for 2h. Lupeol strongly inhibited pro-inflammatory cytokine production in the cells. The bars indicate means ± SEM, (n=3).

24


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TOC Graphic

For Table of Contents Only

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For Table of Contents Only 79x44mm (300 x 300 DPI)


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Figure 1. Schematic diagram depicting the preparatory procedure to the TNF-α detection by using a TNF sensor. (a) Schematic diagram showing the fabrication of a TNF sensor: (i) fabrication of a CNT-FET sensor with five floating electrodes; (ii) modification of the floating electrodes by N-acetyl-L-cysteine; (iii) activation of COOH terminal groups as succinimide ester groups; (iv) immobilization of anti-TNF-α on the floating electrodes via linkers. (b) Stimulation of Raw 264.7 cells secreting TNF-α by LPS: (i) culturing Raw 264.7 cells on a six-well plate; (ii) treatment of the cells with LPS for TNF-α secretions. (c) Detection of TNF-α in the harvested medium solutions using a TNF sensor. The drawing is not to scale. 350x260mm (96 x 96 DPI)



Figure 2. Characterization of a TNF sensor based on a CNT-FET sensor with floating electrodes. (a) AFM images (top) of the surface of a floating electrode before and after anti-TNF-α immobilization and height profile comparison (bottom) of that surface before (blue) and after (red) the immobilization. (b) Fluorescence image of a TNF sensor obtained after the incubation of the sensor with alexa-fluor-488conjugated anti-rabbit IgG. The scale bar represents 50 µm. (c) Liquid gating effect of a TNF sensor before (blue) and after (red) binding TNF-α. Here, a source-drain bias of 0.1 V was maintained during the sweep of gate bias from -0.5 V to 0.5 V. 122x265mm (96 x 96 DPI)


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Figure 3. Response of TNF sensors to the addition of cytokines. (a) Schematic diagram showing the sensing mechanism of a TNF sensor. Once TNF-α binds to anti-TNF-α on floating electrodes, a field effect is induced on the underlying CNTs. (b) Real-time electrical current changes of a TNF sensor during the addition of TNFα solutions. A source-drain voltage bias was maintained at 0.1 V. The addition of TNF-α solutions at concentrations of 1, 10 and 100 pg/L caused the increase of electrical currents by 32, 61 and 83 nA, respectively. (c) Dose-dependent relative conductance changes of TNF sensors in response to the concentration changes of TNF-α. Data are expressed as means ± SEM (standard error of the mean), (n=3). (d) Real-time response of a TNF sensor to various cytokines. The addition of IL-1β and IL-6 resulted in the negligible electrical current changes of the TNF sensor, while TNF-α caused a sharp increase for the currents in the sensor. 250x201mm (96 x 96 DPI)



Figure 4. Monitoring of the TNF-α secretion of Raw 264.7 cells under the stimulation by LPS. (a) Schematic drawing showing the mechanism of the cytokines secretion of Raw 264.7 cells stimulated by LPS (IKK = IκB kinase). (b) Dose response curve for the stimulation of different LPS concentrations on the TNF-α secretion of Raw 264.7 cells. The cells were cultured on six-well plates for 2 days before LPS stimulation. After that, the cells were stimulated by various concentrations of LPS for 2 h. (c) Effect of LPS-stimulated time on the TNF-α secretion of Raw 264.7 cells. Medium solutions harvested at different time after the stimulation of 10 µg/mL LPS. The concentrations of TNF-α were calculated from the relative conductance change of the TNF sensor by using equation (1) and given as means ± SEM, (n=3). 249x204mm (96 x 96 DPI)


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Figure 5. Evaluation of the inhibiting effect of lupeol on LPS-induced NF-κB signaling pathway. (a) Schematic drawing depicting the inhibition mechanism of lupeol on the LPS-induced TNF-α secretion in mouse macrophages. (b) Concentration of TNF-α secreted from lupeol-pretreated Raw 264.7 cells. The cells were pretreated with various concentrations of lupeol for 24h, followed by the stimulation of 10 µg/mL LPS for 2h. Lupeol strongly inhibited pro-inflammatory cytokine production in the cells. The bars indicate means ± SEM, (n=3). 136x193mm (96 x 96 DPI)


Modified Floating Electrode-based Sensors for the Quantitative

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