Electrochemical Immunosensor for TNFÎ±-Mediated Inflammatory

Subscriber access provided by ALBRIGHT COLLEGE

Letter

Electrochemical immunosensor for TNF#mediated inflammatory disease screening Andrea Cruz, Raquel Queirós, Catarina M. Abreu, Catarina Barata, Rosa Fernandes, Rufino Silva, Antonio F. Ambrosio, Ricardo Soares dos Reis, Joana Guimarães, Maria José Sá, João B. Relvas, Paulo P. Freitas, and Inês Mendes Pinto ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00036 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 18 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

1 2

ACS Chemical Neuroscience

Electrochemical immunosensor for TNFα-mediated inflammatory disease screening

3 4 5 6

Authors: Andrea Cruz1, Raquel Queirós1, Catarina M. Abreu1,2, Catarina Barata1,3, Rosa Fernandes4,5, Rufino Silva4,6, Antonio F. Ambrósio4,5, Ricardo Soares-dos-Reis7,8,9, Joana Guimarães7,8,10, Maria José Sá7,11,12, João B. Relvas13, Paulo P. Freitas1 and Inês Mendes Pinto1*

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Affiliations: 1International Iberian Nanotechnology Laboratory, Braga, Portugal. 2Swansea University Medical School, UK. 3Instituto Superior Técnico, University of Lisbon, Portugal. 4Coimbra Institute for Clinical and Biomedical Research, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. 5CNC.IBILI, University of Coimbra, Coimbra, Portugal 6Coimbra University Hospital, Portugal. 7Neurology Department, Centro Hospitalar de São João, Porto, Portugal. 8Department of Clinical Neurosciences and Mental Health, Faculty of Medicine, University of Porto, Portugal. 9Department of Biomedicine, Faculty of Medicine, University of Porto, Portugal 10Center for Drug Discovery and Innovative Medicines (MedInUP), University of Porto, Porto, Portugal 11Energy, Environment and Health Research Unit (FP-ENAS), University Fernando Pessoa, Porto, Portugal 12Faculty of Health Sciences, University Fernando Pessoa, Porto, Portugal 13Institute for Research and Innovation in Health, University of Porto, Portugal.

26 27

*To whom correspondence should be addressed: [email protected].

28

Abstract

29

Inflammation associated to cancer, neurodegenerative, ocular and autoimmune diseases has a

30

considerable impact in public health. Tumor necrosis factor alpha (TNFα) is a key mediator of

31

inflammatory responses, responsible for many of the systemic manifestations during the

32

inflammatory process. Thus, inhibition of TNFα, is a commonplace practice in the treatment of

33

these disorders. Successful therapy requires the ability to determine the appropriate dose of anti-

34

TNFα drugs to be administered in a timely manner, based on circulating TNFα levels.

35

In this article, we report the development of an immunosensor technology able to quantify TNFα

36

at the picogram level in relevant human body fluids, holding the potential to detect inflammation

37

early and monitor TNFα levels during treatment, enabling TNFα-targeted treatments to be tailored

ACS Paragon Plus Environment

ACS Chemical Neuroscience 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

38

according to the immune status of an individual patient. This immunosensor technology is

39

significantly more rapid and sensitive than conventional Enzyme Linked Immunosorbent Assays,

40

maintaining high specificity and requiring small sample volumes. These features might also be

41

advantageous in the context of personalized medicine, as this analytical platform can deliver

42

advanced diagnostics and reduce clinical burden.

43 44

Keywords

45

Electrochemical immunosensor, TNFα, inflammation monitoring, human blood serum, human

46

CSF, human tears

47 48

Inflammation underlies a wide variety of physiological and pathological processes 1. In recent

49

years, increasing evidence has shown a strong association between inflammation and several

50

chronic diseases including many types of cancer 2, autoimmune 3, neurological 4, and ocular

51

disorders

52

therapy monitoring.

53

Tumor necrosis factor alpha (TNFα), a pleiotropic cytokine with distinct functions in homeostasis

54

and disease pathogenesis, has been regarded as one of the major inflammatory mediators 3. TNFα

55

is, in fact, a potential biomarker in Multiple Sclerosis, Parkinson´s disease 4, rheumatoid arthritis

56

6,

57

diseases, mostly rely on the inhibition of the pathological effects of TNFα using anti-TNFα

58

antibodies

59

magnitude of the overall immune activation in a patient and plays a critical role in successful anti-

60

TNFα-based therapies through drug dose adjustment

61

and cerebrospinal fluid (CSF), in the case of neurological diseases 4 and psychiatric disorders 14–

62

17

63

relevance in a wide range of medical fields

64

convenient method of analyzing an accessible body fluid for the investigation of biomarkers in

65

predictive and preventive medicine and for the development of bedside diagnostic tests. As tears

66

have a less complex biological matrix, when compared with blood serum or CSF, research is

5

highlighting the importance of inflammation in disease progression assessment and

as well as, in diabetic retinopathy 5,7,8. Therapeutic interventions in autoimmune TNFα-mediated 3,9,10.

Measurement of blood serum TNFα provides valuable information about the 10–13.

Although examining TNFα in blood

is gaining relevance, its identification in non-invasive body fluids (e.g. tears) might be of 18–20.

Tear fluid sampling potentially provides a


Page 2 of 18

Page 3 of 18 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


67

starting to focus on how neuroinflammatory diseases affect the composition of tear fluids.

68

Although, the chemical composition is not completely known, recent studies showed the existence

69

of high levels of TNFα in tear fluids in patients diagnosed with Parkinson´s disease 21 and diabetic

70

retinopathy

71

Linked Immunosorbent Assay (ELISA). Although ELISA is sensitive, this technique is rather

72

expensive, time-consuming and restricted to clinical and research laboratories, contributing to the

73

difficulty of diagnosing inflammatory diseases rapidly 23.

74

In this work, we report a label free ultrasensitive immunosensor based on an electrochemical

75

impedance spectroscopy (EIS) system comprising a sensing component with functionalized

76

electrodes to transduce TNFα concentrations into electrochemical signals for disease diagnostics

77

and therapeutic monitoring.

22.

Conventionally, TNFα is measured using a labelling-based method - Enzyme

78 79

Results and discussion

80

Impedimetric immunosensor for sensitive TNFα detection.

81

EIS is a very sensitive technique and relies on the measurement of impedance of an

82

electrochemical system subject to alternating current over a range of frequencies. EIS is

83

particularly useful for electrochemical biosensing as it is capable of monitoring changes in

84

electrical properties arising from WE surface modification and biorecognition events. Gold was

85

selected as the material for the WE due to its relatively good stability, favorable electron transfer

86

kinetics with high in-plane conductivity, biocompatibility, and its ability to react with crosslinking

87

agents such as Sulfo-LC-SPDP for anti-TNFα antibodies immobilization onto the WE surface.

88

Bovine Serum Albumin (BSA) was used to prevent non-specific binding (Fig. 1 (inset)).

89

CV and EIS were performed to monitor the electrochemical properties of each immobilization step

90

of the immunosensor (Fig. 2). A solution of 5.0 mmol/L of [Fe(CN)6]3/4- was used as a redox-

91

active probe to observe the electron transfer between the electrolyte and the WE surface. The

92

voltammogram (Fig. 2A) showed quasi-reversible oxidation and reduction wave peaks of

93

[Fe(CN)6]3-/4-. This phenomenon is likely attributed to the formation of a self-assembled

94

monolayer (SAM) and subsequent functionalization steps (anti-TNFα and BSA immobilization)

95

acting as a diffusional barrier to the electron transfer between the redox probe and the WE surface.

96

Conjugation of the anti-TNFα antibody onto the WE surface leads to a decrease in the peak-to-

97

peak current (ΔIp), indicating successful immobilization.



98

Electrochemical impedance spectra was graphically represented by Nyquist plots where both real

99

(Z’) and imaginary (Z’’) values are represented in response to a range of frequencies and after each

100

WE modification (Fig. 2B). The experimental values were fitted using a Randles theoretical model

101

24

102

used parameter to evaluate the concentration of a molecule of interest. Rct increase correlates with

103

electron transfer blockage and therefore the successful modification of the WE surface, as seen in

104

Fig. 2B.

and used to estimate the charge-transfer resistance (Rct) at the WE. Rct, is the most commonly

105 106

Immunosensor analytical performance

107

In healthy individuals, blood serum concentrations of TNFα are reported within the range of 0.7

108

to 20 pg/mL 12,25,26, while in inflammatory diseases TNFα concentrations are above this maximum

109

indicative value 13. TNFα can also be found in other body fluids such as tears, in patients diagnosed

110

with Parkinson´s disease

111

capability to measure different TNFα concentrations. Increasing TNFα concentrations, covering

112

the clinically relevant range of TNFα levels in the human body, from 1 to 50 pg/mL, were spiked

113

in 0.9% NaCl solution and measured in individual sensors.

114

The Nyquist plots (Fig. 3A) clearly demonstrate the effective recognition response of the

115

immunosensor to increasing TNFα concentrations. An increment in the TNFα concentration led to

116

an increase in the Rct values normalized to Rct (BSA average). The linear relationship between the

117

normalized Rct and the log (TNFα) concentration indicates a linear range from 1-25 pg/mL (Fig.

118

3B). The linear regression is given by y=0.6199x+0.02926 with a correlation of R2=0.9852. The

119

calculated LOD was 0.085 pg/mL.

19.

Given these biological ranges, we analyzed the immunosensor

120 121

Immunosensor reproducibility, specificity and selectivity

122

The reproducibility of the immunosensor was assessed in a series of six independent

123

immunosensors. The standard deviation (SD) values obtained were 0.08 and 0.18 for 1 and 10

124

pg/mL TNFα respectively, indicating precision and reproducibility of the immunosensor.

125

To determine the specificity of our immunosensor different concentrations of recombinant IFN

126

and IL-4 cytokines were used. We observed that the Rct variation for IFN and IL-4 were in the

127

same variation range of Rct measurements obtained for BSA immobilization (Fig. 4A and 4B).


Page 4 of 18

Page 5 of 18 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


128

Additionally, we examined the immunosensor ability to detect TNFα in 0.9% NaCl or in a 0.9%

129

NaCl solution with other cytokines (10 pg/mL of IL-4 and IFN). The normalized Rct values

130

obtained for TNFα alone or in a cocktail solution with IL-4 and IFN were not significantly

131

different (Fig. 4B), indicating that the immunosensor is highly selective for TNFα.

132 133

Immunosensor applicability to human sample analysis

134

In order to show the potential applicability of our technology in clinical settings, the immunosensor

135

was tested with non-invasive (tears) and invasive (blood serum and CSF) human samples and

136

compared with conventional ELISA.

137

As shown in Fig. 4C, our immunosensor was not only able to reproduce the ELISA results in

138

human tear samples but also to detect TNFα below the detection limit of ELISA (4 pg/mL

139

eBioscience #88-7346). These results indicate that our immunosensor is more sensitive and

140

accurate than conventional ELISA.

141

The immunosensor was further tested with CSF spiked solution with different concentrations of

142

recombinant TNFα. The Rct variation in response to increasing TNFα concentrations was

143

negligible, suggesting the influence of the complex nature of CSF on the immunosensor

144

performance and the need for sample pre-processing. Undiluted blood serum was also tested,

145

however similar interference was observed. CSF and blood serum samples were further diluted to

146

25% in 0.9% NaCl and spiked with 10 pg/mL of TNFα. The biological matrix effect was

147

minimized and recovered TNFα concentrations were comparable to the ones obtained for spiked

148

0.9% NaCl, as shown in Fig. 4D. Interestingly, TNFα recovery performances (98.5%±25.6 for

149

0.9% NaCl, 88.7±15 for 25% CSF and 109.7±18 for 25% blood serum) obtained with the

150

technology reported in this study are within previously reported values for similar technologies 27.



Page 6 of 18

151

In recent years, efforts have been made towards the development of electrochemical biosensors,

152

for potential biomedical applications, due to their high sensitivity, selectivity, and ease of

153

adaptation for miniaturized formats. A comparative analysis between our technology and

154

previously reported label-free electrochemical sensors

155

performance of our immunosensor able to achieve 10 times lower LOD

156

sample volumes28. Furthermore, our immunosensor is able to detect TNFα in clinically relevant

157

samples (tears, blood serum and CSF) that have never been tested with previously reported sensors

158

which have only been validated in PBS 29 and cell culture media 28.

28–30

highlighted the significant analytical 28

in 20 times smaller

159 160

Conclusion

161

In summary, a new electrochemical label free immunosensor for TNFα quantification was

162

developed based on antibody-antigen interactions at a modified gold-SPE surface. In this work,

163

the functionalization of gold-SPE based on Sulfo-LC-SPDP SAM was shown to be an effective

164

approach

165

functionalization methodology can, in fact, be further explored towards the development of

166

immunosensors capable of detecting other relevant biomarkers.

167

Our immunosensor showed a high consistency with the conventional ELISA for TNFα

168

quantification, yet with a significantly lower LOD. Furthermore, when compared to other

169

biosensing technologies, our immunosensor was the only proven to be effective in detecting TNFα

170

in body fluids such as CSF and tears. The observed limit of TNFα detection in tears was 0.085

171

pg/mL while in CSF and blood serum was 2 pg/mL. Although, CSF and blood serum requires

172

sample dilution prior to biosensor testing, the LOD is still lower than the one obtained with ELISA

173

(4 pg/mL).

174

Overall, the proposed immunosensor holds advantages as of high sensitivity and reproducibility,

175

its ease of use, low processing and signal acquisition times, altogether representing a potentially

176

effective point-of-care strategy for inflammatory disease screening and therapeutic monitoring,

177

particularly relevant in ocular and neurological diseases where detection of TNFα in blood serum,

178

CSF and tears has a potential prognostic value alone or in combination with other relevant

179

biomarkers. Future work envisions the clinical validation of our technology in patient cohorts

180

where the detection of inflammatory mediators are of outmost relevance.

for

antibody

immobilization

and

improved

181


analytical

performance.

This

Page 7 of 18 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


182

Methods

183

Immunosensor design and functionalization.

184

In this study, it was used Gold Screen-printed electrodes (SPEs) (DropSens - C223AT) composed

185

of a printed gold working electrode (WE) (1.6 mm ), gold counter electrode (CE) and a silver

186

pseudo-reference electrode (RE) (Fig. 1). The WE serves as the transduction element in the TNFα

187

and anti-TNFα interaction, while the CE establishes a connection to an electrolytic solution so that

188

a current can be applied to the WE. It should be noted that, with the built-in RE, a desired and

189

stable electrical potential between the WE and electrolyte solution can be maintained during the

190

EIS measurements.

191

Prior to functionalization, the gold-SPEs were pre-cleaned with isopropanol and deionized (DI)

192

water. The sensor was further incubated with a 10 mg/mL solution of Sulfo-LC-SPDP

193

(sulfosuccinimidyl 6- (3'- (2-pyridyldithio) propionamido) hexanoate) (Thermo Fisher) in 10 mM

194

phosphate buffer (PB, pH7.4) with 5% glycerol for 20 minutes at room temperature (RT) and

195

rinsed with PB. The self-assembled monolayer (SAM) was formed through the reaction of the

196

disulfide bond of the Sulfo-LC-SPDP with the gold surface of the WE. Anti-TNFα antibodies

197

(eBioscience #88-7346) were bound to SAM via overnight incubation, at 4ºC, of 0.25 µg/µL

198

antibody solution (in PB) with 5% glycerol followed by washing with PB. 1% BSA solution in PB

199

was incubated at RT for 30 minutes with 5% glycerol to prevent non-specific interactions with the

200

anti-TNFα antibody.

201 202

Cyclic voltammetry and electrochemical impedance spectroscopy measurements.

203

Cyclic voltammetry (CV) was performed during the electrode functionalization process to confirm

204

anti-TNFα antibody immobilization and to characterize the gold WE surface in terms of electron

205

transfer kinetics and redox processes 31. CV measurements were conducted using a potential scan

206

from -0.4 to +0.4 V, at a scan rate 0.05 V/s. EIS characterization was carried out to evaluate the

207

functionalization process and was further used for TNFα detection and quantification tests. EIS

208

measurements were performed using an electrolyte solution with the redox probe [Fe(CN)6]3-/4-, at

209

a fixed potential of +0.125 V, using a sinusoidal perturbation with amplitude of 5 mV and a

210

frequency range of 1E+5-0.1 Hz. Impedance data was fitted to a Randles equivalent circuit

211

[Rs(CPE[RctW])], using the Nova Software. This circuit includes the ohmic resistance of the



Page 8 of 18

212

electrolyte solution (Rs) the Warburg impedance (Zw) resulting from the diffusion of the redox-

213

probe, constant phase element (CPE), and the charge-transfer resistance (Rct). The latter two

214

components, CPE and Rct, represent interfacial properties of the WE electrode, which are highly

215

sensitive to the surface modification. The extracted Rct value was further used for the determination

216

of the calibration curve and subsequent extrapolation of TNFα concentrations.

217

All electrochemical measurements (CV and EIS) were performed using a potentiostat/galvanostat,

218

equipped

219

PGSTAT302N/FRA32M) and controlled by NOVA Software. The electrolyte solution was

220

prepared at concentration of 5.0 mM of [Fe(CN)6]3-/4- in 10 mM phosphate buffered saline (PBS)

221

buffer, pH 7.4. The PBS tablets and Fe(CN)6 redox pair reagents were obtained from Sigma

222

Aldrich-UK.

with

a

Frequency

Response

Analysis

module

(Metrohm

Autolab

-

223 224

Immunosensor optimization and testing for TNFα quantification.

225

The TNFα calibration curve was obtained after testing 1 µl of different concentrations of

226

recombinant TNFα solution (eBioscience #88-7346). TNFα solutions, ranging from 1 pg/mL to 50

227

pg/mL, were prepared by diluting 15 ng/mL standard TNFα solutions in 0.9% NaCl. A period of

228

90 minutes was allowed for antigen/antibody interaction, followed by Milli-Q DI rinsing. The

229

calibration curve was determined based on the linear correlation between the normalized Rct value

230

((Rct

231

concentration. LOD was defined as the TNFα concentration at which the calibration curve

232

corresponds to ((3*SDblank-b)/m), where SDblank corresponds to the standard deviation of BSA

233

Rct values. The m (slope) and b (the Y Intercept) values were obtained from the calibration curve.

234

The specificity and selectivity of the immunosensor was tested by analyzing the Rct values to

235

different concentrations of recombinant IFN and IL-4 cytokines (Preprotech® #400-20 and #400-

236

04, respectively) or in a cocktail solution with TNFα, IFN and IL-4.

(TNFα)

- Rct

(BSA average))/

- Rct

(BSA average))

and the common logarithm (base 10) of TNFα

237 238

Human tears, cerebrospinal fluid and blood serum sample collection and processing.

239

Tear samples were collected from eyes of five healthy volunteers between the age of 23 and 51,

240

upon written informed consent. Inclusion criteria were as follows: no history of diabetes or chronic

241

diseases, no ocular diseases requiring topical ocular treatments, no abnormal lid anatomy or


Page 9 of 18 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


242

blinking function in either eye, no dry eye resulting from scarring, no history of any systemic or

243

ocular disorder, no contact lens wear in the last 8 h. Tear samples were collected in the morning

244

between 8-10 am, by the Investigator, wearing gloves, by placing a sterile ophthalmology

245

diagnostic strip (Dina strip Schirmer-Plus, Dina Hitex) in standardized conditions (strip inserted

246

in the inferior cul-de-sac for 5 min, while subjects closed their eyes without any anesthetic) of each

247

anonymized healthy subject. Following collection, the wet portion of the strip was soaked in 0.9%

248

NaCl for 1h to elute tear proteins 32. After obtaining informed written patient consent, CSF samples

249

were obtained during routine lumbar puncture. CSF was centrifuged for 10 min at 2,000 g, and the

250

supernatant was frozen at -80ºC. After thawing, CSF was diluted in 0.9% NaCl for further analysis.

251

Blood serum were obtained from healthy volunteers by standard venipuncture, upon written

252

consent. Blood serum was frozen at -80ºC. After thawing, blood serum was diluted in 0.9% NaCl

253

for further analysis. The use of human samples was approved by the Ethics Committee of Centro

254

Hospitalar de São João/Faculdade de Medicina da Universidade do Porto.

255 256

ELISA testing for TNFα detection in human samples.

257

TNFα was measured, in human tear samples, using commercially available ELISA kits

258

(eBioscience #88-7346) according to the manufacturer’s specifications.

259 260

Statistical Analysis

261

For the performed statistical analysis, the GraphPad Prism Software vs. 6.0 (GraphPad Software

262

Inc.) was used. Differences between groups were compared using Wilcoxon signed-rank test.

263

Results are expressed as a mean ± standard deviation. Differences were considered significant at

264

p < 0.05.

265 266 267

Author Contributions

268

A.C.: immunosensor surface functionalization, optimization and validation, ELISA testing, data

269

analysis, figures and manuscript preparation; R.Q.: biosensor surface functionalization and

270

optimization, data analysis, manuscript preparation; C.M.A.: immunosensor testing, data analysis,

271

manuscript preparation; C.B.: immunosensor surface functionalization and optimization; R.F.,



272

F.A., R.S., R.S.R., JG and M.J.S: clinical sample collection and manuscript preparation; J.B.R:

273

manuscript preparation; P.P.F.: project supervision and manuscript preparation; I.M.P.: overall

274

project design, supervision and manuscript preparation.

275 276

Acknowledgments

277

The authors acknowledge Sofia Teixeira and Sofia Domingues for critical reading of this

278

manuscript. I.M.P. and A.C. acknowledge the financial support from the Marie Curie COFUND

279

Programme “NanoTRAINforGrowth”, from the European Union’s Seventh Framework

280

Programme for research, technological development and demonstration under grant agreement no

281

600375. This article is a result of the project Nanotechnology based functional solutions (NORTE-

282

01-0145-FEDER-000019), co-financed by Norte Portugal Regional Operational Programme

283

(NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European

284

Regional Development Fund (ERDF).

285 286

Declarations of interest. The authors declare no competing financial interest

287 288

References

289 290

(1)

Medzhitov, R. Origin and Physiological Roles of Inflammation. Nature 2008, 454 (7203), 428–435. https://doi.org/10.1038/nature07201.

291 292 293

(2)

Crusz, S. M.; Balkwill, F. R. Inflammation and Cancer: Advances and New Agents. Nature Reviews Clinical Oncology. Nature Publishing Group October 2015, pp 584–596. https://doi.org/10.1038/nrclinonc.2015.105.

294 295 296

(3)

Kalliolias, G. D.; Ivashkiv, L. B. TNF Biology, Pathogenic Mechanisms and Emerging Therapeutic Strategies. Nat. Rev. Rheumatol. 2016, 12 (1), 49–62. https://doi.org/10.1038/nrrheum.2015.169.

297 298 299 300

(4)

Abreu, C. M.; Soares-dos-Reis, R.; Melo, P. N.; Relvas, J. B.; Guimarães, J.; Sá, M. J.; Cruz, A. P.; Mendes Pinto, I. Emerging Biosensing Technologies for Neuroinflammatory and Neurodegenerative Disease Diagnostics. Front. Mol. Neurosci. 2018, 11, 164. https://doi.org/10.3389/fnmol.2018.00164.

301 302 303 304

(5)

Nalini., M.; Raghavulu, B. V.; Annapurna, A.; Avinash, P.; Chandi, V.; Swathi, N.; Wasim. Correlation of Various Serum Biomarkers with the Severity of Diabetic Retinopathy. Diabetes Metab. Syndr. Clin. Res. Rev. 2017, 11, S451–S454. https://doi.org/10.1016/j.dsx.2017.03.034.

305

(6)

Yamanaka, H. TNF as a Target of Inflammation in Rheumatoid Arthritis. Endocr. Metab.


Page 10 of 18

Page 11 of 18 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


Immune Disord. Drug Targets 2015, 15 (2), 129–134. https://doi.org/10.2174/1871530315666150316121808.

306 307 308 309 310 311

(7)

Boss, J. D.; Singh, P. K.; Pandya, H. K.; Tosi, J.; Kim, C.; Tewari, A.; Juzych, M. S.; Abrams, G. W.; Kumar, A. Assessment of Neurotrophins and Inflammatory Mediators in Vitreous of Patients with Diabetic Retinopathy. Investig. Ophthalmol. Vis. Sci. 2017, 58 (12), 5594–5603. https://doi.org/10.1167/iovs.17-21973.

312 313 314 315

(8)

Preciado-Puga, M.; Malacara, J.; Fajardo-Araujo, M.; Wröbel, K.; Wröbel, K.; Kornhauser-Araujo, C.; Garay-Sevilla, M. Markers of the Progression of Complications in Patients with Type 2 Diabetes: A One-Year Longitudinal Study. Exp. Clin. Endocrinol. Diabetes 2014, 122 (08), 484–490. https://doi.org/10.1055/s-0034-1372594.

316 317 318 319

(9)

Grell, M.; Douni, E.; Wajant, H.; Löhden, M.; Clauss, M.; Maxeiner, B.; Georgopoulos, S.; Lesslauer, W.; Kollias, G.; Pfizenmaier, K.; et al. The Transmembrane Form of Tumor Necrosis Factor Is the Prime Activating Ligand of the 80 KDa Tumor Necrosis Factor Receptor. Cell 1995, 83 (5), 793–802. https://doi.org/10.1016/0092-8674(95)90192-2.

320 321 322

(10)

Zänker, M.; Becher, G.; Arbach, O.; Maurer, M.; Stuhlmüller, B.; Schäfer, A.; Strohner, P.; Brand, J. Improved Adalimumab Dose Decision with Comprehensive Diagnostics Data. Clin. Exp. Rheumatol. 2018, 36 (1), 136–139.

323 324 325 326

(11)

Takeuchi, T.; Miyasaka, N.; Tatsuki, Y.; Yano, T.; Yoshinari, T.; Abe, T.; Koike, T. Baseline Tumour Necrosis Factor Alpha Levels Predict the Necessity for Dose Escalation of Infliximab Therapy in Patients with Rheumatoid Arthritis. Ann Rheum Dis 2011, 70 (7), 1208–1215. https://doi.org/10.1136/ard.2011.153023.

327 328 329 330

(12)

Scully, P.; McKernan, D. P.; Keohane, J.; Groeger, D.; Shanahan, F.; Dinan, T. G.; Quigley, E. M. Plasma Cytokine Profiles in Females With Irritable Bowel Syndrome and Extra-Intestinal Co-Morbidity. Am. J. Gastroenterol. 2010, 105 (10), 2235–2243. https://doi.org/10.1038/ajg.2010.159.

331 332 333 334 335

(13)

Martínez-Borra, J.; López-Larrea, C.; González, S.; Fuentes, D.; Dieguez, A.; Deschamps, E. M.; Pérez-Pariente, J. M.; López-Vázquez, A.; de Francisco, R.; Rodrigo, L. High Serum Tumor Necrosis Factor-Alpha Levels Are Associated with Lack of Response to Infliximab in Fistulizing Crohn’s Disease. Am. J. Gastroenterol. 2002, 97 (9), 2350–2356. https://doi.org/10.1111/j.1572-0241.2002.05990.x.

336 337 338 339

(14)

Wu, W.; Guan, Y.; Zhao, G.; Fu, X.-J.; Guo, T.-Z.; Liu, Y.-T.; Ren, X.-L.; Wang, W.; Liu, H.-R.; Li, Y.-Q. Elevated IL-6 and TNF-α Levels in Cerebrospinal Fluid of Subarachnoid Hemorrhage Patients. Mol. Neurobiol. 2016, 53 (5), 3277–3285. https://doi.org/10.1007/s12035-015-9268-1.

340 341 342 343 344

(15)

Trenova, A. G.; Slavov, G. S.; Draganova-Filipova, M. N.; Mateva, N. G.; Manova, M. G.; Miteva, L. D.; Stanilova, S. A. Circulating Levels of Interleukin-17A, Tumor Necrosis Factor-Alpha, Interleukin-18, Interleukin-10, and Cognitive Performance of Patients with Relapsing-Remitting Multiple Sclerosis. Neurol. Res. 2018, 40 (3), 153–159. https://doi.org/10.1080/01616412.2017.1420522.

345 346 347

(16)

Kouchaki, E.; Kakhaki, R. D.; Tamtaji, O. R.; Dadgostar, E.; Behnam, M.; Nikoueinejad, H.; Akbari, H. Increased Serum Levels of TNF-α and Decreased Serum Levels of IL-27 in Patients with Parkinson Disease and Their Correlation with Disease Severity. Clin.



Neurol. Neurosurg. 2018, 166, 76–79. https://doi.org/10.1016/j.clineuro.2018.01.022.

348 349 350 351 352

(17)

Ruland, T.; Chan, M. K.; Stocki, P.; Grosse, L.; Rothermundt, M.; Cooper, J. D.; Arolt, V.; Bahn, S. Molecular Serum Signature of Treatment Resistant Depression. Psychopharmacology (Berl). 2016, 233 (15–16), 3051–3059. https://doi.org/10.1007/s00213-016-4348-0.

353 354 355 356 357

(18)

Bellagambi, F. G.; Baraket, A.; Longo, A.; Vatteroni, M.; Zine, N.; Bausells, J.; Fuoco, R.; Di Francesco, F.; Salvo, P.; Karanasiou, G. S.; et al. Electrochemical Biosensor Platform for TNF-α Cytokines Detection in Both Artificial and Human Saliva: Heart Failure. Sensors Actuators, B Chem. 2017, 251, 1026–1033. https://doi.org/10.1016/j.snb.2017.05.169.

358 359 360

(19)

Çomoğlu, S. S.; Güven, H.; Acar, M.; Öztürk, G.; Koçer, B. Tear Levels of Tumor Necrosis Factor-Alpha in Patients with Parkinson’s Disease. Neurosci. Lett. 2013, 553, 63–67. https://doi.org/10.1016/J.NEULET.2013.08.019.

361 362 363 364

(20)

Lee, S. Y.; Han, S. J.; Nam, S. M.; Yoon, S. C.; Ahn, J. M.; Kim, T.-I.; Kim, E. K.; Seo, K. Y. Analysis of Tear Cytokines and Clinical Correlations in Sjögren Syndrome Dry Eye Patients and Non–Sjögren Syndrome Dry Eye Patients. Am. J. Ophthalmol. 2013, 156 (2), 247–253.e1. https://doi.org/10.1016/J.AJO.2013.04.003.

365 366 367

(21)

Comoglu, S. S.; Guven, H.; Acar, M.; Ozturk, G.; Kocer, B. Tear Levels of Tumor Necrosis Factor-Alpha in Patients with Parkinson’s Disease. Neurosci. Lett. 2013. https://doi.org/10.1016/j.neulet.2013.08.019.

368 369 370 371

(22)

Costagliola, C.; Romano, V.; De Tollis, M.; Aceto, F.; Dell’Omo, R.; Romano, M. R.; Pedicino, C.; Semeraro, F. TNF-Alpha Levels in Tears: A Novel Biomarker to Assess the Degree of Diabetic Retinopathy. Mediators Inflamm. 2013, 2013, 1–6. https://doi.org/10.1155/2013/629529.

372 373 374

(23)

Tze Sian Pui; Tushar Bansal; Patthara Kongsuphol; Sunil K. Arya. Highly Sensitive Label Free Biosensor for Tumor Necrosis Factor. Int. J. Medical, Heal. Biomed. Bioeng. Pharm. Eng. 2012, 6 (9), 443–446.

375 376

(24)

Randles, J. E. B. Kinetics of Rapid Electrode Reactions. Faraday Discuss. 1947, 1, 11–19. https://doi.org/10.1039/DF9470100011.

377 378 379 380

(25)

Todd, J.; Simpson, P.; Estis, J.; Torres, V.; Wub, A. H. B. Reference Range and Shortand Long-Term Biological Variation of Interleukin (IL)-6, IL-17A and Tissue Necrosis Factor-Alpha Using High Sensitivity Assays. Cytokine 2013, 64 (3), 660–665. https://doi.org/10.1016/j.cyto.2013.09.018.

381 382 383 384

(26)

Arican, O.; Aral, M.; Sasmaz, S.; Ciragil, P. Serum Levels of TNF-α, IFN-γ, IL-6, IL-8, IL-12, IL-17, and IL-18 in Patients with Active Psoriasis and Correlation with Disease Severity. Mediators Inflamm. 2005, 2005 (5), 273–279. https://doi.org/10.1155/MI.2005.273.

385 386 387 388

(27)

Xing, Y.; Feng, X. Z.; Zhang, L.; Hou, J.; Han, G. C.; Chen, Z. A Sensitive and Selective Electrochemical Biosensor for the Determination of Beta-Amyloid Oligomer by Inhibiting the Peptide-Triggered in Situ Assembly of Silver Nanoparticles. Int. J. Nanomedicine 2017, 12, 3171–3179. https://doi.org/10.2147/IJN.S132776.


Page 12 of 18

Page 13 of 18 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


389 390 391 392

(28)

Pui, T. S.; Kongsuphol, P.; Arya, S. K.; Bansal, T. Sensors and Actuators B : Chemical Detection of Tumor Necrosis Factor ( TNF- α ) in Cell Culture Medium with Label Free Electrochemical Impedance Spectroscopy. Sensors Actuators B. Chem. 2013, 181, 494– 500. https://doi.org/10.1016/j.snb.2013.02.019.

393 394 395 396

(29)

Baraket, A.; Lee, M.; Zine, N.; Yaakoubi, N.; Trivella, M. G.; Elaissari, A.; Sigaud, M.; Jaffrezic-renault, N.; Errachid, A.; Analytiques, S.; et al. A Flexible Label-Free Biosensor Sensitive and Selective to TNF- Α : Application for Chronic Heart Failure. Sensors and Transducers 2014, 27 (May), 15–21.

397 398 399 400

(30)

Mazloum-Ardakani, M.; Hosseinzadeh, L.; Taleat, Z. Synthesis and Electrocatalytic Effect of Ag@Pt Core-Shell Nanoparticles Supported on Reduced Graphene Oxide for Sensitive and Simple Label-Free Electrochemical Aptasensor. Biosens. Bioelectron. 2015, 74, 30–36. https://doi.org/10.1016/j.bios.2015.05.072.

401 402

(31)

Bariya, M.; Nyein, H. Y. Y.; Javey, A. Wearable Sweat Sensors. Nat. Electron. 2018, 1 (3), 160–171. https://doi.org/10.1038/s41928-018-0043-y.

403 404 405

(32)

Denisin, A. K.; Karns, K.; Herr, A. E. Post-Collection Processing of Schirmer StripCollected Human Tear Fluid Impacts Protein Content. Analyst 2012, 137 (21), 5088. https://doi.org/10.1039/c2an35821b.

406 407 408 409 410



411

Figures:

412

413 414 415 416 417 418 419 420

Fig. 1. Schematic representation of the immunosensor biofunctionalization process. The gold working electrode (WE) of the electrochemical system was functionalized in order to provide specific TNFα biorecognition. The sulfo-LC-SPDP crosslinker interacted with the gold surface to improve anti-TNFα binding to the WE. Subsequently, potential unspecific binding sites were blocked with BSA to reduce interference. TNFα detection was performed by electrochemical impedance spectroscopy (EIS) analysis.

421 422 423 424 425 426 427 428 429


Page 14 of 18

Page 15 of 18 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


430 431 432 433 434 435 436 437 438

Fig. 2. Cyclic voltammogram and electrochemical impedance spectra of the biofunctionalization process. (A) Cyclic voltammetry was used to monitor the [Fe(CN)6]3-/4oxidation and reduction peak during the functionalization process upon application of a potential sweep between -0.4V and +0.4V. (B) Nyquist plots of the functionalization process were obtained in 5.0 mM [Fe(CN)6]3-/4- PBS buffer pH 7.4 using a sinusoidal potential perturbation of 5 mV over a frequency range of 1E+5-0.1 Hz

439 440 441 442



443 444 445 446 447 448 449 450 451 452

Fig. 3. Calibration curve of the TNFα immunosensor. (A) Nyquist plots were obtained in 5.0 mM [Fe(CN)6]3-/4- PBS buffer pH 7.4, previously incubated in increasing concentrations of TNFα using a sinusoidal potential perturbation of 5 mV over a frequency range of 1E+5-0.1 Hz. Randles circuit (inset) was used to fit the electrochemical impedance data in which Rs, CPE, W and Rct represent electrolyte resistance, constant phase element, Warburg element and charge transfer resistance, respectively. (B) Rct values (n=6) were extracted from the Nyquist plots shown in (A), normalized to BSA and plotted against the logarithm (log) of the concentration of TNFα. Error bars correspond to SD.

453 454 455


Page 16 of 18

Page 17 of 18 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


456 457 458 459 460 461 462 463 464 465 466 467 468

Fig. 4. TNFα immunosensor specificity, selectivity and applicability to human sample analysis. (A) TNFα immunosensor specificity was tested against increasing concentrations of interferon gamma (IFN) and interleukin-4 (IL-4) (n=3) (B) Immunosensor selectivity and specificity was analyzed by comparing normalized Rct values obtained for 10 pg/mL of TNFα spiked in 0.9% NaCl; in a cocktail solution of 10 pg/mL TNFα, IL-4, IFN each and in 0.9% NaCl solution spiked with 10 pg/mL of IFN or IL-4. Per each condition, a sample size of 3 was considered. n.s.: not significant. (C) TNFα quantification in tear samples of 5 healthy individuals using the TNFα immunosensor and conventional ELISA. Two replicas of each individual were tested. SD corresponds to Standard Deviation and n.d. corresponds to not detected. (D) TNFα recovery tests in human cerebral spinal fluid (CSF) and blood serum diluted at 25% in 0.9% NaCl 0.9% (n=4 and 3, respectively).

469 470 471



472 473

For Table of Contents Only

474

475 476


Page 18 of 18

Electrochemical Immunosensor for TNFÎ±-Mediated Inflammatory

Recommend Documents