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: ines.m.pinto@inl.int.
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
ACS Paragon Plus Environment
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
ACS Chemical Neuroscience
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.
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
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).
ACS Paragon Plus Environment
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
ACS Chemical Neuroscience
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.
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
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
ACS Paragon Plus Environment
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
ACS Chemical Neuroscience
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
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
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
ACS Paragon Plus Environment
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
ACS Chemical Neuroscience
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.,
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
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.
ACS Paragon Plus Environment
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
ACS Chemical Neuroscience
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.
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
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.
ACS Paragon Plus Environment
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
ACS Chemical Neuroscience
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
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
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
ACS Paragon Plus Environment
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
ACS Chemical Neuroscience
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
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
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
ACS Paragon Plus Environment
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
ACS Chemical Neuroscience
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
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
472 473
For Table of Contents Only
474
475 476
ACS Paragon Plus Environment
Page 18 of 18