Piezoelectric immunosensor based on antibody recognition of immobilized open-tissue transglutaminase: An innovative perspective on diagnostic devices for celiac disease

Accepted Manuscript Title: Piezoelectric immunosensor based on antibody recognition of immobilized Open-tissue transglutaminase: An innovative perspective on diagnostic devices for celiac disease Author: Anita Manfredi Monica Mattarozzi Marco Giannetto Maria Careri PII: DOI: Reference:

S0925-4005(14)00537-1 http://dx.doi.org/doi:10.1016/j.snb.2014.05.018 SNB 16892

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

25-2-2014 21-3-2014 4-5-2014

Please cite this article as: A. Manfredi MattarozziM. GiannettoM. Careri Piezoelectric immunosensor based on antibody recognition of immobilized Open-tissue transglutaminase: an innovative perspective on diagnostic devices for celiac disease, Sensors and Actuators B: Chemical (2014), http://dx.doi.org/10.1016/j.snb.2014.05.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

* Correct Manuscript

1

Piezoelectric immunosensor based on antibody recognition of immobilized Open-tissue

2

transglutaminase: an innovative perspective on diagnostic devices for celiac disease

3 4

Anita Manfredia, Monica Mattarozzia,b*, Marco Giannettoa,b, Maria Careria,b

5 6

a

7

Parma

8

b

9

181/A, 43124, Parma

ip t

Dipartimento di Chimica, Università degli Studi di Parma, Parco Area delle Scienze 17/A, 43124,

cr

Centro Interdipartimentale SITEIA.PR, Università degli Studi di Parma, Parco Area delle Scienze

10

*Corresponding author.

12

Phone: +39 0521 905446. Fax: +39 0521 905557. E-mail: [email protected]

us

11

an

13 14

Abstract

31

Keywords: Quartz crystal microbalance (QCM) immunosensor, Open-tissue transglutaminase,

32

Anti-tissue tranglutaminase antibodies, Flow-through cell, Nanogold amplification, Celiac disease

M

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Ac

ce pt

ed

A piezoelectric immunosensor was developed for the first time for direct detection of anti-tissue transglutaminase antibodies (anti-tTG), very specific biomarkers for reliable and early diagnosis of celiac disease. Since the inflammation processes associated to the pathology’s occurence involve tTG structural changes from closed to open conformation as well the extended structure has been demonstrated to have higher diagnostic accuracy if compared with closed conformation, the new strategy undertaken in this study was based on the immobilization of tTG enzyme in its open conformation as receptor on immunosensor surface. Ten nm-sized gold nanoparticles conjugated with secondary antibodies were exploited for signal amplification. Liquid phase detection conditions using a laminar flow cell were properly selected in order to have a good signal stability both in dynamic and in static modes. Optimization of the operating conditions, by experimental design on mouse anti-tTG antibodies in serum, allowed us to obtain a model for the realization of a reliable piezoelectric immunosensor with high potential as diagnostic device for the determination of human autoantibodies of celiac patients.

33 34 35 36 37 38 39 1 Page 1 of 23

ce pt

ed

M

an

us

cr

ip t

1. Introduction In recent years, biosensors have been explored for their application in food [1-3], environmental [4,5] and biological [6,7] fields, with particular attention to the development of devices for rapid and reliable quality control and medical diagnosis. Concerning the transducers, quartz crystal microbalance (QCM)-based sensors are gaining an increasing interest as competitive tools for biosensing applications and clinical bioassays in liquid phase due to high sensitivity, low cost and real-time output. QCM-devices are based on the relationship between mass deposited/adsorbed on the crystal surface and the resonant frequency variations. By proper crystal surface functionalization, it is possible to tune sensor selectivity in order to obtain a layer interacting with specific analytes [8-12]. Non-viscoelastic small mass added to the surface can be quantified at ng levels using the linear Sauerbray equation, which is applicable only in gas phase analysis [13]. Otherwise, in liquid-phase measurements two indistinguishable contributions have to be considered for the total frequency decrease: one is due to the interacting bounded mass and the other originates from the viscoelastic properties of the liquid phase and the overlayed material [14]. In fact, solution effectively adds a mass component to the oscillating crystal and the frequency varies with the square root of liquid density and viscosity, as expressed in Kanazawa and Gordon’s equation [1517]. Therefore, for reliable QCM biosensor applications, the properties of the liquid phase should be taken under strict control and should not significantly change during analysis. In addition, in the automated systems, the use of a cell operating in laminar flow conditions allows to minimize mechanical perturbation and to stabilize the recorded signal. Piezoelectric immunosensors have been proposed as valid alternative to classic colorimetric immunoassay, such as ELISA, for a wide variety of application areas, relying upon the antigen-antibody interaction to carry out molecular recognition of small molecules [18,19], biomolecules [20-22] and pathogens [23,24]. Celiac disease (CD) is an autoimmune disorder that occurs in genetically predisposed people after the ingestion of prolamins found in common grains. The rapidly increasing prevalence of CD, now recognized as one of the most common autoimmune diseases in the world, has focused research interest towards its early and reliable diagnosis. Some biomarkers are involved in the disease and specific autoantibodies, such as anti-tissue transglutaminase (anti-tTG), anti-endomysium and antideaminated gliadin peptide antibodies, are used for disease diagnosis. In particular, assays based on detection of anti-tTG, which is present at high concentration in celiac patients, have been demonstrated very sensitive and specific for CD diagnosis. Although the high level of IgA anti-tTG in most CD patients, it has to be taken into account that in 1.7-2.6 % of cases an IgA deficiency occurs, leading to false negative results, so that IgG antibodies screening should be performed [25]. Recently, it has been demonstrated that tTG enzymatic activity is tightly regulated, requiring in particular the presence of Ca2+ ions as well as an intra-molecular disulfide bond to induce tTG structural changes from closed to open conformation. In this way, the extended structure exposes the site involved in the catalytic activity, thus increasing autoantibody binding [26-29]. Actually, the open and active tTG presents a higher diagnostic accuracy with respect to the closed conformation: the latter form is generally present in healthy tissue, whereas the extended one is more prominent during inflammation [30]. As for the biosensors recently developed for anti-tTG detection, all of them are based on electrochemical or optical transduction mechanisms and on antibody recognition of tTG antigen immobilized in closed conformation [31-38]. In this context, the innovative aspects of the study lie in the first application of piezoelectric transduction and exploitation of Open-tTG specific bioreceptor, as a new and accurate diagnostic tool for celiac

Ac

40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83

2 Page 2 of 23

disease. The frequency change arising from the antibody-antigen interaction on the sensor surface was amplified by using gold nanoparticles conjugated with secondary antibodies in order to improve sensitivity. 2. Materials and methods 2.1 Chemicals

2.2 QCM system

ce pt

ed

M

an

us

cr

ip t

Human tissue transglutaminase recombinantly produced in insect cells and stabilized in the open conformation (Open-tTG™) and mouse monoclonal IgG antibody to human tissue transglutaminase were purchased from Zedira (Darmstadt, Germany). Anti-Mouse IgG-Gold 10 nm colloidal gold produced in goat (Ab-AuNPs), bovine serum albumin (BSA), tris(hydroxymethyl)aminomethane hydrochloride (TRIS-HCl), N-hydroxysuccinimide (NHS), sodium chloride (NaCl), potassium phosphate monobasic (KH2PO4), sodium phosphate dibasic dodecahydrate (Na2HPO4·12H2O), 11mercaptoundecanoic acid (MUA), DL-dithiothreitol (DTT), potassium chloride (KCl), Tween 20, 2-propanol, calcium chloride hexahydrate (CaCl2·6H2O), glycine (Gly), hydrochloric acid (HCl) and human serum were purchased from Sigma Aldrich (St. Louis, MO, USA). 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC), absolute ethanol, sulfuric acid (H2SO4) and urea were from Fluka (St. Louis, MO, USA). Ethylenediaminetetraacetic acid (EDTA), sodium hydroxide (NaOH) and hydrogen peroxide (H2O2) were from Riedel-de Haën (St. Louis, MO, USA). The composition of the enzyme buffer (EB), used during Open-tTG immobilization, was the following: 0.02 M TRIS-HCl, 0.3 M NaCl, 0.001 M EDTA, 0.001 M DTT, 0.01 M CaCl2·6H2O (pH = 7.2). Phosphate buffer saline (PBS 10×) was prepared according to the following composition: 1.37 M NaCl, 0.027 M KCl, 0.015 M KH2PO4, 0.08 M Na2HPO4·12H2O (pH = 7.4). Diluted phosphate buffer saline (PBS 1×) was prepared by dilution of PBS 10× in water. Phosphate buffer salineTween (PBS-T) was prepared as follows: Tween 20 (0.05 %, m/v) and NaCl (to a final concentration of 0.3M) were added to PBS 1×. Piranha solution for final sensor cleaning was prepared as follows: 30 % H2O2 and concentrated H2SO4 (1:3) (caution!), requiring specific safety equipment and extremely careful handling. Milli-Q water was used for the preparation of the buffered solutions (Milli-Q element A10 System, Millipore, San Francisco, CA, USA).

Ac

84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127

Measurements were performed using an Eureka instrument (BioAge s.r.l., Lamezia Terme, CZ, Italy), consisting of a base containing the measurement electronics and a standard measure chamber which contains the quartz crystal. As for the crystal, a 10 MHz AT-cut piezoelectric quartz crystal chips with gold electrodes on both sides (crystal diameter, 13.9 mm; crystal thickness, 160 µm; gold electrodes diameter, 6.0 mm) was exploited. Eureka is self-powered only by the PC USB port and all measurements were performed at 25°C in a flow-through mode under flow laminar conditions using a peristaltic pump (3 rpm). The acquisition speed was set to 2 measurements/s. Morphological analysis were carried out by using an environmental scanning electron microscope (ESEM) QuantaTM 250 FEG (FEI Company, Oregon, USA) on untreated crystal surface.

3 Page 3 of 23

2.3 QCM immunosensor fabrication

ce pt

ed

M

an

us

cr

ip t

Prior to functionalization, each quartz crystal was cleaned as follows: it was sonicated in 2-propanol for 3 min, then immersed in 1.2 M NaOH for 10 min and in 1.2 M HCl for 5 min. The rinsed crystal was mounted in a QCM off-line cell in order to perform the subsequent treatments only on the selected gold surface. This gold surface was exposed to a drop of concentrated HCl (37 %, v/v) for 30 s, rinsed with deionized water again and finally washed with ethanol for three times. Selfassembled monolayer (SAM) was deposed by exposition to 0.014 M solution of MUA in absolute ethanol for 20 h. The SAM was characterized by cyclic voltammetry, scanning the potential three times at 50 mV/s from -1.5 to 0 V in aqueous solution of 0.5 M NaOH. Then it was activated by incubation with 1.25 ml of 0.2 M EDC and 0.05 M NHS solution in absolute ethanol for 30 min. After the SAM deposition the coated electrode was assembled in a flow cell. First the crystal was washed with deionized water and then the EB was fluxed for 3 min. The electrode was finally kept under static conditions for further 3 min in order to get a stable baseline signal. The linking of the Open-tTG enzyme to the carboxylate functionalities of the SAM was performed for 1 h in EB buffer, exploiting EDC and NHS as coupling reagents. Then, the gold electrode was washed and incubated with EB for 30 min to quench the remaining reactive free functionalities of MUA, by exploiting the primary amino group of TRIS-HCl. In order to minimize the nonspecific binding, predominantly caused by hydrophobic interactions, Tween 20 was used as surfactant to PBS 1×. Thus, the QCM was washed with PBS-T for 10 min and then incubated with 2 % (m/v) BSA solution for 1 h. After the blocking reaction, PBS-T was fluxed consecutively into the flow cell for 10 min each. Finally, anti-tTG antibodies in human serum (diluted 1:100 in PBS-T) were introduced in the cell and incubated for 1 h. Then the washing step with PBS-T was performed for 10 min. After each washing a steady state step was achieved for 10 min in order to measure the stabilized frequency shift. For the amplification of the enzyme-antibody binding reaction, a solution of Ab-AuNPs was injected and then incubated for 4 h. The washing step was performed by fluxing PBS-T for 10 min, followed by a stop-flow mode for further 10 min. The frequency change (Df [Hz]) was calculated by the difference between the final baseline frequency, evaluated at the end of the washing step involving PBS-T in static mode, and the frequency of PBS-T steady-state step just before antibody incubation. The attempt of regeneration of QCM was performed under constant flow (9 rpm) of 8 M urea in 20 mM TRIS-HCl for 20 min, followed by a stop-flow step for 10 min; finally the crystal was exposed to a constant flow of Gly-HCl for 10 min in order to cleave the analyte-enzyme bond. After regeneration, PBS-T solution was again flown-through for 20 min (9 rpm). The frequency of the new stable baseline was measured with the aim of testing sensor reusability. Once each assay was finished, the final cleaning of the sensing surface was performed in the offline cell, by exposing the gold surface to piranha solution for 10 s (3×) and then rinsing with H2O and ethanol.

Ac

128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171

2.4 QCM immunosensor optimization A 22 two-levels full factorial design (FFD) [39] was performed to investigate the effects of OpentTG concentration (COpen-tTG) and Ab-AuNPs dilution (CAb-AuNPs) on signal-to-noise (S/N) ratio. 4 Page 4 of 23

3. Results and discussion

ce pt

3.1 Immunosensor setup strategy

ed

M

an

us

cr

ip t

Low (level -1) and high (level +1) levels were the following: COpen-tTG =15-45 µg/ml and CAb-AuNPs =1:20-1:5 as dilution factor of the commercial gold nanoparticle suspension. Anti-tTG concentration was maintained at 10 µg/ml. The order of experiments was randomized and three replicates were carried out for each experiment. Firstly, a two-way ANOVA with interaction analysis was performed in order to assess if the investigated factors and their interaction were significant; then, on the basis of this information, the final regression model was calculated. All statistical analyses were carried out by using the statistical package SPSS 16.0 (IBM inc. New York, US). Concerning the assessment of immunosensor performance, the relationship between frequency shift and anti-tTG concentration was investigated over the 0-20 µg/ml range. Limit of detection (LOD) was calculated as 3.3×Sy/b, where Sy is regression residual standard deviation and b is the slope calculated from the calibration curve. Analogously, limit of quantification (LOQ) was calculated as 10×Sy/b. Linearity of calibration curve was investigated in the LOD-20 µg/ml range of anti-tTG in serum (eight concentration levels, three independent replicates for each level) and was evaluated by Mandel’s fitting test. The significance of the intercept (significance level of 5 %) was established by running a t test. As for accuracy, precision and trueness were assessed. Precision was evaluated in terms of intra-crystal and inter-crystal reproducibility, a parameter directly influencing the reusability of the device. The first was calculated as relative standard deviation (RSD %) from three independent measurements from the same crystal subjected to cleaning after each analysis. The latter was calculated as RSD % from three replicated measurements from different functionalized crystals and incubated with the same concentration of anti-tTG. Trueness was evaluated as percent ratio between calculated and spiked anti-tTG amounts on two concentration levels (LOQ and 10 µg/ml), performing three replicates.

The immobilization of bio-receptors on SAMs allows a highly ordered surface functionalization with the aim of improving biosensor reproducibility and sensitivity. In this study, the gold electrode was derivatized with carboxyl-reactive MUA, suitable for the subsequent covalent linking of the amine functionalities of the Open-tTG to previously activated SAM [40]. An important issue is to retain the open conformation of the tTG enzyme in order to simulate the in vivo conditions occurring during immune response in celiac patients. For this purpose the immobilization of OpentTG was carried out by exploitation of proper conditions, according to producer’s recommendation. With this aim, the composition of the EB buffer used during enzyme immobilization included DTT as reducing agent for thiol sites, Ca2+ ions to assure the proper interaction with anti- tTG antibodies, and EDTA as chelating agent in order to regulate the amount of Ca2+ ions. The linking of Open-tTG to the SAM layer can be controlled by recording the continuous frequency decrease over time, as long as the system reaches a plateau indicating the exhaustive functionalization of the sensor surface. As for frequency variation observed upon Open-tTG linking to the SAM, a mean value of 163±24 Hz (n=30) was obtained. Once functionalized, the biosensor was exposed to anti-tTG analyte and the interaction with immobilized Open-tTG caused a frequency variation. Taking into account that the recorded values of frequency shift were not high enough to reach the requested

Ac

172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215

5 Page 5 of 23

cr

ip t

sensitivity for accurate diagnostic purposes, the response of the sensor was enhanced by final exposition to gold nanoparticles conjugated with secondary antibodies. The capacity of the secondary antibodies conjugated with AuNPs to recognize the anti-tTG Fc region, which is not involved in the interaction with the receptor, allowed to obtain an enhanced mass variation, which was recorded as an improved frequency shift proportional to the amount of immobilized analyte. More precisely, the effectiveness of the frequency shift amplification, due to the use of Ab-AuNPs, was proved by calculation of signal enhancement rate for each concentration level explored for calibration purpose. The results are reported in Table 1. In addition, the sensitivity improvement was assessed by comparing the slope values calculated with and without Ab-AuNPs, obtaining a sensitivity increased by 84%. A schematic representation of the developed QCM immunosensor is shown in Figure 1.

us

3.2 Immunosensor development

ce pt

ed

M

an

The aspects related to i) the functionalization mode of the gold electrode crystal surface, ii) the sensitive, specific and reproducible detection of the analyte in liquid phase and iii) the regeneration of the sensor surface are fundamental parameters to be carefully studied and optimized for the development of a functional bioimmunosensor based on piezoelectric transduction. It is important to remark that liquid phase can cause severe signal instability over variation of solution composition during the experiment. Indeed, preliminary experiments performed on non-functionalized quartz crystal resonators pointed out that they are sensitive to viscosity and ionic strength of solution, requiring a proper choice and a strict control of experimental conditions for measurements in liquid phase. Buffer solutions used for measurements were selected in order to have a good signal stability both in dynamic and in static mode as well as proper conditions for the exploited biological interactions. In particular, it was observed that high NaCl concentrations lead to noisy frequencygrams under dynamic mode, but these ionic strength conditions resulted necessary to guarantee the correct reactivity, avoiding aggregation phenomena of Ab-AuNPs, as also reported in the producer’s data sheet. However, these conditions did not negatively affect sensor performance, so buffer composition was uniformed during all the analysis. Sensor reliability depends strictly on the possibility of ascribing the measured signals only to specific antibody-antigen interactions. In order to verify detection specificity, some experiments were carried out without exposition to the analyte (Figure 2) or without tTG immobilization (Figure 3), obtaining a Df mean value of 25±4 (n=5) in the first case and a mean value not significantly different from zero (p value > 0.05; n=5) in the second case. The recorded frequency variations proved the absence of non-specific interactions, thus attesting suitability of immunosensing strategy.

Ac

216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258

3.3 Immunosensor optimization A 22 two-level factorial design was used to evaluate the significance of the main and interaction effects of the parameters investigated. The experimental domain was defined on the basis of some preliminary experiments taking into account the need to minimize the amount of biomaterials used for each analysis, while ensuring efficiency of biosensor functionalization and detection. By applying the two-way ANOVA with interaction analysis, the following equation was calculated by

6 Page 6 of 23

an

us

cr

ip t

multiple regression (Eq. (1), R2=0.996), demonstrating that both factors and their interaction were significant (p<0.05): Df= 103.8 (± 8.1) – 3156.9 (± 564.5) COpen-tTG + 929.2 (± 55.4) CAb-AuNPs + (1) + 11372.5 (± 3872.2) COpen-tTG × CAb-AuNPs The optimal conditions found by calculating the maximum of the regression function resulted the level -1 for COpen-tTG and the level +1 for CAb-AuNPs. It was observed that the low Open-tTG concentration induces a slow frequency decrease before reaching the plateau: probably the reaction kinetics, slower than that observed with high Open-tTG level, permits a more ordinated immobilization of the enzyme to the SAM, leading to a more efficient and reproducible sensor functionalization. The level +1 of Ab-AuNPs guarantees to prevent their stoichiometric defect especially in the case of high analyte concentrations. The frequencygrams obtained by analyzing serum spiked with anti-tTG at LOD and 10 µg/ml levels are shown in Figures 4 and 5, respectively. In order to verify and control the binding of the nanoparticles to the sensor, ESEM microscopy was used to record the images of SAM/Open-tTG/anti-tTG/Ab-AuNPs crystal surface (Figure 6), demonstrating a uniform Ab-AuNPs distribution without aggregation phenomena, with dimensions compatible with the declared diameter. 3.4 Anti-tTG Antibodies determination

ce pt

ed

M

The optimized conditions were used to assess analytical sensor performance in human serum. A good precision in terms of intra-crystal and inter-crystal reproducibility was obtained, with RSD % values lower than 17 % and 13 % (n=3), respectively. LOD and LOQ values were estimated as 1.3 and 4.0 µg/ml, respectively. Linearity was assessed in the LOD-12 µg/ml range and the resulting calibration curve was described by the equation y = 12.5(±0.6) x + 85.2(±4.8) (y, ‫׀‬Df‫ ;׀‬x, anti-tTG concentration in µg/ml) R2=0.991, n=18 (Figure 7); trueness corresponding to 92(±5) % recovery rate was also calculated. The assessed analytical parameters evidenced as the frequencymetric transduction, associated to the use of Open-tTG as diagnostically powerful bioreceptor, is useful for the development of reliable and accurate diagnostic tools in celiac disease. In particular, the good reproducibility and accuracy, as well as the enhanced sensitivity thanks to the exploitation of Ab-AuNPs, proved that the developed approach is suitable for anti-tTG determination in serum matrix.These findings attest that the piezoelectric immunosensor, devised and optimized by using mouse antibodies, represents a good platform and model to be extended and optimized for the determination of human IgG and IgA anti-tTG antibodies in serum. The surface regeneration represents a very important issue to be addressed, because it allows to perform repeated analyses by reutilizing the same SAM/Open-tTG functionalized crystal, reducing analysis time and costs. Concerning this aspect, different approaches were investigated, based on the exploitation of denaturing agents as urea, sodium dodecyl sulphate (SDS) [41] and glycine-HCl solution (pH=2.1) [42,43], as previously reported in other literature studies. Using the protocol indicated in the experimental section, the Ab-AuNPs are not completely removed, suggesting that stronger conditions are required for the displacement of bounded nanoparticles (Figure 8). Finally, a complete cleaning of the quartz crystal was obtained by a treatment with piranha solution, allowing the complete removal of all the organic components deposited over the gold crystal surface, which resulted to be ready for a further SAM deposition.

Ac

259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302

7 Page 7 of 23

4. Conclusions

us

cr

ip t

In this study, a new approach based on piezoelectric immunosensing and Open-tTG immobilization was demonstrated very promising for the determination of anti-tTG antibodies involved in celiac disease. The exploitation of Ab-AuNPs for signal amplification guarantees good sensitivity, suitable for diagnostic purposes. Optimization of a model based on mouse antibodies in human serum opens the way to a reliable strategy for the determination of anti-tTG autoantibodies in clinical diagnosis. Reusability of the immunosensor needs further investigations to minimize the time required for immunosensor set up. As for the practical usefulness of the device, the hypothesis that auto-antibodies to the Open-tTG may have higher specificity for the inflammatory intestinal process active in celiac disease [30], would make diagnostic testing more efficient. In fact, the use of the recently discovered Open-tTG antigen allows early diagnosis also in patients with lesser degrees of inflammation as well as detection of dietary not-compliance [30].

an

5. References

ce pt

ed

M

[1] P.-T. Chu, C.-S. Lin, W.-J. Chen, C.-F. Chen, H.-W. Wen, Detection of gliadin in foods using a quartz crystal microbalance biosensor that incorporates gold nanoparticles, J. Agric. Food. Chem. 60 (2012) 6483-6492. [2] T.F. McGrath, C.T. Elliott, T.L. Fodey, Biosensors for the analysis of microbiological and chemical contaminants in food, Anal. Bioanal. Chem. 403 (2012) 75-92. [3] K. Thandavan, S. Gandhi, S. Sethuraman, J.B.B. Rayappan, U.M. Krishnan, Development of electrochemical biosensor with nano-interface for xanthine sensing - A novel approach for fish freshness estimation, Food. Chem. 139 (2013) 963-969. [4] M. Giannetto, E. Maiolini, E.N. Ferri, S. Girotti, G. Mori, M. Careri, Competitive amperometric immunosensor based on covalent linking of a protein conjugate to dendrimer-functionalised nanogold substrate for the determination of 2,4,6-trinitrotoluene, Anal. Bioanal. Chem. 405 (2013) 737-743. [5] L.S. Wong, Y.H. Lee, S. Surif, Performance of a cyanobacteria whole cell-based fluorescence biosensor for heavy metal and pesticide detection, Sensors 13 (2013) 6394-6404. [6] Z.-Q. Shen, J.-F. Wang, Z.-G. Qiu, M. Jin, X.-W. Wang, Z.-L. Chen, J.-W. Li, F.-H., Cao, QCM immunosensor detection of Escherichia coli O157:H7 based on beacon immunomagnetic nanoparticles and catalytic growth of colloidal gold, Biosens. Bioelectron. 26 (2011) 3376-3381. [7] M. Giannetto, L. Elviri, M. Careri, A. Mangia, G. Mori, A voltammetric immunosensor based on nanobiocomposite materials for the determination of alpha-fetoprotein in serum, Biosens. Bioelectron. 26 (2011) 2232-2236. [8] C.I. Cheng, Y.-P. Chang, Y.-H. Chu, Biomolecular interactions and tools for their recognition: focus on the quartz crystal microbalance and its diverse surface chemistries and applications, Chem. Soc. Rev. 41 (2012) 1947-1971. [9] X. Fan, B. Du, Selective detection of trace p-xylene by polymer-coated QCM sensors, Sens. Actuat. B-Chem. 166-167 (2012) 753-760.

Ac

303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344

8 Page 8 of 23

ce pt

ed

M

an

us

cr

ip t

[10] J.-F. Ju, M.-J. Syu, H.-S. Teng, S.-K. Chou, Y.-S. Chang, Preparation and identification of βcyclodextrin polymer thin film for quartz crystal microbalance sensing of benzene, toluene, and pxylene, Sens. Actuat. B-Chem. 132 (2008) 319-326. [11] X. Fan, B. Du, Selective detection of trace 1-butanol by QCM sensor coated with copolymer P(HEMA-co-MA), Sens. Actuat. B-Chem. 160 (2011) 724-729. [12] Z. Cao, J. Guo, X. Fan, J. Xu, Z. Fan, B. Du, Detection of heavy metal ions in aqueous solution by P(MBTVBC-co-VIM)-coated QCM sensor, Sens. Actuat. B-Chem. 157 (2011) 34-41.[13] M.C. Dixon, Quartz crystal microbalance with dissipation monitoring: enabling real-time characterization of biological materials and their interactions, J. Biomol. Techn. 19 (2008) 151-158. [14] B. Du, D. Johannsmann, Operation of the quartz crystal microbalance in liquids: derivation of the elastic compliance of a film from the ratio of bandwidth shift and frequency shift, Langmuir 20 (2004) 2809-2812. [15] M.V. Voinova, M. Jonson, B. Kasemo, 'Missing mass' effect in biosensor's QCM applications, Biosens. Bioelectron. 17 (2002) 835-841. [16] K.A. Marx, Quartz Crystal Microbalance: A useful tool for studying thin polymer films and complex biomolecular systems at the solution-surface interface, Biomacromolecules 4 (2003) 10991120. [17] B. Du, I. Goubaidoulline, D. Johannsmann, Effects of laterally heterogeneous slip on the resonance properties of quartz crystals immersed in liquids, Langmuir 20 (2004) 10617-10624. [18] C. March, J.J. Manclús, Y. Jiménez, A. Arnau, A. Montoya, A piezoelectric immunosensor for the determination of pesticide residues and metabolites in fruit juices, Talanta 78 (2009) 827-833. [19] N.A. Karaseva, T.N. Ermolaeva, A piezoelectric immunosensor for chloramphenicol detection in food, Talanta 93 (2012) 44-48. [20] J. Zhou, N. Gan, T. Li, H. Zhou, X. Li, Y. Cao, L. Wang, W. Sang, F. Hu, Ultratrace detection of C-reactive protein by a piezoelectric immunosensor based on Fe3O4@SiO2 magnetic capture nanoprobes and HRP-antibody co-immobilized nano gold as signal tags, Sensor. Actuat. B-Chem. 178 (2013) 494-500. [21] G. Shen, M. Liu, X. Cai, J. Lu, A novel piezoelectric quartz crystal immnuosensor based on hyperbranched polymer films for the detection of α-Fetoprotein, Anal. Chim. Acta 630 (2008) 7581. [22] M. Salmain, M. Ghasemi, S. Boujday, J. Spadavecchia, C. Técher, F. Val, V. Le Moigne, M. Gautier, R. Briandet, C.-M. Pradier, Piezoelectric immunosensor for direct and rapid detection of staphylococcal enterotoxin A (SEA) at the ng level, Biosens. Bioelectron. 29 (2011) 140-144. [23] X. Guo, C.-S. Lin, S.-H. Chen, R. Ye, V.C.H. Wu, A piezoelectric immunosensor for specific capture and enrichment of viable pathogens by quartz crystal microbalance sensor, followed by detection with antibody-functionalized gold nanoparticles, Biosens. Bioelectron 38 (2012) 177-183. [24] T. Xu, J. Miao, Z. Wang, L. Yu, C.M. Li, Micro-piezoelectric immunoassay chip for simultaneous detection of Hepatitis B virus and α-fetoprotein, Sensor. Actuat. B 151 (2011) 370376. [25] M. Vives-Pi, S. Takasawa, I. Pujol-Autonell, R. Planas, E. Cabre, I. Ojanguren, M. Montraveta, A.L. Santos, E. Ruiz-Ortiz, Biomarkers for diagnosis and monitoring of celiac disease, J. Clin. Gastroenterol. 47 (2013) 308-313. [26] E.B. Roth, K. Sjoberg, P. Stenberg, Biochemical and immuno-pathological aspects of tissue transglutaminase in coeliac disease, Autoimmunity 36 (2003) 221-226.

Ac

345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388

9 Page 9 of 23

ce pt

ed

M

an

us

cr

ip t

[27] D.M. Pinkas, P. Strop, A.T. Brunger, C. Khosla, Transglutaminase 2 undergoes a large conformational change upon activation, PLoS Biol. 5 (2007) 2788-2796. [28] J. Stamnaes, D.M. Pinkas, B. Fleckenstein, C. Khosla, L.M. Sollid, Redox regulation of transglutaminase 2 activity, J. Biol. Chem. 285 (2010) 25402-25409. [29] B.-G. Han, J.-W. Cho, Y.D. Cho, K.-C. Jeong, S.-Y. Kim, B.I. Lee, Crystal structure of human transglutaminase 2 in complex with adenosine triphosphate, Int. J. Biol. Macromol. 47 (2010) 190195. [30] K. Pallav, D.A. Leffler, M. Bennett, S. Tariq, H. Xu, T. Kabbani, A.C. Mos, M. Dennis, C.P. Kelly, D. Schuppan, Open conformation tissue transglutaminase testing for celiac dietary assessment, Digest. Liver Dis. 44 (2012) 375-378.[31] M.I. Pividori, A. Lermo, A. Bonanni, S. Alegret, M. del Valle, Electrochemical immunosensor for the diagnosis of celiac disease, Anal. Biochem. 388 (2009) 229-234. [32] S. Dulay, P. Lozano-Sánchez, E. Iwuoh, I. Katakis, C.K. O’Sullivan, Electrochemical detection of celiac disease-related anti-tissue transglutaminase antibodies using thiol based surface chemistry, Biosens. Bioelectron. 26 (2011) 3852-3856. [33] T. Balkenhohl, F. Lisdat, Screen-printed electrodes as impedimetric immunosensors for the detection of anti-transglutaminase antibodies in human sera, Anal. Chim. Acta 597 (2007) 50-57. [34] M.M.P.S. Neves, M.B. González-García, H.P.A. Nouws, A. Costa-García, Celiac disease detection using a transglutaminase electrochemical immunosensor fabricated on nanohybrid screenprinted carbon electrodes, Biosens. Bioelectron. 31 (2012) 95-100. [35] G. Adornetto, G. Volpe, A. De Stefano, S. Martini, G. Gallucci, A. Manzoni, S. Bernardini, M. Mascini, D. Moscone, An ELIME assay for the rapid diagnosis of coeliac disease, Anal. Bioanal. Chem. 403 (2012) 1191-1194. [36] S.V. Kergaravat, L. Beltramino, N. Garnero, L. Trotta, M. Wagener, M.I. Pividori, S.R. Hernandez, Electrochemical magneto immunosensor for the detection of anti-TG2 antibody in celiac disease, Biosens. Bioeletron. 48 (2013) 203-209. [37] S.V. Kergaravat, L. Beltramino, N. Garnero, L. Trotta, M. Wagener, S.N. Fabiano, M.I. Pividori, S.R. Hernandez, Magneto immunofluorescence assay for diagnosis of celiac disease, Anal. Chim. Acta 798 (2013) 89-96. [38] M.M.P.S. Neves, M.B. González-García, C. Delerue-Matos, A. Costa-García, Multiplexed electrochemical immunosensor for detection of celiac disease serological markers, Sens. Actuat. BChem. 187 (2013) 33-39. [39] G.E.P. Box, W.G. Hunter, J.S. Hunter, Statistic for Experimenters: an Introduction to Design, Data Analysis and Model Building, Wiley, New York, 1978. [40] M. Giannetto, L. Mori, G. Mori, M. Careri, A. Mangia, New amperometric immunosensor with response enhanced by PAMAM-dendrimers linked via self assembled monolayers for determination of alpha-fetoprotein in human serum, Sens. Actuat. B-Chem. 159 (2011) 185-192. [41] J. Ramos-Jesus, K.A. Carvalho, R.A.S. Fonseca, G.G.S. Oliveira, S.M. Barrouin Melo, N.M. Alcantara-Neves, R.F. Dutra, A piezoelectric immunosensor for Leishmania chagasi antibodies in canine serum, Anal. Bioanal. Chem. 401 (2011) 917-925. [42] S. Storri, S. Santoni, M. Minunni, M. Mascini, Surface modifications for the development of piezoimmunosensors, Biosens. Bioelectron. 13 (1998) 347-357.

Ac

389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430

10 Page 10 of 23

431 432 433 434 435

[43] A. Ruchi, A. Satlewal, M. Chaudhary, A. Verma, R. Singh, A.K. Verma, R. Kumar, K.P. Singh, Rapid detection of cadmium-resistant plant growth promotory Rhizobacteria: A perspective of ELISA and QCM-based immunosensor, J. Microbiol. Biotechnol. 22 (2012) 849-855.

436

ip t

437 438

cr

439

us

440 441

an

442 443

M

444 445

ed

446 447

450 451 452 453 454

Ac

449

ce pt

448

455 456 457 458 459 11 Page 11 of 23

460

Figure captions

461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489

Fig. 1. Schematic depiction of the immunosensor set up. The model is not in scale

491 492

cr

ip t

Fig. 3. QCM sensorgram (frequency vs. time) without Open-tTG immobilization for the evaluation of non-specific interactions (anti-tTG concentration, 10 µg/ml; Df not significantly different from zero). Inset: zooming out of anti-tTG incubation. (Q, quenching; B, blocking; HS, human serum; (s), static mode; (d), dynamic mode)

us

Fig. 4. QCM sensorgram (frequency vs. time) of anti-tTG antibody in human serum at LOD level (Df=70 Hz). Inset: zooming out of anti-tTG incubation. (Q, quenching; B, blocking; HS, human serum; (s), static mode; (d), dynamic mode)

M

an

Fig. 5. QCM sensorgram (frequency vs. time) of anti-tTG antibody in human serum at 10 µg/ml (Df=199 Hz). Inset: zooming out of anti-tTG incubation. (Q, quenching; B, blocking; HS, human serum; (s), static mode; (d), dynamic mode)

ed

Fig. 6. ESEM images of the top view of QCM gold surface. (a) SAM/Open-tTG/anti-tTG; (b) SAM/Open-tTG/anti-tTG/Ab-AuNPs

ce pt

Fig. 7. Plot of the frequency shift as a function of anti-tTG concentration. (●) Data points of the calibration curve. (♦) Data point corresponding to blank level (no anti-tTG antibody exposition), not interpolated for calculation of calibration curve equation Fig. 8. QCM sensorgram (frequency vs. time) with regeneration (anti-tTG concentration, 10 µg/ml; Dfreg=78). (Q, quenching; B, blocking; HS, human serum; (s), static mode; (d), dynamic mode)

Ac

490

Fig. 2. QCM sensorgram (frequency vs. time) without exposition to anti-tTG antibody for the evaluation of non-specific interactions (Df=25 Hz). Inset: zooming out of blank human serum incubation. (Q, quenching; B, blocking; HS, human serum; (s), static mode; (d), dynamic mode)

493 494 495 496 497 498 12 Page 12 of 23

Biographies Anita Manfredi was born in Parma, Italy in 1986. In 2012 she graduated (M.Sci) in Molecular Biology at University of Parma. From 2013 she is PhD student in Chemistry at University of Parma. Her scientific research is focused on methodological aspects and applications of biosensors and MS-based analytical methods for identification and determination of biomarkers, combined with chemometric techniques for optimization of operating conditions.

an

us

cr

ip t

Monica Mattarozzi was born in Cremona, Italy in 1983. In 2007 she graduated (M.Sci) in Chemistry at University of Parma. In 2011 she achieved a PhD fellowship in Chemistry at University of Parma. She worked as researcher at University of Parma and in 2014 she achieved a researcher fellowship at University of Parma. Her current research interests include the study and application of innovative biosensors, the morphological and compositional characterization of micro/nano structures of materials by environmental scanning electron microscopy as well as the development and validation of innovative methods in proteomics by using mass spectrometry-based techniques.

ed

M

Marco Giannetto was born in Messina, Italy in 1973. In 1996 he graduated (M.Sci) in Chemistry at University of Messina. In 2000 he achieved a PhD fellowship in Chemistry at University of Parma. From 2001 he is researcher at University of Parma. His scientific activity is focused on the realization and characterization of new chemical sensors and biosensors based on innovative recognition materials and different transduction mechanisms. The scientific production is documented by more than 30 scientific papers published on peer-reviewed international journals.

ce pt

Maria Careri has been Full Professor of Analytical Chemistry at the University of Parma, Italy, since 2001, and Director of the Department of Chemistry since 2012. She is a member of the Editorial Board of Analytical and Bioanalytical Chemistry, Current Analytical Chemistry, and Journal of Chromatography A. Her research activities have centered on the development of novel materials for solvent-free extraction, and innovative methods for structural and functional proteomics using MS-based techniques. Her current research interests include the study of innovative biosensors and the development of novel materials for desorption electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry.

Ac

499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530

13 Page 13 of 23

Table 1

cr

ip t

Table 1 Ab-AuNPs signal enhancement: frequency shifts of the QCM immunosensor, with and without AbAuNPs, at different anti-tTG antibody concentration levels, n=3. anti-tTG Concentration Signal │Dfanti-tTGa│(Hz) ± S.D. │Dfb│(Hz) ± S.D. (µg/ml) enhancementc (%) 0 5±1 26 ± 4 81 1.3 (LOD) 24 ± 3 99 ± 11 76 4 (LOQ) 31 ± 2 139 ± 6 78 6 34 ± 1 164 ± 6 79 8 36 ± 1 180 ± 4 80 10 38 ± 1 204 ± 5 81 12 50 ± 4 240 ± 7 79 a

frequency shift caused by only anti-tTG antibody linking. frequency shift caused by anti-tTG and Ab-AuNPs linking (Df signals used for calibration purposes). c calculated as: (1-(Dfanti-tTG/Df))*100.

Ac

ce pt

ed

M

an

us

b

Page 14 of 23

Figure 1

Au

Ab-AuNP

anti-tTG

ip t

Open-tTG

BSA

us

BSA

cr

SAM

Ac

ce pt

ed

M

an

Fig. 1.

Page 15 of 23

Figure 2

PBS-T(d) HS

PBS-T(d)

PBS-T(d) PBS-T(s)

PBS-T(s)

Open-tTG

TE(d)

PBS-T(d) Q

B Ab-AuNPs HS

ip t

PBS-T(d)

PBS-T(s)

PBS-T(s)

PBS-T(s)

an

us

PBS-T(s)

cr

Df

Ac ce p

te

d

M

Fig. 2.

Page 16 of 23

Figure 3

anti-tTG HS

PBS-T(d)

PBS-T(d) Ab-AuNPs

B anti-tTG HS

cr

TE(d) PBS-T(d) Q

PBS-T(d)

us

TE(s)

PBS-T(d)

ip t

PBS-T(s) PBS-T(s)

PBS-T(s) PBS-T(s)

PBS-T(s)

an

PBS-T(s)

Df

Ac ce p

te

d

M

Fig. 3.

Page 17 of 23

Figure 4

PBS-T(d) anti-tTG HS PBS-T(d)

PBS-T(d)

PBS-T(s)

Open-tTG TE(d) PBS-T(d) Q

B

PBS-T(d) Ab-AuNPs

PBS-T(s) PBS-T(s)

Df

PBS-T(s)

an

us

PBS-T(s)

cr

anti-tTG HS

ip t

PBS-T(s)

Ac ce p

te

d

M

Fig. 4.

Page 18 of 23

Figure 5

PBS-T(d)

anti-tTG HS PBS-T(d) PBS-T(d)

PBS-T(s)

TE(d)

Open-tTG

PBS-T(s)

B

Q

Ab-AuNPs

PBS-T(s)

cr

anti-tTG HS

ip t

PBS-T(d)

PBS-T(d)

PBS-T(s)

Df

PBS-T(s)

an

us

PBS-T(s)

Ac ce p

te

d

M

Fig. 5.

Page 19 of 23

Figure 6

b

cr

ip t

a

Ac

ce pt

ed

M

an

us

Fig. 6.

Page 20 of 23

cr

ip t

Figure 7

Ac

ce pt

ed

M

an

us

Fig. 7.

Page 21 of 23

Figure 8

PBS-T(d)

TE(d)

PBS-T(d) Q PBS-T(d)

B anti-tTG HS PBS-T(s)

cr

Ab-AuNPs

ip t

Open-tTG

PBS-T(d)

us

PBS-T(s)

Dfreg

PBS-T(s)

PBS-T(s)

an

PBS-T(s)

Ac ce p

te

d

M

Fig. 8.

Page 22 of 23

Research Highlights

Highlights

ce pt

ed

M

an

us

cr

ip t

Determination of anti-tissue transglutaminase antibodies involved in celiac disease Immobilization of tissue transglutaminase enzyme in its open conformation Good performance for diagnostic purposes Biosensor optimization by experimental design

Ac

   

Page 23 of 23