This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see: https://dx.doi.org/10.1021/acs.analchem.9b03407. A smart chip for visual detection of bacteria using the electro- chromic properties of polyaniline Saba Ranjbar†,‡, Mohammad Amin Farahmand Nejad†,‡, Claudio Parolo†, Saeed Shahrokhian‡, Arben Merkoçi†, ∥* †Nanobioelectronics and Biosensor Group, Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, The Barce- lona Institute of Science and Technology, Campus UAB, Bellaterra, 08193, Barcelona, Spain ‡Department of Chemistry, Sharif University of Technology, Tehran 11155-9516, Iran ∥ Catalan Institution for Research and Advanced Studies (ICREA), Pg. Lluís Companys 23, 08010 Barcelona, Spain ABSTRACT: Finding fast and reliable ways to detect pathogenic bacteria is crucial for addressing serious public health issues in clinical, environmental and food settings. Here, we present a novel assay based on the conversion of an electrochemical signal into a more convenient optical read out for the visual detection of E. coli. Electropolymerizing polyaniline (PANI) on indium tin oxide screen printed electrode (ITO SPE), we achieved not only the desired electrochromic behavior, but also a convenient way to modify the electrode surface with antibodies (taking advantage of the many amine groups of PANI). Applying a con- stant potential to the PANI-modified ITO SPE induces a change in their oxidation state, which in turn generates a colour change on the electrode surface. The presence of E. Coli on the electrode surface in- creases the resistance in the circuit affecting the PANI oxidation states, producing a different electro- chromic response. Using this electrochromic sensor, we could measure concentrations of E. Coli spanning four orders of magnitude with a limit of detection of 102 CFU mL-1. In this work we show that merging the sensitivity of electrochemistry with the user-friendliness of an optical read out can generate a new and powerful class of biosensors, with potentially unlimited applications in a variety of fields. Intorduction for the accurate and ubiquitous analysis of fresh products and The highest number of annual hospitalizations and deaths water samples. Electrochromic sensors14-15 provide exactly this: within the United States are attributed to water resources con- an optical readout that depends on the applied potential (which taminated with bacteria.1 Therefore, water monitoring for bac- can be achieved with a device smaller and considerably cheaper teria contamination is a number one priority for public health. than a fully portable potentiostat).16-17 This type of sensors har- With current technologies, testing for every single bacteria at nesses the different colors that different oxidation states of elec- the point of care is not feasible, that is why the United States trochromic materials (ECMs).18 ECMs are classified into three Food and Drug Administration (FDA) suggested the use of the categories including transition metal oxides (molybdenum, “generic” Escherichia Coli (E. Coli) level as an indicator for the tungsten, titanium, iridium), single molecular materials (Prus- overall bacterial contamination in fresh products and water sian blue, Viologen) and π conjugated polymer films (polyani- samples.2 Current methods for the detection of E. Coli can be line, polypyrole, polythiophen).19-20 Considering the sensitivity divided into laboratory-based tests (such as bacteria cultures of human eyes to the color change rather than color intensity, and counting,3 enzyme-linked immunosorbent assay4 and poly- multicolor ECMs (such as π conjugated polymer films and their merase chain reaction5) and point-of-care sensors6-8 (with the nanocomposites) provide the most user friendly platform for most common ones being lateral flow immunoassay – LFIA -9 point of care sensing.20-21 In this regard, polyaniline (PANI) is a and electrochemical sensors10). While the former methods pro- unique ECM, having a fast response at closed applied potentials vide high sensitivity and specificity they also require long, com- with a wide range of colors (yellow, green and blue)(Fig. 1). plicated, multi-step protocols (often involving expensive equip- Besides its electrochromic behavior, PANI also provides high ment) that prevent their use in applications where a fast detec- electrical conductivity, facile synthesis, high stability, simple tion is paramount.11The latter instead are convenient to use at (bio)functionalization (thanks to its –NH2 groups) and strong the point of care, but often times lack the sensitivity and speci- adhesion to the electrode surface, qualities that make it an ideal ficity required by most applications (i.e. LFIA); or they require material for the development of point of care sensors.22-24 a portable equipment (potentiostat), which considerably in- In the present study, we developed a novel electrochromic crease their overall cost (i.e. electrochemical sensors).12-13 immunosensor based on the use of PANI and indium tin oxide A method that combines the advantages of a simple optical (ITO) electrodes for the visual and fast detection of E.Coli in read out and the portability of LFIA with the analytical capabil- different water samples. ities of electrochemical sensors would provide a powerful tool Figure 1. Schematic illustration of different PANI oxidation states with their relative colors (penigraniline/blue-violet, emeraldine base/blue, emeraldine salt/green, leucoemeraldine salt/yellow) and how they can be related to different E. Coli concentrations. Experimental section All of the images were taken by a Samsung Galaxy J 5 pro, Chemical and Apparatus in manual mode, manual focus in ambient light conditions and images were analyzed with the ImageJ software. Aniline (99%), sulphuric acid, potassium chloride, potassium hexacyanoferrate (III), potassium hexacyanoferrate (II), E. coli Scanning electron microscopy (SEM) images were obtained O157:H7 lyophilized cell, bovine serum albumin (BSA), glu- using a FEI Quanta™ 650 field emission gun scanning electron taraldehyde (GA, 25%), conductive indium tin oxide microscope (FEI, USA) to surface characterization of the elec- (ITO)/polyethylene terephthalate substrates (Rct = 60 Ω cm-1), trode. XRD diagrams were collected on an X’Pert PRO MRD ethanol, acetone, MES buffer and phosphate buffered saline analytical diffractometer (Panalytical) at 45 kV, 40 mA using tablets were obtained from Sigma-Aldrich (Madrid, Spain). CuKα radiation (λ = 1.5406 Å). Fourier transform infrared (FT- Aniline was purified under vacuum distillation to obtain a col- IR) spectra were recorded on a Bruker Tensor 27FTIR spec- orless liquid and the other chemicals were used as received trometer equipped with a Golden Gate diamond attenuated total without further purification. Fresh Milli-Q water (Millipore) reflection (ATR) cell, in transmittance mode at room tempera- was used to prepare all aqueous solutions. Salmonella typhi- ture. AFM experiments were carried out with a Veeco CP Re- murium (CECT 722T, S. typhimurium) strain was obtained search instrument using Si cantilever. from the Colección Española de Cultivos Tipo (CECT, Valen- cia, Spain). ITO Screen-Printed Electrodes assembling Serial dilutions of bacteria were prepared in sterile fresh The well-cleaned ITO slides (1 cm × 2.5 cm) were obtained Milli-Q water and stored at 4 °C for one week. An E. coli sero- by pretreating slides in an ultrasonic bath for 15 min in each of type O/K Polyclonal antibody (PA1-7213, 4-5 mg/mL) was the following solvents: soapy water, Milli-Q water, acetone, purchased from Thermo Fisher Scientific and diluted to 0.5 and ethanol sequentially. Then, the slides were rinsed with mg/mL with sterile MES buffer (0.1M, pH= 5.5). Milli-Q water and dried with a stream of compressed air. In fol- DEK 248 screen-printing machine was used to prepare home- lowing, the ITO SPEs were assembled by attachment of the ITO made screen-printed electrodes (SPEs) containing carbon ink as slides instead of the working electrode on the polyethylene ter- the counter electrode (CE), AgCl ink as the reference electrode ephthalate (PET) substrates containing counter electrode, a ref- (RE) and the insulating ink as a dielectric layer. The electro- erence electrode, and dielectric layer by using double sided chemical experiments were performed by PGSTAT 302N con- scotch tape. trolled by a NOVA 1.11 software for voltammetric experiments and FRA software for impedimetric experiments. The optical Immunosensor design and fabrication study was conducted by applying different constant potentials to the PANI/ITO SPE, while absorption spectra were collected Prior to fabrication of electrochromic immunosensor, the ITO in 350-800 nm with Cary 4000 UV−vis spectrophotometer. SPE was conditioned in PBS (pH= 7.4, 0.01M) for 10 cycles using a potential range between 0.0 V to 1.0 V at the scan rate of 0.1V s−1. The electrochromic thin film of polyaniline was ob- E. coli O157:H7 detection tained by drop casting of aniline monomer (0.25 M in 0.5 M The visual detection of E. coli O157:H7 was conducted by H2SO4) at the surface of ITO SPE and electropolymerization of applying constant potential (0.2 V) to the electrochromic im- aniline with cyclic voltammetry (CV) technique in the potential munosensor and following the PANI color changes in the pres- range of -0.8 V to 1.0 V under the scan rate of 0.1V s −1 during ence the different concentration of E. coli O157:H7. Due to ste- 6 cycle (optimum conditions). To improve the sensitivity of the ric hindrance occurred on the surface of PANI/ITO SPE by detection system, the leuocoemeraldine form of polyaniline forming the antibody-antigen complex, the color change of (yellow color) was stabilized by casting 50 µL of H2SO4 (0.5 working electrode disrupted during apply constant potential. M) at the surface of the electrode and applying the fixed poten- The polyaniline color changes in wide color range from yellow tial of -0.4 V during 50 s. Afterward, the antibody was cova- to blue were followed by increasing the concentration of E. coli lently conjugated to the PANI using glutaraldehyde (GA) as a O157:H7 by the naked eye and smartphone and also analyzed cross-linking agent. For this purpose, after washing the elec- with ImageJ software to the detection of E. coli O157:H7. trode with Milli-Q water carefully, the 5 µL of GA (0.25%) was dropped at the surface of PANI/ITO SPE to activate the amine groups of polyaniline for 30 min followed by rinsing with Milli- Real sample preparation Q water to remove unbound GA. Next, 10 µL of anti-E. coli Applicability of the designed electrochromic immunosensing antibody (0.5mg/mL) was incubated at the surface of platform was confirmed by testing of electrodes in different wa- GA/PANI/ITO SPEs and kept 2h in room temperature, then the ter samples. These experiments were conducted by spiking 10 2 electrode was blocked using 5 µL of 1% BSA casted at the sur- and 104 CFU mL-1 of E. coli O157:H7 in collected Barcelona face of anti-E. coli/GA/PANI/ITO SPE for 30 min to minimize tap water and Ter river water (three replicates for each bacterial the nonspecific binding which then rinsed with Milli-Q water concentration in each sample). again to remove unbounded BSA. Finally, the prepared im- munosensor was immersed in different concentration of E. coli for 60 min for antigen-antibody bonding. Figure 2: Characterization of PANI/ITO screen printed electrodes. (A) Cyclic voltamograms showing the formation of the thin film of PANI. The three redox couples A1/C1, A2/C2, and A3/C3 correspond respectively to: lucoemeraldine base, emeraldine salt and emeraldine base. (B) The diferent absorbance spectra further confirm the formation of the thin film of PANI. In the basnece of PANI, ITO SPEs do not show any peak, indicating their transparency, while the application of different potential to the PANI thin film produces characteristic peaks: the one at 450 represent the yellow color of lucoemeraldine base and the one at 650 nm represents emeraldine. (C) Chronoamperogram showing the change in oxidation and reduction state over time applying a fixed potential. (D) FESEM image showing the fibrous morphology of the PANI thin film. Results and discussion PANI peaks the XRD spectrum of PANI obtained by the same The thin film of polyaniline (PANI) was obtained on the ITO procedure on SPE and shown in Fig. S2 C). 26 SPE with cyclic voltammetry (CV) technique using a solution In order to extend the applications of polyaniline as an elec- containing 0.25 M aniline monomer in 0.5 M H 2SO4 during 6 trochromic material for using in biosensing platform, the elec- cycles. By scanning the potential from -0.8 V to 1.0 V during trochromic feature of PANI was evaluated in phosphate buffer electropolymerization of aniline on the ITO SPE (Fig. 2A), we saline (PBS, 0.1 M, pH = 7.4) as a biological electrolyte. 27 Be- observe three peaks (corresponding to three redox couples ing electrochromic properties pH and saline dependent, it is no A1/C1, A2/C2, and A3/C3) of three different aniline oxidation surprise that the PANI behavior in PBS differs from the one in states: lucoemeraldine base (LB, fully reduced) to emeraldine 0.5 M H2SO4. In particular, we observed that the LB form of salt (ES, partially oxidized) and to emeraldine base (EB, fully PANI is less stable in PBS than in H2SO4, thus requiring a lower oxidized).19 Thanks to its electrochromic properties, we can potential to switch to the EB form (Fig. S2). The effect of the follow the PANI film electropolymerization directly on the higher pH of PBS, also speed up the conversion of LB to ES working electrode surface, which changes color from pale yel- and EB form, which would also happen at lower potentials. We low to green and blue by scanning to more positive potentials, believe that this different behavior is due to the deprotonation while it changes from blue to pale yellow toward a negative di- of aniline ring in high pH values.28 rection (movie 1, clip of PANI film formation in S.I.). As the PANI shows electrochromic behavior just when in the The main reason why we decided to use ITO electrodes is thin film conformation, we produced different electrodes with because of their transparency, which greatly facilitates the dif- different thicknesses, obtained by changing the number of CV ferent colors recognition (besides providing good electrical cycles used for the electropolymerization. We found that the conductivity and a cheap and easy manufacturing). As expected electrochromic behavior of PANI appears on the fabricated ITO ITO electrodes without PANI is fully transparent, thus failing SPE after 4 CV cycles, but it reaches the appropriate stability in producing any absorption peak, while different oxidation and wide color range at 6 cycles. Increasing the number of cy- states of the PANI film formation produced specific absorption cles over 6 decreases the color range, since it stabilizes the em- spectra. The bands at 450 nm is related to π-π* transition of eraldine form impeding the formation of the lucoemeraldine benzene rings, characteristic of LB form from yellow to yellow- (yellow color).28 Once optimized the thin film fabrication for ish green in the potential range of -0.4 V to 0.2 V. As the applied our purpose, we characterized it using Atomic force microscopy potential increased, this characteristic peak disappeared and the (AFM) (Fig. S3), finding that the final thickness, using the 6 new one observed at 650 nm, demonstrate formation of emer- cycles CV deposition, is around 36.4 ± 4.4 nm. The nm-size aldine which contains conjugated rings.25 The coloration thickness causes a considerable increase in the active surface switching response of PANI is followed by recording chrono- area for the electrode to improve the immobilization of anti- amperogram of PANI/ITO SPE in 0.5 M H2SO4 during a redox body. reaction. Fig. 2C reports the current-time events by applying - 0.4 V (movie 2. clip of reduction reaction in S.I.) and +1.0 V (movie 3. clip of oxidation reaction in S.I.). The time of switch- ing between fully reduced (LB) and fully oxidized (EB) is found to be 1.25s, meaning that the average switching time between three PANI redox states (LB to ES to EB) is approximately 0.4s, which is suitable for the fabrication of the fast multicolor elec- trochromic device. To further confirm the electropolymerization of aniline at the surface of the working electrode, we characterized the resulting ITO SPE using Field Emission Scanning Electron Microscope (FESEM), Energy-dispersive X-ray spectroscopy (EDS), X-ray powder diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR) analysis. Fig. 2D shows the fibrous mor- phology for PANI with a uniform distribution of chains over the surface of ITO SPE, which were further confirmed by detecting carbon, nitrogen, and sulphur in EDS data (Fig. S1 A) and by the FTIR spectra (Fig. S1 B). In particular, the well-resolved peaks at 1495 and 1575 cm-1 corresponded to the C=C stretch- ing vibration of benzenoid and quinonoid rings, the peak at 1304 cm-1 corresponds to the C-N stretching vibration with ar- omatic conjugation, the peak observed at 1153 cm-1 corresponds to Q=NH+-B stretching (Q and B are quinonoid and benzene rings respectively) and, especially, the peak between 2500-3500 cm-1 corresponds to the =N-H stretching vibration.25 We also studied the polymer crystal structure by XRD (Fig. S1 C), Figure 3. Effect of the applied potential (A) and its applica- whose data show peaks at 2θ=22° and 2θ=26° that are origi- tion time (B) to the signal generation for different E. Coli con- nated from (020) and (200) crystalline phase of PANI. As com- centrations. The use of high potentials limits the response range pared with the highly crystalline polyethylene terephthalate (we selected 0.2V as the optimal parameter). Instead the time (PET) of bare ITO SPE, the peaks of PANI are relatively small does not affect much the response (we thus selected 10 sec) and merged with PET peaks (however, for better illustration of The amount of antibody immobilized on the surface of the affecting PANI electrochromic behavior, saturating the system electrode is a key factor to efficiently capture the analyte. Thus, even before the capture of the bacteria. The middle concentra- we tested three different amounts of antibody (using solutions tion was the only one that provided enough binding capability with 2.5, 5.0 and 10.0 µg/mL of antibody), which we immobi- without affecting the sensor before the binding of the bacteria lized using glutaraldehyde as cross-linking agent. We found happens. It provided the full color range from yellow to green that the smallest concentration failed in providing enough bind- and blue. These results are depicted in Fig. S4 A. The interac- ing capability to accumulate enough bacteria to affect the elec- tion time between E. coli and anti-E. coli antibody was opti- trochromic properties of PANI (the color was always dark blue mized using a concentration of 104 CFU/mL of E. coli. We for all concentration of E. coli O157:H7 we tested). Instead, the found that the optimal incubation time to observe a color change highest concentration was so high that the antibody itself was corresponded to 60 min (Fig. S4 B). Figure 4: Schematic and performance of the proposed electrochromic sensor. A) Scheme of sensor fabrication and utilization. B) Sensor output for different concentrations of E. Coli. C) Sensor selectivity using S. typhymorium as interferent. D) Real samples results using tap water and Ter river water spiked with E. Coli. One of the most important parameters in an electrochromic Conclusion sensor is the choice of the applied potential, since it defines the In summary, here we presented a novel electrochromic sensor dynamic range of the sensor and its sensitivity. In our case we for the convenient optical detection of bacteria in water sample tested one blank and three different target concentrations (10 2, at the point of care. The outstanding analytical performances in 104, 106 CFU/mL) vs five different potentials (0.2. 0.4, 0.6, 0.8, terms of sensitivity, specificity and ease of use make of this sen- 1 V). The results (Fig. 3A) show how the smallest potential ap- sor a great tool for environmental and food-safety applications, plied (0.2 V) was the one that provided the highest sensitivity that go beyond the detection of E. Coli. and color change, allowing the discrimination of the three dif- ferent E. coli concentrations. Using higher potential lead to a ASSOCIATED CONTENT minor sensitivity due to the fact the complex antibody/bacteria was not enough to disrupt the PANI electrochromic properties. Supporting Information We also tested different times for the applied potential (Fig. Supporting information includes: EDS, XRD and FTIR analysis of 3B), but this parameter did not affect much the system, thus we synthesized PANI; The absorbance spectra obtained for PANI in decided to use the shortest time of 10 sec. PBS; The AFM images of PANI thin film; Optimization of anti E. coli antibody amount and E. coli incubation time; Cyclic voltam- Having an insight into the fabrication and detection process mograms and Nyquist plots in each steps of immunosensor fabri- can be very handy when preparing a new biosensing device (i.e. cation. checking for potential issues before running the sample can save The Supporting Information is available free of charge on the ACS both time and money, besides it can confirm what happens on Publications website. the electrode surface). Taking advantage of the electrochemical capabilities of our system we managed to do just that, using cy- clic voltammetry (CV) and electrochemical impedance spec- AUTHOR INFORMATION troscopy (EIS) methods in the presence of redox probe (0.1 M KCl containing 1 mM Fe(CN)63-/4-). First we confirmed the for- Corresponding Author mation of PANI thin film onto the SPE electrode surface. *Arben Merkoçi, Tel.: +34 937374604 Thanks to the high conductivity and surface area of PANI, once E-mail address: arben.merkoci@icn2.cat.. the thin film is produced we observed both an increase in the Author Contributions peak current as well as a decrease in the charge transfer re- sistance (Rct). Then we verified the correct surface functional- The manuscript was written through contributions of all authors. / ization with anti-E. Coli antibody observing a decrease in the All authors have given approval to the final version of the manu- peak current and an increase in the Rct. This behavior is due to script. / the steric hindrance that antibodies produce on the electrode Notes Any additional relevant notes should be placed here. surface, limiting in fact the contact between the electrode and the redox probe. Similarly, the addition of E. Coli produced a ACKNOWLEDGMENT further peak decrease and increased Rct, due to an even higher steric hindrance effect produce by the bacteria on the surface The ICN2 is funded by the CERCA Programme / Generalitat de (Fig. S5). Catalunya. The ICN2 is supported by the Severo Ochoa program of the Spanish Ministry of Economy, Industry and Competitiveness After the optimization and characterization of our electro- (MINECO, grant No. SEV-2017-0706 and MAT2017-87202-P). chromic sensor, we proceeded to evaluate its analytical perfor- C.P. acknowledges Marie Skłodowska-Curie Actions Individual mances for the detection of E. Coli. First we challenged it with Fellowship (Funding scheme: MSCA-IF-EF-RI, Proposal number: concentrations of E Coli from 101 to 106 CFU ml-1, showing that 795635). The Iranian authors gratefully acknowledge the Iranian we can detect up to 102 CFU ml-1 by naked eye (Fig. 4). 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