www.acsami.org Research Article Laser Reduced Graphene Oxide Electrode for Pathogenic Escherichia coli Detection Lei Zhao, Giulio Rosati,* Andrew Piper,* Cecilia de Carvalho Castro e Silva, Liming Hu, Qiuyue Yang, Flavio Della Pelle, Ruslán R. Alvarez-Diduk, and Arben Merkoçi* Cite This: ACS Appl. Mater. Interfaces 2023, 15, 9024−9033 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Graphene-based materials are of interest in electro- chemical biosensing due to their unique properties, such as high surface areas, unique electrochemical properties, and biocompatibility. How- ever, the scalable production of graphene electrodes remains a challenge; it is typically slow, expensive, and inefficient. Herein, we reported a simple, fast, and maskless method for large-scale, low-cost reduced graphene oxide electrode fabrication; using direct writing (laser scribing and inkjet printing) coupled with a stamp-transferring method. In this process, graphene oxide is simultaneously reduced and patterned with a laser, before being press-stamped onto polyester sheets. The transferred electrodes were characterized by SEM, XPS, Raman, and electrochemical methods. The biosensing utility of the electrodes was demonstrated by developing an electrochemical test for Escherichia coli. These biosensors exhibited a wide dynamic range (917−2.1 × 107 CFU/mL) of low limits of detection (283 CFU/mL) using just 5 μL of sample. The test was also verified in spiked artificial urine, and the sensor was integrated into a portable wireless system driven and measured by a smartphone. This work demonstrates the potential to use these biosensors for real-world, point-of-care applications. Hypothetically, the devices are suitable for the detection of other pathogenic bacteria. KEYWORDS: Graphene electrodes, Fabrication, Biosensing, Nanomaterials, Bacteria detection ■ INTRODUCTION Electrochemical biosensors have many inherent advantages over graphene electrodes with different surface compositions and defect densities. The patterning of graphene electrodes is important for other sensing modalities, including quantitative output, low cost, electrochemical sensor fabrication.4,16,17 Lithographic techni- and high sensitivity.1−3 Graphene-based electrodes have shown ques are commonly used to pattern low defect graphene;17,18 great potential for developing sensitive electrochemical sensors however, the need for expensive equipment and clean room due to the tunable electrical conductivity, high surface area, facilities makes these methods inaccessible to most researchers. versatile functionality, and biocompatibility, with numerous Template based methods, such as vacuum filtration,19 chemical applications in the field of health care, food safety, and covalent bonding,20 and contact transfer printing,21 have also environmental monitoring.4−6 Low defect graphene is usually been used to pattern graphene, but they require physically or produced by physical exfoliation, in which expensive/toxic chemically patterned templates, which are laborious to prepare solvents (N-methyl-2-pyrrolidone, dimethyl sulfoxide, and N,N- and can require harmful chemicals, limiting their scalability. dimethylformamide) are used,6 or chemical vapor deposition Printing approaches have been developed to realize the scalable (CVD),7 which involves high temperatures and tedious production of low-cost graphene electrodes.22 However, in transferring process and has low yields, limiting the real-world order to make the graphene printable, additives and fillers are applicability of CVD graphene electrodes. To address these often needed to adjust the rheological properties of the inks; this issues, “functionalized” graphene,8−10 such as graphene oxide can affect the electrochemical performance of the final printed (GO), has emerged as an alternative for graphene electrode fabrication. However, the poor conductivity of GO restricts its Received: November 21, 2022 use in electronic devices.11 Reduced graphene oxide (rGO) Accepted: February 1, 2023 displays good conductivity and a high electron transfer rate and Published: February 14, 2023 preserves enough oxygen groups for further functionalization.12 Chemical,13 thermal,14 and electrochemical15 methods to reduce GO to rGO have all been reported. Each results in © 2023 The Authors. Published by American Chemical Society https://doi.org/10.1021/acsami.2c20859 9024 ACS Appl. Mater. Interfaces 2023, 15, 9024−9033 ACS Applied Materials & Interfaces www.acsami.org Research Article electrodes. Likewise, the substrates may require chemical and artificial urine (AU), with a commercial potentiostat and a modification to have stable and uniform ink adhesion. portable, wireless system driven by a smartphone,36 displaying Additionally, high temperature thermal annealing is often its capability for point-of-care applications. required after printing,23 which can affect the material properties of the fabricated devices and limit the choice of substrates to temperature resistant materials. Since printing is a solution- based fabrication process, restacking of graphene flakes is also ■ EXPERIMENTAL SECTION Materials. GO solution (GO water dispersion, 10 mg/mL) was purchased from Graphenea (San Sebastian, Spain). Polyester (PE) possible during sintering.9 This restacking, driven by van der sheets (AUTOSTAT HT1802168201) were obtained from MacDer- Waals forces and π−π stacking, reduces the graphene surface mid Autotype Ltd. (Wantage, UK). Phosphate buffered saline tablets, area. Tween 20, and Tryptic Soy Agar were ordered from Merck Life Science One promising method of patterning graphene is with lasers, S.L.U (Madrid, Spain). Phosphate buffered saline buffer (PBS, 10 mM, to create laser-induced graphene (LIG) electrodes.24−26 It is an pH 7.4) was prepared by dissolving PBS tablets in ultrapure water. PBS easy, efficient, low-cost, chemical, and mask-free method of with Tween 20 (PBST) buffer was prepared by adding Tween 20 (final simultaneously reducing and patterning GO (or alternatively the concentration 0.05 wt %) in PBS buffer (10 mM). Polyclonal anti-E. coli in situ creation of graphene). Previous works have been reported antibody (PA1−7213) was obtained from Fisher Scientific (Madrid, Spain). HRP labeled antibodies (ab20425) and 3,3′,5,5′-tetramethyl- by the Kaner group,27,28 in which LIG electrodes were fabricated benzidine (TMB) substrate (ab171523) were purchased from Abcam using a standard LightScribe DVD driver; the total cost of this plc (Cambridge, UK). Escherichia coli (E. coli, CECT 4972) and process, including purchasing the laser, was less than $20. Lin et Staphylococcus aureus subsp. Aureus (SA, CECT 5190) were purchased al.29 were the first to produce porous graphene via the direct from CECT (Valencia, Spain), and Salmonella typhimurium (ST, laser scribing of a commercial polyimide sheet with a CO2 laser ATCC 14028) from ATCC (Virginia, USA). in 2014. Ever since, polyimide substrates have dominated the LRGO Electrode Production. PE substrates were washed with field, although there has been much research into alternative isopropanol (IPA) and dried with nitrogen or compressed air. materials, including other polymers,16 carbon, and natural Afterward, 5 mL of GO solution was drop-casted on an 8 × 8 cm2 square defined on the PE sheet with paper tape (to prevent the solution materials (such as wood and leaves) produced by different laser from spreading), shaken gently by hand, and then dried at 60 °C for 2 h. types.16,30,31 There have been various works in the literature that A CO2 laser engraver (Rayjet 50 Laser Engraver, Trotec Laser GmbH, focus on not only electrode fabrication but also biosensor Marchtrenk, Austria) was used to scribe the GO film to produce laser development on electrodes formed by laser scribing polyimide reduced graphene oxide (LRGO). The laser settings were as follows: films.16,24,26 However, at the time of writing, the authors are power = 2.07 W and speed = 1.5 m/s. The LRGO was placed face down unaware of any publications showing biosensor development on on the desired substrate, fixed tightly in place with scotch tape, electrodes created by the laser-induced reduction of GO. The sandwiched between four sheets of A4 paper (2 layers above and 2 main difficulties that prevent the development of biosensors by layers below the sample), and placed in a hydraulic press (SPECAC this method are as follows. (1) The reduction happens in situ, manual hydraulic press 15 ton, UK). A 6-ton force was applied for 1 min to transfer the LRGO onto the hosting substrate, forming both the meaning that there will be GO and rGO in the same plane. Since working electrode (WE) and counter electrode (CE). Silver contacts GO is highly soluble in water, it will dissolve in any biological were inkjet printed with a Dimatix 2831 (FUJIFILM Dimatix, USA. media.9,32 (2) LIG is very fragile, even being damaged by gentle Print temperature 30 °C, plate temperature 60 °C, drop space 25 μm, 3 gas flows.33 To overcome these issues, the Nam group layers), using a silver ink (Sicrys I40DM-106, PV Nano Cell Ltd., developed a method of transferring LIG from polyethylene Migdal Ha’Emek, Israel) and then oven-cured at 160 °C for 2 h. terephthalate (PET), but this method is only applicable to Polydimethylsiloxane (PDMS, SYLGARD 184 silicon elastomer kit, substrates that do not absorb the excitation laser.34 The Merkoçi Dow corning, MI, USA) was used as an insulating layer; a schematic group recently reported a novel, stamp transfer method, capable summary of the insulation process has been included in Figure S1. Ag/ of fabricating rGO electrodes on a wide variety of substrates.35 AgCl pseudoreference electrodes (RE) were obtained by chlorinating the printed Ag trace with 0.4% NaClO for 3 min.37 In this method, a GO film is formed on a polyvinylidene fluoride Characterizations of LRGO. Cyclic voltammetry (CV) was done filter membrane by vacuum filtration, before being simulta- with a commercial potentiostat (PalmSens4, PalmSens BV, Houten, neously patterned and reduced by a LightScribe DVD driver. Netherlands), in an aqueous solution of 0.5 mM potassium The LIG was then transferred to various substrates with a ferrocyanide, 0.5 mM potassium ferricyanide, and 0.1 M KCl at scan mechanical press. In this process, the rGO is transferred and the rate of 50 mV/s. For defining the electrochemical active surface area GO is not, while the LIG is compressed, improving its (ECSA), CVs were performed in 0.1 M NaClO4 (in ethanol) by using a mechanical properties. This proof-of-concept work only verified 3 mm diameter LRGO disk as the WE, four LRGO squares (Each 1 that the transfer stamped LIG electrodes were capable of cm2) as CEs, and a commercial Ag/AgCl electrode (CHI111, CH Instruments, Inc.) as the RE. The height of the films was measured by electrochemical sensing, not biosensing. In addition, the process an optical profilometer (Profilm 3D, Filmetrics Inc.) equipped with a required a vacuum pump, could only produce small batch sizes, 10× objective and using a stage movement speed of 0.12 mm/s. required expensive filter membranes, and was relatively slow. Scanning electron microscopy (SEM) was performed with a Quanta Herein we report advancements on our previous work on laser 650 (UK) at a working distance of 10 mm and voltage 10 kV; the reduced graphene oxide (LRGO) electrode fabrication, to make samples were mounted on aluminum stubs and sputtered with Au prior the production simpler and more scalable, as well as showing to imaging. The sheet resistance was obtained by measuring square (1 × their biosensing ability. The LRGO working electrodes were 1 cm2) samples with a Keithley 2400 using a four-point probe system stamped onto polyester (PE) sheets ,and silver inks were inkjet- (van der Pauw method). Raman spectra were obtained with a Raman printed to create silver contacts and Ag/AgCl reference microscope (Alpha300 R-Raman Imaging Microscope, Oxford Instru- ments) using a 488 nm laser with a spot size of approximately 3 μm. The electrodes. A LRGO electrode based biosensor is demonstrated parameters used were as follows: laser power = 1 mW, grating = 600 gr/ for the first time; as a proof-of-concept, it was developed for the nm, objective = 50×, exposure time = 10 s, accumulations = 2, detection of Escherichia coli (E. coli) by creating an electro- waveform range = 500−3500/cm. X-ray photoelectron spectroscopy chemical enzyme linked immunosorbent assay (ELISA). The (XPS) measurements were performed at room temperature with a sensor was validated in both phosphate buffered saline (PBS) SPECS PHOIBOS 150 hemispherical analyzer (SPECS GmbH, Berlin, 9025 https://doi.org/10.1021/acsami.2c20859 ACS Appl. Mater. Interfaces 2023, 15, 9024−9033 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 1. (a) Schematic of the laser reduced graphene oxide (LRGO) electrode fabrication process. (i) A graphene oxide (GO) film was formed on a polyester (PE) sheet by drop casting GO solution in a defined area and drying at 60 °C; (ii) laser scribing to reduce and pattern the GO film, creating LRGO; (iii) LRGO was placed face down on the desired substrate; (iv) pressure was applied to stamp the LRGO; (v) after separation, a mirror image of the LRGO had been transferred; (vi) inkjet printing of silver connections; (vii) polydimethylsiloxane (PDMS) was cast as a dielectric layer; (vii) NaClO was used to convert Ag to Ag/AgCl as reference electrode. (b) Photographs corresponding to each of the steps shown in (a); (ix) magnification of the final electrode, consisting of LRGO working electrode (black dot), LRGO counter electrode (black arc) and Ag/AgCl pseudoreference electrode (dark brown arc) and PDMS insulation layer inside the dashed square. Germany) at a base pressure of 5 × 10−10 mbar using monochromatic Al Bacteria Preparation. Bacteria were cultured overnight in Tryptic Kα radiation (1486.74 eV) as the excitation source, operated at 300 W. Soy Agar at 37 °C. Afterward, colonies were picked and suspended in The energy resolution as measured by the full width at half-maximum PBS. Then the bacterial solutions were diluted to obtain an OD600 (fwhm) of the Ag 3d 5/2 peak for a sputtered silver foil, and was found value as close to 109 CFU/mL as possible.38 Finally, the bacteria were to be 0.62 eV. The fitting of the obtained spectra was done with heat-killed at 65 °C for 20 min.39 XPSPEAK 41 with a Shirley background correction. Electrochemical Bacterial Detection. To perform the bacteria LRGO Electrode Functionalization. The LRGO working detection, 5 μL of the bacteria-containing solution was pipetted onto electrode was activated with 1-pyrenebutanoic acid succinimidyl ester the WE, left for 30 min, and then washed twice with PBST and twice solution (PBASE, 1 μL of 2 mg/mL in dimethylformamide) at room with PBS. Directly after cleaning, 4 μL of the detection antibody solution (dAb, ab20425, 2 μg/mL) was pipetted onto the WE and left temperature for 4 h. The electrode was then cleaned twice with IPA and for 30 min, and then washed twice with PBST and 4 times with PBS. four times with ultrapure water. The capture antibody (cAb, 15 μg/mL Subsequently, 50 μL of the TMB ELISA solution was added, and the in 10 mM PBS) was spontaneously immobilized by incubating 5 μL of chronoamperometric sensing started. Chronoamperometry (CA) was the solution on the WE (4 °C, overnight). Ethylamine (4 μL, 0.1 M in performed at +0.125 V, vs the on-board Ag/AgCl RE, with both the PBS) was put on the WE for 30 min to block any unreacted PBASE, and PalmSens4 and a wireless smartphone driven potentiostat developed in then the electrode was further blocked with bovine serum albumin house and fabricated according to a previous publication.36 (BSA, 5 μL, 5% in PBS) at 37 °C for 1 h to prevent nonspecific fouling ELISA Bacterial Detection. The ELISA test was performed in complex media. Between each step, the electrode was washed twice according to a previously established protocol with minor modifica- with PBST and twice with PBS; all the steps above were performed in a tions.40 First, 100 μL of capture antibody (PA1−7213, 2 μg/mL in humidity chamber. Carbonate-Bicarbonate Buffer, pH 9.6) was aliquoted into a 96-well 9026 https://doi.org/10.1021/acsami.2c20859 ACS Appl. Mater. Interfaces 2023, 15, 9024−9033 ACS Applied Materials & Interfaces www.acsami.org Research Article ELISA plate (44−2404−21, MaxiSorp, Fisher Scientific, Spain) and which causes the surface to rapidly expanded and blister, this is incubated at 4 °C overnight. Second, 200 μL of BSA (3% in PBS) was proposed as the root cause for the changes in surface added into the wells and incubated at 37 °C for 1 h, to prevent morphology in these experiments. A large pressure is applied nonspecific fouling of the wells. Third, 100 μL of bacterial samples was to the film so it is pressed flat during transferring, hence the added and kept at room temperature for 1h. The wells were washed three times with PBST between each step, and then incubated with 100 decrease in thickness to 0.4−1.3 μm. μL of HRP-labeled detection antibodies (HRP-dAb, 0.33 μg/mL GO, LRGO and T-LRGO were characterized via XPS, in all ab20425, in PBS) for 1 h at room temperature. Finally, the ELISA wells these samples the C and O peaks dominated (Figure 3a). The were washed 5 times with PBST. Afterward, 100 μL of the TMB substrate solution was added into each well and left for 10 min at room temperature. Finally, 50 μL of 2 M H2SO4 was added into the wells and then the absorbance of each well was detected at 450 nm using a SpectraMax iD3Multi-Mode Microplate Reader (Molecular Devices, USA). ■ RESULTS AND DISCUSSION In this work, all the digital designs were created with AutoCAD 2018 (Autodesk, USA) and then sent to the laser engraver/ inkjet printer to produce the patterns, this direct writing strategy along with the stamp-transferring method enables the simple, fast, maskless and large-scale production of LRGO electrodes (Figure 1). SEM image of the GO film surface (Figure 2a), Figure 3. (a) XPS survey and high-resolution C 1s spectra of (b) GO, (c) LRGO, and (d) T-LRGO. The survey spectra show a significant decrease in O content after laser radiation; C 1s spectra of GO reveals an abundance of oxidized carbon C−O (286.4 eV) and C�O (287.7 eV); the dominance of the C−C (284.4 eV) bond and presence of π−π* (291.2 eV) in LRGO and T-LRGO proves GO has been reduced by the laser. increase in the C/O ratio when the GO (2.7) is reduced to LRGO (22.0) is expected and confirms that the laser reduction was successful. During the stamp-transferring step, the surface of the LRGO is placed in contact with the new substrate and the Figure 2. (a) SEM image of the boundary between the LRGO and GO film, showing the morphological and topographical changes in the surface of the T-LRGO is, what was once, the bulk of the LRGO. graphene upon reduction. (b) Magnified SEM image of the LRGO Therefore, the similar C/O ratio between T-LRGO (23.4) and showing the flake-like structure. (c) SEM image of the LRGO after LRGO (22.0) indicates that the laser-induced reduction is not a transferring onto a polyester (PE) sheet, dubbed T-LRGO. (d) surface confined process but penetrates into the bulk of the GO. Magnified image of the compressed flake-like structure of T-LRGO on The high-resolution C 1s spectra of GO (Figure 3b) display PE; inset is a high magnification of the same region to evidence the flake peaks at 284.4, 286.4, and 287.7 eV, which can be assigned to sp2 structures. hybridized carbon in the graphene lattice (C−C), sp3 carbons in the form of epoxide and hydroxyl carbons (C−O) and sp3 shows that the surface has the bumps and ravines typical of carbons in the form of carbonyl groups (C�O), respec- graphene-based materials.9,32,41 After laser treatment, the film tively.28,42,43 Further analysis shows the relative peak areas are becomes a foam-like, porous structure, composed of flakes 10s of 39.0% (284.4 eV), 46.8% (286.4 eV), and 13.9% (287.7 eV), microns long (Figure 2a and b). Additionally, the GO changed Table S1; the high proportion of oxygen containing functional from a brown color to black (Figure 1, i, ii) upon reduction, in groups shows the degree of oxidation of the GO. In the C 1s agreement with the literature.35 Although the LRGO surface is spectra of LRGO (Figure 3c), the presence of a disordered smoother and less porous after the stamp-transferring process, it carbon peak at 285.0 eV (25.6%)28,43 with a small C−O peak still exhibits a highly porous 3D framework (Figure 2c and d). (8.8%) and the disappearance of the C�O peak further proves The data from the optical profilometer measurements, Figure the effective reduction of the GO by the laser scribing. The S2, show that the GO film is 3−4.5 μm thick; after laser increase in sp2 hybridized carbon content (C−C, 56.4%) and irradiation, the surface becomes much more heterogeneous but significant increase in the π−π* (at 291.2 eV, 9.2%; and 0.3% in noticeably increases to a maximum height of 18.2 μm in some the case of GO) satellite peak shows that the graphene lattice has areas. The laser scribing of GO is a photothermal process; been restored upon reduction of the GO. The data from the although the exposure time is only microseconds, the LRGO and T-LRGO samples are the same within error, showing instantaneous temperature at the surface is over 1000 °C,16 that the reduction is uniform to the transferred graphene depth 9027 https://doi.org/10.1021/acsami.2c20859 ACS Appl. Mater. Interfaces 2023, 15, 9024−9033 ACS Applied Materials & Interfaces www.acsami.org Research Article and that the transferring has no effect on the chemistry of the measurement so the GO lying on the surface of the electrode graphene. The degree of reduction GO reduction is clearly very dominates the signal. This was not observed in the XPS since it high for this process, given all the above, but was further proven has much larger sampling area (spot size 100s μm, and 3 μm in by the improved conductivity of the materials, the GO had a the case of Raman) and probes the chemistry up to a certain sheet resistance of >20 MΩ/sq,27 which decreased to 90.6 ± 3.9 depth (depending on the substrate and experimental con- Ω/sq for LRGO (the thickness was assumed to be 1 μm). ditions), reporting the average chemical composition of a The Raman spectroscopy data (Figure 4a) shows that the volume at the surface. We can therefore surmise that there is a characteristic D (∼1345 cm−1) and G (∼1580 cm−1) bands of thin layer of GO on the surface of the T-LRGO. The surface area, roughness, and porosity of electrodes can directly influence the performance of electrochemical sen- sors.1,25 To ascertain the electrochemically active surface area (ECSA) of the LRGO electrodes, non-Faradaic CV was performed in 0.1 M NaClO4 in ethanol. The CVs of the electrodes at scan rates from 20 to 200 mV/s are provided in Figure 4c. Plotting current density (j) against the scan rate (υ) (Figure 4d), a linear relationship was obtained, in which the slope of the linear plot is equal to the area specific double layer capacitance (CS), and the CS of the electrode is directly related to its ECSA through eqs 1−3:48 (1) (2) (3) where S is the geometric surface area of the electrode; υ is scan rate; I is the current; j is current density; Cref is the area specific Figure 4. (a) Representative Raman spectra of GO, LRGO, and T- double-layer capacitance of a flat reference surface. LRGO on PE, showing the typical D (∼1345 cm−1) and G (∼1580 Equationss 1−3 and the data in Figures 4c,d and S3 were used cm−1) bands of graphene; the 2D band and decreased ID/IG ratio in to calculate the ECSA of the electrodes based on the Cref of LRGO are indicative of GO reduction by laser scribing. (b) Average 2D graphite (∼20 μF/cm2);49,50 the average electrode area from band from 5 measurements of LRGO (shaded area refers to the three repeats was found to be 0.781 ± 0.022 cm2. The ECSA is standard deviation of the average), showing that the LRGO contains more than 10 times larger than the geometric area (0.07 cm2), only a few graphene layers, i.e., is not multilayered graphene. (c) Cyclic voltammograms obtained in a solution of 0.1 M NaClO4 in ethanol vs a which is expected given the highly rough and porous nature of LRGO CE (4 cm2) and a Ag wire pseudoreference electrode, at a series the electrodes. The standard deviation of the three repeats of scan rates shown in the inset. (d) Current density from (c) plotted as speaks to the reproducibility of the LRGO working electrodes. a function of the scan rate, to determine the electrochemical active Inkjet-printed silver was used to create electrode contacts and surface area (ECSA) of the electrodes. The black dots represent raw to fabricate the reference electrode.37 Open circuit potential data and red line is a linear fit to the data. measurements of the printed quasi-reference electrodes (N = 4) vs a commercial Ag/AgCl electrode (CHI111, CH Instruments, graphene are present in all three samples, with intensity ratios Inc.) exhibit potential fluctuations of less than 4 mV over 200 s, (ID/IG) of 1.00 ± 0.02 for GO, 0.41 ± 0.07 for LRGO and 0.99 ± and a potential difference of less than 14 mV (Figure S4a), 0.01 for T-LRGO (n = 6). The 2D band, at ∼2700 cm−1, was verifying the functionality of the prepared reference electrodes. only present in the LRGO samples. Previous publications41,44,45 Figure 1b, ix shows a photograph of the final electrodes, have assigned the D peak to the primary in plane vibrations of consisting of LRGO working and counter electrodes, and Ag/ graphene, so it can be used to estimate the degree of defects and AgCl quasi-reference electrode. The electrodes are made in disorder within the hexagonal lattice. The 2D peak is attributed batches; it is therefore prudent to assess the reproducibility of to second-order in-plane vibrations of graphene. The significant the electrochemical signal obtained from multiple batches. To decrease of ID/IG after laser exposure is therefore indicative of do this, 3 batches of electrodes were fabricated and 6 electrodes rGO formation, which is in accord with the XPS and sheet from each batch selected at random for testing (a total of 18 resistance findings. The normalized average 2D peaks from 5 electrodes were used). These were first modified with PBASE to LRGO spectra recorded at random locations on a single working make them hydrophilic, and CVs were recorded in 0.5 mM electrode surface, Figure 4b, show small standard deviations; this [Fe(CN)6]4−/[Fe(CN)6]3− with 0.1 M KCl. The peak indicates a homogeneous reduction of GO across the electrode potentials were identical for all the electrodes tested, which surface. The full width at half maximum (FWHM) of the 2D further shows the reproducibility of the quasi-reference bands were extracted using the Lorentzian function (Figure 4b), electrode fabrication (Figure S4b). The relative standard for the LRGO the FWHM was found to be 74.97 ± 2.93 cm−1, deviation (RSD) of the anodic peak current shows the indicating that the LRGO film consists of only a few graphene intrabatch variation is 4.2%, and the interbatch variation is 5.6%. layers.28,46,47 The Raman spectra of the T-LRGO are the same To prove that the electrodes have the potential to be used for within error as that of the GO. It is proposed that there are biosensor development, an immunoassay for the detection of residual amounts of unreduced GO on the surface of the pathogenic bacteria was developed on them. E. coli was used as a transferred electrodes. Raman spectroscopy is a surface model bacteria in these proof-of-concept tests, since their 9028 https://doi.org/10.1021/acsami.2c20859 ACS Appl. Mater. Interfaces 2023, 15, 9024−9033 ACS Applied Materials & Interfaces www.acsami.org Research Article detection is important for medical, food safety, and environ- mental monitoring.39,51 First a conventional ELISA was run to select the best capture and detection antibody pairs and act as a reference with which the performance of the T-LRGO electrodes could be compared. The control ELISA was capable of detecting E. coli between 9.35 × 104−9.06 × 106 CFU/mL and had a limit of detection (LOD) of 1.30 × 104 CFU/mL (Figure S5a). The same antibodies used in the conventional ELISA were then used to functionalize the T-LRGO and optimized to create a point of care biosensing system, that could be wirelessly driven by a smartphone, Figure 5a (see electric scheme in Scheme Figure 6. (a) Calibration curve of E. coli concentration vs normalized current response from the chronoamperometry at +0.125 V; the black stars were recorded with a commercial potentiostat in PBS, the green diamonds were recorded with the smartphone driven potentiostat developed in house, and the blue circles were obtained with a commercial potentiostat in artificial urine (AU). (b) Selectivity studies showing the current response without bacteria (orange), with Escherichia coli (green), Staphylococcus aureus (purple), Salmonella typhimurium (yellow), Escherichia coli and Staphylococcus aureus (blue), and Escherichia coli and Salmonella typhimurium (pink). In all cases, each bacterial concentration was 104 CFU/mL, with a 50:50 mix of each in the last two conditions. (c) Control experiments run either without the cAb, E. coli or without both. (d) Data from the stability studies of the sensors stored in different conditions for 1 month, measuring the current response to 105 CFU/mL of E. coli. Figure 5. (a) Smartphone controlled portable wireless system for E. coli highest concentrations of the analytes based on 10% and 90% of detection (not to scale). (b) Illustration showing the working the maximum signal respond on the calibration curve). The mechanism of the electrochemical enzyme linked immunosorbent improved sensing performance of the LRGO electrode based assay (ELISA). (c) Cyclic voltammograms of TMB on the function- platform compared with the standard optical ELISA(Table S2), alized T-LRGO electrode. (d) Representative current vs time transients is in accord with other reported electrochemical ELISA of various E. coli concentrations, recorded at +0.125 V vs Ag/AgCl. platforms.52 The performance of the sensor is suitable for the detection of E. coli in urine at clinically relevant concentrations S1).36 Figure 5b shows a schematic representation of the (103 − 105 CFU/mL)54,55 and it is advantageous that all the electrochemical sensing mechanism. In brief, HRP catalyzes the results were obtained from 5 μL samples. Ultrasensitive generation of OH radicals from H2O2, and these radicals oxidize detection, even down to single cell, has been demonstrated,56−58 TMB. The oxidized TMB (oxTMB) can be detected optically or but tedious material synthesis and complicated sensor electrochemically. When the oxTMB is reduced, the reductive construction are typically required (Table 1). Our platform current generated is proportional to the amount of oxTMB showed comparable performance with other electrochemical present. Given that the amount of oxTMB is determined by the sensors,59−61 and could be improved with further optimization amount of dAb, which is proportional to the amount of E. coli of the antibody concentrations (this work is mainly focused on bound to the electrode surface, the reductive current gives a the fabrication and characterization of the electrodes, with the quantitative evaluation of the amount of E. coli in the sample. In sensor construction being merely a proof-of-concept). other words, the amount of oxTMB is a directly proportional of To test the selectivity of the sensors, Salmonella enterica the amount of E. coli bound to the electrode. serovar Typhimurium (ST) and Staphylococcus aureus subsp. Figure 5c displays a CV of TMB on the functionalized LRGO Aureus (SA) were run in place of and alongside E. coli. The electrode. Oxidation peaks at +0.231 and +0.411 V and results of these tests are given in Figure 6b, where it can be seen reduction peaks at +0.166 and +0.367 V can all be easily that neither of these bacteria gave a signal significantly above distinguished (all potentials vs the on-chip Ag/AgCl electrodes). that of the blank. Likewise, when run alongside E. coli, the The current time transients obtained after incubating the responses are the same within error as those of E. coli alone. The functionalized electrodes with E. coli concentrations between 0 same trends were seen in the ELISA tests, Figure S5b; proving and 109 CFU/mL are provided in Figure 5d. As expected, the that the sensor is specific and that specificity is attributable to the normalized dose−response curve in Figure 6a shows a sigmoidal antibodies. Other control experiments, Figure 6c, showed that response. With the 4-Parameter Logistic model fitting52,53 the the signal response is negligible (−0.083 ± 0.034 μA, N = 4) in LOD was calculated to be 283 CFU/mL (blank + 3SD) with a the absence of E. coli and cAb; it increases to −0.349 ± 0.010 μA detection range between 917−2.1 × 107 CFU/mL (lowest and when only E. coli is excluded (blank control, N = 4), indicating 9029 https://doi.org/10.1021/acsami.2c20859 ACS Appl. Mater. Interfaces 2023, 15, 9024−9033 ACS Applied Materials & Interfaces www.acsami.org Research Article Table 1. Representative Nanomaterial-Based Biosensors for E. coli Detection Reported in Literaturea platform technique detection range (CFU/mL) LOD (CFU/mL) response time (min) sample volume (μL) ref metal NPs-LIG EIS 1 × 102−1 × 108 102 30 59 silica NPs CV 8 × 104−8 × 106 2 × 103 30 10 61 Au NPs LFA 104−106 104 10 150 39 Fe3O4@SiO2/ polymer/fluorescein fluorescent 4−4 × 108 3 75 1000 56 MWCNTs/chitosan/thionine CV 102−109 102 180 200 60 N-GQDs/MIP ECL 10−107 5 140 57 rGO/Al2O3/Au NPs FET 1−100 single cell 50 s 1 58 MoS2/Au/optical fiber SPR 103−8 × 109 94 ∼15 62 LRGO CA 917−2.1 × 107 283 60 5 this work a NPs = nanoparticles; N-GQDs = nitrogen dopped graphene quantum dots; MWCNTs = multiwall carbon nanotubes. that there is some cross-reaction between the antibodies, which sources with wavelengths ranging from 248 nm to 10.6 μm that is common when working with polyclonal antibodies.63,64 When can be used for LRGO formation.16,25,27,35,69 The printing of Ag only the cAb was omitted, the current (−0.391 ± 0.108 μA, N = connections and the reference electrodes can also be realized by 4) was the same within error as the blank control, indicating much cheaper methods, such as consumer inkjet printers, insignificant nonspecific binding of E. coli to the electrodes. screen/stencil printing, or simply painting silver inks. These The stability of the sensors was tested in different storage features mean that T-LRGO based sensing platforms can be conditions. After functionalization, batches of the sensors were produced in most research laboratories around the world. stored in humidity chambers placed at room temperature (taken as 20 °C), in the fridge (4 °C) and in a freezer (−20 °C), all under N2. The E. coli detection was performed at several time ■ CONCLUSIONS AND FUTURE WORK In this work, we reported a cheap and scalable method of points over 30 days, Figure 6d (N = 4). The signal variability at fabricating LRGO-based electrodes on polyester substrates. The 20 and 4 °C was found to be similar (RSD ≤ 7.6%) within the biosensing potential of the electrodes was proven by first 10 days, comparable to that of the variation in the electrodes functionalizing them for the detection of E. coli in PBS and calculated earlier (5.6%); even stored for 30 days, the sensors artificial urine. This proof-of-concept sensing platform could showed similar performance in all cases (RSD of the current detect E. coli between 917 and 2.1 × 107 CFU/mL, with a LOD response, 12.8% for 4 °C, 13.0% for 20 °C, 14.0% for −20 °C); of 283 CFU/mL, within the clinically relevant range for E. coli in these data show that the sensors are relatively stable for 30 days human urine. We believe that the platform can be utilized for the and the storage temperature makes no significant difference to detection of other bacteria or biomarkers, simply by changing the sensor stability. The slightly higher RSD for the sensors the antibodies. stored at −20 °C may come from the freeze−thawing cycles Being able to transfer these graphene-based sensors onto when taking them in and out of the freezer, which could damage different substrates would be of interest to other sensing the antibodies.65 applications, such as wearable sensing.6,36,44 An initial study The efficacy of the sensors was further evaluated in spiked showed that these LRGO films could be transferred onto a artificial urine. The results of these tests are shown in Figure 6a variety of other substrates, Figure S6, demonstrating the (the blue circles) and correlate very well with those obtained in potential applicability of these electrodes. It is also of note PBS. The recovery rates obtained are 119% for 500 CFU/mL, that the maximum resolution of the laser will define the 88% for 105 CFU/mL, and 90% for 106 CFU/mL of E. coli, resolution with which electrodes can be patterned. With our showing that the system has the potential to be used in real world equipment this was down to the micron scale, making matrices. microelectrode development with this technology possible. In order to create a point-of-care (POC) test, there is a need Future work will include the development of microelectrode for sensitive, inexpensive, wireless, portable detection plat- arrays by this method and the optimization of printing and forms.6,66−68 A previously established smartphone-based wire- stamping onto other substrates for different applications. less system36 was used to make this test more suitable for POC applications. Three concentrations of E. coli in PBS were tested with said wireless system and compared to a commercial ■ * ASSOCIATED CONTENT sı Supporting Information potentiostat. Movie S1 shows the real-time response of the The Supporting Information is available free of charge at system. The current responses obtained for three E. coli https://pubs.acs.org/doi/10.1021/acsami.2c20859. concentrations are the same within error as those recorded with a commercial potentiostat (Figure 6a, green diamonds). Illustration of PDMS insulation process; one-dimensional The scalability of the electrode fabrication has been proven by surface profile; XPS C 1s elemental composition; current the production of ∼500 electrodes in 1 day, although the whole density at different scan rates obtained with 0.1 M fabrication process for a single batch of 12 electrodes takes ∼4.5 NaClO4 in ethanol of two T-LRGO electrodes; open h. In fact, more than 2000 electrodes could be made under ideal circuit potential measurements of the as-prepared conditions, as calculated from Table S3. It is also worth pseudoreference electrodes vs a commercial Ag/AgCl mentioning that the cost of GO is estimated at € 0.06 per electrode, CV measurements of PBASE functionalized electrode, 4 times cheaper than polyimide. The total materials electrodes; ELISA calibration curve of E. coli and cost per electrode is € 0.09, and the whole sensor costs less than selectivity test by ELISA; 4-Parameter Logistic model € 0.12 (Table S4). While the CO2 lasers used herein are not fitting parameters for the stander optical ELISA and as- common laboratory equipment, there are other low-cost laser prepared electrochemical ELISA; the timetable of 9030 https://doi.org/10.1021/acsami.2c20859 ACS Appl. Mater. Interfaces 2023, 15, 9024−9033 ACS Applied Materials & Interfaces www.acsami.org Research Article electrode fabrication in detail; detailed material costs for University and Research (MIUR) and European Social Fund the device fabrication; electric scheme of the wireless (ESF) for the PON R&I 2014-2020 program, action 1.2 ’AIM: electrochemical measurement unit; T-LRGO patterns on Attraction and International Mobility’ (AIM1894039-3). different substrates (PDF) Cecilia C. C. Silva acknowledges funding through CAPES − Real-time response of the portable wireless system (MP4) PRINT (Programa Institucional de Internacionalização; grant #88887.310281/2018-00 and 88887.467442/2019-00) and ■ AUTHOR INFORMATION Corresponding Authors Mackpesquisa-UPM. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 825694. Views and Arben Merkoçi − Catalan Institute of Nanoscience and opinions expressed are however those of the author(s) only and Nanotechnology (ICN2), 08193 Bellaterra, Barcelona, Spain; do not necessarily reflect those of the European Union. The Catalan Institution for Research and Advanced Studies European Union can not be held responsible for them. We (ICREA), 08010 Barcelona, Spain; orcid.org/0000-0003- acknowledge Miguel Angel Aroca and Johann F. Osma (both 2486-8085; Email: arben.merkoci@icn2.cat from Department of Electrical and Electronic Engineering, Giulio Rosati − Catalan Institute of Nanoscience and Universidad de los Andes, Bogotá, Colombia) for the wireless Nanotechnology (ICN2), 08193 Bellaterra, Barcelona, Spain; smartphone-controlled system used in this work. The authors orcid.org/0000-0002-0227-4561; Email: giulio.rosati@ wish to express thanks to Marcos Rosado Iglesias, Antonio Pablo icn2.cat Pérez, Elena Del Corro García, Guillaume Sauthier and Raúl Andrew Piper − Catalan Institute of Nanoscience and Pérez (all Catalan Institute of Nanoscience and Nano- Nanotechnology (ICN2), 08193 Bellaterra, Barcelona, Spain; technology, CSIC and The Barcelona Institute of Science and Email: andrew.piper@icn2.cat Technology, Barcelona, Spain) for their expert assistance with Authors SEM, Raman spectroscopy, XPS and surface profile, respec- Lei Zhao − Catalan Institute of Nanoscience and tively. Nanotechnology (ICN2), 08193 Bellaterra, Barcelona, Spain; Department of Chemical Engineering, School of Engineering, Universitat Autònoma de Barcelona, 08193 Bellaterra, ■ ABBREVIATIONS GO, graphene oxide; rGO, reduced graphene oxide; LIG, laser- Barcelona, Spain induced graphene; E. coli, Escherichia coli; LRGO, laser reduced Cecilia de Carvalho Castro e Silva − MackGraphe-Mackenzie graphene oxide; T-LRGO, transferred laser reduced graphene Institute for Research in Graphene and Nanotechnologies, oxide; PDMS, Poly dimethylsiloxane; WE, working electrode; Mackenzie Presbyterian University, 01302-907 São Paulo, CE, counter electrode; RE, reference electrode; PBASE, 1- Brazil; orcid.org/0000-0003-3933-1838 pyrenebutanoic acid succinimidyl ester solution; cAb, capture Liming Hu − Catalan Institute of Nanoscience and antibody; dAb, detection antibody; LOD, limit of detection; CV, Nanotechnology (ICN2), 08193 Bellaterra, Barcelona, Spain; cyclic voltammetry; CA, chronoamperometry; SA, Staph- Department of Chemical Engineering, School of Engineering, ylococcus aureus subsp. Aureus; ST, Salmonella typhimurium; Universitat Autònoma de Barcelona, 08193 Bellaterra, XPS, X-ray photoelectron spectroscopy; SEM, scanning electron Barcelona, Spain; orcid.org/0000-0002-8666-9287 microscope; AU, artificial urine Qiuyue Yang − Catalan Institute of Nanoscience and Nanotechnology (ICN2), 08193 Bellaterra, Barcelona, Spain; Department of Material Science, Universitat Autònoma de ■ REFERENCES (1) Lahcen, A. A.; Rauf, S.; Beduk, T.; Durmus, C.; Aljedaibi, A.; Barcelona, 08193 Bellaterra, Barcelona, Spain Timur, S.; Alshareef, H. N.; Amine, A.; Wolfbeis, O. S.; Salama, K. N. Flavio Della Pelle − Faculty of Bioscience and Technology for Electrochemical Sensors and Biosensors Using Laser-Derived Gra- Food, Agriculture and Environment, University of Teramo, phene: A Comprehensive Review. Biosens. Bioelectron. 2020, 168, 64100 Teramo, Italy; orcid.org/0000-0002-8877-7580 112565. Ruslán R. 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Bioelectron. 2020, 150, 111896. 9033 https://doi.org/10.1021/acsami.2c20859 ACS Appl. Mater. Interfaces 2023, 15, 9024−9033