3D-printed graphene direct electron transfer enzyme biosensors

Journal Pre-proof 3D-printed graphene direct electron transfer enzyme biosensors Adaris M. López Marzo, Carmen C. Mayorga-Martinez, Martin Pumera PII:

S0956-5663(19)31057-7

DOI:

https://doi.org/10.1016/j.bios.2019.111980

Reference:

BIOS 111980

To appear in:

Biosensors and Bioelectronics

Received Date: 10 October 2019 Revised Date:

17 December 2019

Accepted Date: 19 December 2019

Please cite this article as: López Marzo, A.M., Mayorga-Martinez, C.C., Pumera, M., 3D-printed graphene direct electron transfer enzyme biosensors, Biosensors and Bioelectronics (2020), doi: https:// doi.org/10.1016/j.bios.2019.111980. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.

3D-Printed Graphene Direct Electron Transfer Enzyme Biosensors Adaris M. López Marzoa, Carmen C. Mayorga-Martineza, Martin Pumeraa,b,c,d* a

Center for Advanced Functional Nanorobots, Department of Inorganic Chemistry, University of Chemistry and Technology Prague, Technická 5, 166 28 Prague 6, Czech Republic

b

Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro, Seodaemungu, Seoul 03722, Korea

c

Department of Medical Research, China Medical University Hospital, China Medical University, No. 91 Hsueh-Shih Road, Taichung, Taiwan

d

Future Energy and Innovation Laboratory, Central European Institute of Technology, Brno University of Technology, Purkyňova 656/123, Brno, CZ-616 00, Czech Republic *Corresponding author: [email protected]

Abstract Three-dimensional (3D) printing technology offers attractive possibilities for many fields. In electrochemistry, 3D printing technology has been used to fabricate customized 3D-printed electrodes as a platform to develop bio/sensing and energy generation and storage devices. Here, we use a 3D-printed graphene/polylactic (PLA) electrode made by additive manufacturing technology and immobilize horseradish peroxidase (HRP) to create a direct electron transfer enzyme-based biosensors for hydrogen peroxide detection. Gold nanoparticles are included in the system to facilitate heterogeneous electron transfer. This work opens a new direction for the fabrication of third-generation electrochemical biosensors using 3D printing technology, with implications for applications in the environmental and biomedical fields. Keywords:: 3D-printed electrodes, graphene/polylactic acid, horseradish peroxidase, direct electron transfer 1. Introduction Three-dimensional (3D) printing technology has dramatically changed the methodological landscape for device prototyping and fabrication in the industrial and academic realms (Guo and Leu, 2013). For academic research, many possibilities for engaging this technology have arisen in the fields of biochemistry and medicine, and in the fabrication of microfluidic and sensor devices (Bhattacharjee et al., 2016; Palenzuela and Pumera, 2018); moreover, it opens the possibility to manufacture customized structures tailorable to the desired purpose in a fast and low-cost manner (Ambrosi and Pumera, 2016; Symes et al., 2012). 1

In electrochemistry, 3D printing technology has been used to produce flow cells (O’Neil et al., 2019), microfluidic sensors (Bhattacharjee et al., 2016; Rusling, 2018), and electrodes (Ambrosi et al., 2016; Rohaizad et al., 2019; Rymansaib et al., 2016) with the desired shape and composition. Some studies have reported 3D-printed metal electrodes for sensing (Lee et al., 2017; Loo et al., 2017) and energy devices (Ambrosi et al., 2016; Zhao et al., 2014). However, these 3D metallic electrodes present disadvantages such as a high production cost by selective laser melting, a post-production coating to avoid the underlying material’s poor performance, and the instability of some metals to pH and ambient conditions (Ambrosi and Pumera, 2016; Palenzuela and Pumera, 2018). In contrast, 3D carbon/polymer electrodes have gained in preference due to their cheaper manufacturing process by fused deposition modeling (FDM) printer, better stability, and wide range of working potentials for electrochemical applications (González-Sánchez et al., 2018; Rymansaib et al., 2016; Vernardou et al., 2017). These 3D carbon/polymer (polylactic acid (PLA) or acrylonitrile butadiene styrene) electrodes present poor electrochemical properties and require activation treatments after printing. Both organic solvent digestion and electrochemical treatments have been reported as effective routes of activation of 3D graphene/PLA electrodes (Browne et al., 2018; Palenzuela et al., 2018). Different geometries of 3D graphene/PLA electrodes have been applied in electrochemical sensing (Foo et al., 2018; Palenzuela et al., 2018) and evaluated for future energy device applications (Foo et al., 2018; Foster et al., 2017; Gusmão et al., 2019; Vernardou et al., 2017). Here, we investigate the potential application of 3D printing technology to an enzymatic biosensor. The development of third-generation biosensors is a crucial topic of huge potential for biosensors, biocatalysts, and biofuel cells in the current era of flexible wearable electronics (Das et al., 2016; Ran et al., 2011; Xu et al., 2010; Zhang et al., 2012). Third-generation biosensors obviate the need for mediators and are well suited for real-world biosensing applications. For the fabrication of third-generation biosensors, fast electron transfer between the redox enzymes and the electrode is crucial. Efficient direct electron transfer on carbon (El Kaoutit et al., 2008; Li et al., 2011; Sun et al., 2004) and nanoparticles-modified electrodes (Medina-Sánchez et al., 2016; Negahdary et al., 2012; Santos et al., 2005; Silva et al., 2012; Wan et al., 2013; Xu et al., 2010; Yi et al., 2000; Yin et al., 2009; Zhao et al., 2016) have been reported for horseradish peroxidase (HRP), an enzyme with an iron redox center and widely used in biosensors (Ferri et al., 1998; Kohri, 2014; Rahmanian et al., 2018; Veitch, 2004; Zhao et al., 2016). 2

Here, we demonstrate a new concept of a 3D-printed enzymatic graphene-PLA electrode for direct electron transfer using peroxidase enzyme for hydrogen peroxide (H2O2) detection. The direct electron transfer of horseradish peroxidase (HRP) immobilized directly onto the chemically and electrochemically activated 3D-printed electrode is highly efficient. Further modification of the 3D-printed graphene/PLA electrode with gold nanoparticles (AuNPs) is evaluated to confirm whether the direct electron transfer between the HRP and 3D-printed graphene-PLA electrode enhances it performance. This work opens great possibilities for 3D-printed enzymatic systems to detect glucose and other biomarkers in real samples without the use of electron mediators and binder polymers. 2. Materials and methods 2.1 Chemical and electrochemical 3D electrode activation Chemical and electrochemical treatments (DMF-EC) were accomplished to achieve 3D graphene/PLA electrode activation. For the chemical treatment, the 3D electrodes were completely immersed in DMF and sonicated for 5 min on a Fisherbrand FB11203 ultrasonic bath at a frequency of 37 KHz and 100% potency. Next, they were washed by sonicating for 2 min with water/acetone/water by independent batch. Afterward, a constant potential of 2 V for 150, 300, and 450 s was applied for the electrochemical treatment. 2.2 Preparation of the enzyme-based 3D graphene-PLA electrode Once total activation (chemical and electrochemical, DMF-EC) of the 3D electrodes was accomplished, they were modified with AuNPs by overnight incubation in 8 nM (500 µL) at 7°C and then washed with water. For the subsequent immobilization of the enzyme, 5 mg/mL (500 µL, PBS pH 7.2) of the HRP were also incubated overnight at 7°C with the 3D electrode/AuNPs followed by a slight washing in PBS. The as-prepared 3D graphene-PLA electrode/AuNPs/HRP biosensor was kept in PBS solution at 7°C until use. A similar process, but excluding the AuNPs modification, was carried out to obtain the 3D graphene-PLA electrode/HRP biosensor. Details of materials, reagents, and instruments used in this work are described in supporting information as well as a description of electrochemical measurements.

3

3. Results and discussion Graphene/PLA filament was selected to produce the 3D electrodes. PLA is an insulator polymer used to bind the graphene sheets and it creates a mechanically improved material for printing proposes while graphene adds conductivity to the filament. In addition, the graphene/PLA filament allows the cost-efficient and mass production of 3D-printed electrodes. 3D electrodes were first printed using graphene/PLA filament material (Fig. 1A) and then exposed to a chemical and electrochemical treatment to activate them (Fig. 1B) (Browne et al., 2018; Palenzuela et al., 2018). Subsequently, the electrodes were modified with HRP by electrostatic interactions for H2O2 detection (Figs. 1C and 1E). In addition, another biosensing system that incorporates AuNPs was implemented (Figs. 1D and 1F). AuNPs are well known and reported for this kind of biosensor. The enhanced biosensor performance confirmed the direct electron transfer between the electrode and the enzyme.

4

Fig. 1. Scheme of 3D graphene-PLA biosensor fabrication: (A) 3D-printing of the electrode; (B) activation in DMF and by electrochemistry; (C) modification of 3D-printed electrode with HRP enzyme; and (D) modification of the 3D-printed electrode with gold NPs and, subsequently, with HRP enzyme. (E) and (F) are corresponding mechanisms of H2O2 detection.

SEM images were recorded to monitor each step and study the changes in the surface morphology (Fig. S1). In the surface of the as-fabricated 3D electrodes, the carbon fibers are compressed by the PLA polymer as observed in the SEM image (Fig. S1A). However, a notable change was observed in the SEM image of the DMF-EC-activated electrodes (Fig. S1B). Figures S1C and S1E show the 3D-printed electrode modified with AuNPs, with the uniform distribution of AuNPs on the electrode surface clearly visible.

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Fig. 2. (A) Cyclic voltammograms of 3D-printed graphene-PLA electrode before (black line) and after chemical (orange line) and electrochemical (blue line) activations, scan rate of 0.1 V/s. Inset: CV of the as-printed 3D electrode. (B) RCT values of the 3D graphene/PLA electrode at different modification stages. Inset: corresponding Nyquist curves and Randles circuit used for the fitting. Frequency range from 0.1 to 106 Hz using amplitude of 0.01 V. Conditions: in 1 mM Fe(CN)63-/4-/0.1 M KCl.

Electrochemical performance of 3D-printed electrodes before and after the chemical and electrochemical activation processes was studied and is illustrated in Fig. 2A. The activation process of the electrode improved notably the heterogeneous electron transfer (HET) as demonstrated by the decreased peak-to-peak separation (∆Ep), which is transduced in an enhancement of reversibility of the CV of the Fe(CN)63-/4- redox probe. In addition, each activation step increased the electrode conductivity as demonstrated by the significant increase of current intensity of the peaks. Figure S2A shows the CVs in Fe(CN)63-/4- solution of the 3D electrodes during the electrochemical activation performed at different times (150, 300, and 450 s) by applying 2 V. The duration of the electrochemical activation from 150 to 450 s was associated with a profit in conductivity (Fig. S2B) and fast electron transfer (Fig. S2C) of the 3D electrode, similar to previous studies (Browne et al., 2018). This increase of conductivity in the 3D-printed electrodes was related to the formation of reduced graphene oxide functionalized with O–C=O, C=O, and C–O groups after the activation process. This was demonstrated by high resolution X-ray photoelectron spectra reported previously by Browne et al. (Browne et al., 2018, Al-Gaashani et al., 2019, Sheng-Eng et al., 2013). On the other hand, the rate constant of the heterogeneous electron transfer of the activated 3D electrode in 1 mM Fe(CN)63-/4- solution was 6

3.7 x 10−3 cm/s (Lavagnini et al., 2004; Muhammad et al., 2016) and the active area was 0.5 cm2 (Muhammad et al., 2016). Activated 3D-printed electrodes here were simply modified by electrostatic interactions with HRP (system 1) as well as with AuNPs and HRP (system 2) during overnight incubation (Jeon and Lee, 2011; Zhang et al., 2012; Zhang et al., 2015). Different HRP concentrations (1, 2.5, and 5 mg/mL) and different incubation times (1, 3, and 18 h (overnight)) were tested (data not shown). Next, experiments were conducted with the best response observed being the highest concentration of HRP (5 mg/mL) and longest incubation time (18 h). The electrochemical characterizations of the activated 3D-printed electrode modified with AuNPs (DMF-EC/AuNPs) and with AuNPs plus HRP (DMF-EC/AuNPs/HRP) were carried out by electrochemical impedance spectroscopy (EIS) (Fig. 2B) and CV (Fig. S3) in Fe(CN)63-/4- solution. Nyquist plots were fitted using a simplified Randles circuit (inset in Fig. 2B) to obtain the Rct values. A decrease of Rct across the just activated, activated/AuNPs, and activated AuNPs/HRP-modified electrodes was observed (Fig. 2B) although the activated 3D-printed electrodes (bare) and modified with AuNPs (DMF-EC/AuNPs) and with AuNPs/HRP (DMF-EC/AuNPs/HRP) seem to have high impedance and already show enhanced capacitive behavior. The enhanced capacitive behavior of the 3D-printed electrodes after chemical activation with DMF was reported previously by Gusmão et al. (2019). Cyclic voltammograms of the activated 3D-printed electrode, and following its modification with HRP and AuNPs/HRP, showed a reduction in the ∆Ep values after each modification step (Figs. S3A and S3B) as could be inferred from the impedance measurements (Silva et al., 2012; Wan et al., 2013; Xu et al., 2010; Yin et al., 2009). The catalytic activity of HRP toward H2O2 reduction relies on the redox iron center in its structure. HRP in its reductive form [heme(FeIII), R] can be chemically oxidized [heme(0=FeIV), R+*] in the presence of H2O2 and, consequently, the reduction of H2O2 takes place (Eq. 1). After that, the oxidized intermediate [heme(0=FeIV), R+*] is reduced by applying negative potentials close to zero, coming back to the initial HRP oxidation state (Eq. 2): [heme(FeIII), R] + H2O2 → [heme(0=FeIV), R+*] + H2O,

(1)

[heme(0=FeIV), R+*] + 2e-+ 2H+ → [heme(FeIII), R] + H2O,

(2)

where the [heme(FeIII), R] is native ferric enzyme HRP and the [heme(0=FeIV), R+*] is its oxidized intermediate (Negahdary et al., 2012; Yi et al., 2000).

7

Figure 3A shows the effect of the electrode activation on the electron transfer from the HRP to the electrode during H2O2 detection. CVs are carried out using the 3D electrodes modified with HRP and AuNPs/HRP before (as-printed) and after (DMF-EC) chemical and electrochemical activation. It was found that the electrocatalytic activity of the enzyme toward H2O2 detection was positively influenced by the electrode activation. The voltammogram of the DMF-EC/HRP biosensor showed enhanced current intensities and a reductive peak at negative potentials close to zero in the presence of H2O2, whereas in the control experiments using a non-activated 3D electrode (as-printed/HRP) this peak was not distinguished by the low current intensities observed. This peak corresponds to the reduction of HRP from [heme(0=FeIV), R+*] to [heme(FeIII), R] in Eq. (2) and this signal corresponds to the direct electron transfer between the HRP and the activated 3D-printed electrode. To confirm that the observed signal came from this assertion, we introduced AuNPs into the system (see orange line, Fig. 3A). As expected, the signal slightly enhanced when AuNPs were incorporated (DMF-EC/AuNPs/HRP) and its cyclic voltammogram presented the same shape of the DMF-EC/HRP, and no additional peaks were observed. In this way, we probed the chemical mechanism of this system based on the enzyme’s direct electron transfer with the 3D-printed electrode. Au-related materials were reported as an efficient platform for the direct electron transfer of the HRP (Yin et al., 2000, 2009; Xu et al., 2010). Furthermore, Fig. 3B shows the CVs of the DMF-EC/AuNPs/HRP biosensor in PBS pH 7.2 containing different H2O2 concentrations. These CV profiles evidence the electro-reduction of the HRP toward H2O2 detection in the range of potential from −0.3 to +0.1 V. The inset shows a magnified view of the CVs. In addition, the calibration curves obtained at different H2O2 concentrations for both biosensors (DMF-EC/HRP and DMF-EC/AuNPs/HRP) and their controls using not activated electrodes (as-printed/HRP and as-printed/AuNPs/HRP) are displayed in Fig. S4. The calibration curves at Fig. S4 and its parameters in Table S1 show a clear enhancement of sensitivity for both biosensors when the activated 3D-printed electrodes were used. Therefore, activation of the 3D-printed electrodes (Browne et al., 2018; Palenzuela et al., 2018) was crucial in order to obtain an efficient direct electron transfer from the enzyme to the electrode.

8

A 500

B 600

DMF-EC/HRP DMF-EC/AuNPs/HRP

300

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Current (µA)

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DMF-EC/AuNPs -50

160 120 DMF-EC/HRP 80

Current (µA)

ICurrentI (µA)

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-100 0 µM H2O2 -150 500 µM H2O2

DMF-EC 40

-200 -50

150

350 [H2O2] (µM)

550

-0.5

0 0.5 Potential (V)

1

Fig. 3. (A) CVs at 100 µM H2O2 of the as-printed and activated 3D electrodes functionalized with HRP and AuNPs/HRP. (B) CVs of activated 3D-printed graphene-PLA electrodes functionalized with AuNPs/HRP in the presence of 0, 100, 300, and 500 µM H2O2. (C) Calibration curves at 0 V of activated 3D-printed graphene-PLA electrode bare (blue line), activated 3D-printed graphene-PLA electrode functionalized only with AuNPs (red line) or HRP (black line), and functionalized with AuNPs/HRP (orange line). Conditions: in PBS pH 7.2 and scanning from −1.5 to 1.5 V at scan rate 0.1 V/s.

Finally, the calibration curves of the biosensors (DMF-EC/HRP and DMF-EC/AuNPs/HRP) as well as the activated bare electrode (DMF-EC) and the electrode modified with AuNPs (DMFEC/AuNPs) are shown in Fig. 3C. The small current changes manifested for the DMF-EC and DMF-EC/AuNPs electrodes illustrate that neither the activated bare electrode nor AuNPs can electrocatalyze the H2O2. The significant change of current observed for the DMF-EC/HRP and DMF-EC/AuNPs/HRP under the H2O2 additions demonstrated that the current intensity change observed was caused by the enzyme’s electrocatalytic activity and its interaction with the 3Dprinted DMF-EC activated electrode. This electrocatalytic activity for HRP observed for the DMF-EC/HRP biosensor was slightly enhanced when the HRP was immobilized onto AuNPs in 9

the DMF-EC/AuNPs/HRP electrode. This signal intensification was in good agreement with previous reports using glassy carbon (Silva et al., 2012; Yin et al., 2009) and gold electrodes (Xu et al., 2010; Wan et al., 2013). The chronoamperometric measurements carried out under successive additions of H2O2 for the DMF-EC, DMF-EC/AuNPs, DMF-EC/HRP, and DMF-EC/AuNPs/HRP 3D-printed electrodes (Figs. 4A and 4B) showed similar behavior to their corresponding CVs. Direct electron transfer between

HRP

and

the activated

3D-printed

electrode

was

also

demonstrated

by

chronoamperometry. As can be seen in Fig. 4 (black line), the 3D-printed electrode/HRP (DMFEC/HRP) biosensor shows a very elegant signal, with a very well-defined response after each H2O2 addition, demonstrating that the chemically and electrochemically activated 3D-printed electrode is by itself a good platform for direct electron transfer of HRP.

Fig. 4. (A) Chronoamperometry responses under successive additions of H2O2 and their respective calibration curves (B) for the 3D-printed graphene-PLA electrode bare (blue line), 3D-printed graphenePLA electrode functionalized only with AuNPs (red line) or HRP (black line), and functionalized with AuNPs and modified with HRP (orange line) obtained at 0 V in PBS pH 7.2.

This catalytic effect of the 3D DMF-EC graphene/PLA electrode toward the redox center of the HRP is promoted by the graphene. It is well known that graphene is an excellent electron carrier and is used in biosensors to enhance the electron transfer at the electrode surface (Lawal, 2018; Zhang et al., 2012). Here, a 3D-printed electrode as a direct electron transfer platform to develop mediator-free enzymatic biosensors is presented for the first time. The development of HRP10

based biosensors with direct electron transfer is difficult to achieve because few materials are capable and most are precious metals like gold (Gu et al., 2009; Negahdary et al., 2012) or metal oxides (Gu et al., 2009; Xu et al., 2010; Yi et al., 2000). Moreover, in our 3D-printed graphene/PLA electrode, after the chemical and electrochemical activation, the exposed functional groups of the graphene allow enzyme immobilization without any other binder reagents such as glutaraldehyde, chitosan, etc. This is a great achievement because biosensor mechanisms based on direct electron transfer are important for their simplicity by avoiding the use of mediators and suitable for real sample analysis. Two lineal ranges with different slopes (0 to 100 µM and 150–600 µM of H2O2) were delimited in the calibration curves of the DMF-EC/HRP and DMF-EC/AuNPs/HRP biosensors (obtained by both cyclic voltammetry and chronoamperometry). This behavior was observed previously in enzymatic systems at high analyte concentrations as a consequence of H2O2 saturation at the enzyme interphase. The chronoamperometric detection of H2O2 using the DMF-EC/HRP and DMF-EC/AuNPs/HRP biosensors displayed straight linearity (r = 0.994 and r = 0.996) with LOD of 11.1 and 9.1 µM and LOQ of 37.0 and 30.4 µM, respectively, calculated for the low concentrations of the calibration curve. These values of LOD and LOQ are comparable to similar systems reported before (see Table S2) and, in our case, metal decoration was not required. A more detailed description of the analytical performance of the DMF-EC/HRP and DMF-EC/AuNPs/HRP biosensors is supplied in Table 1. The biosensors showed repeatability for independent 3D device fabrication between 7–9% for 50 µM H2O2 and about 10–11% for all the concentrations analyzed on different days.

Table 1. Analytical performance of DMF-EC/HRP and DMF-EC/AuNPs/HRP biosensors. The relative standard deviation (RSD) corresponds to three replicates (n = 3) for different electrodes at 50 µM of H2O2. Moreover, the average of the total relative standard deviations corresponds to 0–100 µM H2O2, considering n = 3 for each concentration. 11

Biosensor DMF-EC/HRP

Analytical Range (µM) 25–100

Linearity (r) 0.994

LOD (µM) 11.1

LOQ (µM) 37.0

RSD (%) at 50 µM 9.4

RSD (%) Average 11.1

DMF-EC/AuNPs/HRP

25–100

0.996

9.1

30.4

6.6

10.1

A 5

B 120

DMF-EC/AuNPs/HRP DMF-EC/HRP

100

AA

UA

0

D

50 µM H2 O 2

-2.5

80

25 µM

1

60 40 20

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-1 -3 -5

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560

125

DMF-EC/HRP DMF-EC/AuNPs/HRP

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80

470

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50 µM 75 µM 100 µM 125 µM

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6

7

0

1

5 Days

6

7

Fig. 5. (A) Selectivity assay of 3D DMF-EC/AuNPs/HRP electrode for H2O2 analysis in the presence of 50 µM of common interferences in human plasma such as uric acid (UA), ascorbic acid (AA), and dopamine (D). (B) Accuracy assay of 3D DMF-EC/HRP and 3D DMF-EC/AuNPs/HRP biosensors in human serum samples for H2O2 detection. (Inset) Chronoamperometric responses in PBS and serum of the DMF-EC/AuNPs/HRP biosensor. Long-term stability response in PBS of 3D DMF-EC/HRP and 3D DMF-EC/AuNPs/HRP biosensors for 25 (C) and 50 (D) µM H2O2 detection.

The specificity of the biosensors was evaluated by spiked PBS solution with ascorbic and uric acids and dopamine in their usual concentrations in human plasma. The results in Fig. 5A 12

demonstrate that the biosensor has good selectivity because the initial and final H2O2 additions (at 25 µM) generated current changes of about 1.4 and 1.8 µA, respectively. The interferent species evaluated produced oxidative currents while reductive currents were measured for H2O2 detection. In this way, the small oxidative currents generated by uric acid (0.14 µA), ascorbic acid (0.08 µA), and dopamine (0.04 µA) from our point of view are negligible. Moreover, the working potential used in this system is 0 V and it is well-known that at this potential the systems are very selective (Wang, 2001). The response of these biosensors (DMF-EC/HRP and DMF-EC/AuNPs/HRP) in human serum was evaluated as well (Fig. 5B). The recoveries of the DMF-EC/HRP and DMF-EC/AuNPs/HRP biosensors were around 98 and 87.5% for 25 and 50 µM H2O2, respectively, reaching above 65% for the highest studied concentration. The long-term stability was assessed up to seven days for both biosensors (DMF-EC/HRP and DMF-EC/AuNPs/HRP) in the presence of 25 (Fig. 5C) and 50 µM H2O2 (Fig. 5D). The stability of both biosensors after five days was about 84% of the initial response for 25 and 50 µM H2O2. After seven days, the percentages of recoveries of the DMF-EC/HRP biosensor retained 82% and 80% of its initial current for 25 and 50 µM H2O2 concentrations, respectively. However, for the DMF-EC/AuNPs/HRP biosensor, its percentages of recoveries decreased after seven days to 64% and 69% for 25 and 50 µM H2O2 concentrations, respectively. The DMF-EC/HRP biosensor displayed better stability along this time, thus demonstrating that the enzyme immobilization was efficient and its bioactivity was mostly held after seven days. Conclusions 3D-printed graphene/PLA electrodes were used to immobilize easily horseradish peroxidase enzyme, thereby allowing an efficient direct electron transfer for H2O2 detection. The immobilization of HRP onto these electrodes modified with AuNPs confirmed that the detection mechanisms of these biosensors developed were related to the direct electron transfer between the HRP and the activated electrode. Moreover, the presence of AuNPs enhanced this direct electron transfer activity. Finally, the analytical performance of these biosensor systems for H2O2 detection was demonstrated. These results are of great value because we are illustrating here the biocompatibility of 3D-printed graphene electrodes for immobilizing enzymes to produce simple third-generation biosensors that avoid electron mediators and are suitable for real sample 13

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Highlights

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3D-printed graphene/polylactic (PLA) electrode, new platform for direct electron transfer enzyme biosensors

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3D-activated graphene/PLA electrode modified with AuNPs/HRP showed high sensitivity and selectivity for H2O2 detection

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The electrocatalysis of H2O2 was basically promoted by horseradish peroxidase (HRP) and its interaction with 3D electrode

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The activation of the 3D-printed electrodes significantly enhanced the biosensor performance, as well as, its modification with AuNPs

A.M.L.M. performed the mesurements and analyzed the data, C.C.M.M.analyzed the data and co=sipervised the project; M.P. conceptualized the idea and supervised the project. All authors contributed to writing.

A. M. L. Marzo: Data curation; Formal analysis; Writing - original draft; C. C. Mayorga-Martinez: Supervision; Validation; Writing - original draft; M. Pumera: Conceptualization, Funding acquisition Project administration; Resources; Supervision; Writing - review & editing.

Authors declare no conflict of interests.