Electronic Supplementary Material s27

Electronic Supplementary Material

Electrochemical enzyme sensor arrays for the detection of the biogenic amines histamine, putrescine and cadaverine using magnetic beads as immobilisation supports

Sandra Leonardo, Mònica Campàs*

IRTA, Carretera de Poble Nou, km 5.5, 43540 Sant Carles de la Ràpita, Spain * e-mail: [email protected]

Electrochemical protocol for optimisation of the H202 detection

Cyclic voltammetry was used to characterise the response of the electrodes towards H 2O2 (for the subsequent development of mono-enzymatic biosensors). The protocol was as follows: 45 µL of 0.1 M potassium phosphate buffer, 0.1 M KCl, pH 7.2, were placed on the screen-printed electrode and a cyclic voltammogram was recorded between -0.2 and +0.8 V vs. Ag (potential window depended on the electrode and the electrochemical transduction approach) at 5 mV/s; 5 µL of 10 mM H2O2 solution were then added and cyclic voltammograms were recorded again.

Cyclic voltammetry was also used to characterise the response of the HRP-modified electrodes towards H2O2 (for the subsequent development of bi-enzymatic biosensors). The protocol was as follows: 1 µL of 2 mg/mL HRP solution in water or Os-wired HRP stock solution was placed on the DRP-110 working electrode and allowed to dry for 30 min. When using HRP, 45 µL of 0.5 mM N-methylphenazonium methyl sulphate (MPMS) in 0.1 M potassium phosphate buffer, 0.1 M KCl, pH 7.2, were then placed on the screen-printed electrode and a cyclic voltammogram was recorded between 0 and -0.4 V at 5 mV/s; 5 µL of 10 mM H2O2 solution were then added and, after 1 min, a cyclic voltammogram was recorded again. When using Os-wired HRP, 45 µL of 0.1 M potassium phosphate buffer, 0.1 M KCl, pH 7.2, were placed on the screen-printed electrode and a cyclic voltammogram was recorded between +0.5 and 0 V at 5 mV/s; 5 µL of

10 mM H2O2 solution were then added and, after 1 min, a cyclic voltammogram was recorded again. Optimisation of the H202 detection

Several electrochemical strategies for the detection of H202, either directly (on carbon, single- walled carbon nanotubes/carbon, graphene/carbon and gold electrodes), through immobilised redox mediators (on Co(II)-phthalocyanine/carbon, Prussian Blue/carbon) or by HRP-modified electrodes, have been tested previous the use of DAO-MB conjugates for the subsequent development of mono-enzymatic and bi-enzymatic biosensors for BAs detection. The aim of this work was seek the most sensitive and optimal detection for a BAs amperometric biosensor based on the use of magnetic beads as DAO immobilisation supports.

First, the direct detection of H2O2 on different electrodes was investigated. As expected, cyclic voltammograms of carbon, single-walled carbon nanotubes/carbon, graphene/carbon and gold screen-printed electrodes did not show any redox peak in the potential range from 0 to

+0.8 V (results not shown). The H2O2 addition did not cause any effect on carbon electrodes, but resulted in an increase of the oxidation current starting at +0.4 V on single-walled carbon nanotubes/carbon electrodes, graphene/carbon and gold electrodes (Fig. S1A).

Inorganic redox mediators were added to the electrochemical transduction strategy to investigate if it was possible to detect H2O2 at lower working potentials and thus avoid possible electrochemical interferences. The cyclic voltammetry of Co(II)-phthalocyanine/carbon electrodes did not show any oxidation or reduction peak (Fig. S1B, black line), but the addition

3+ 2+ of H2O2 resulted in the chemical reduction of the immobilised mediator (from Co to Co ) and the subsequent electrochemical re-oxidation, which started at +0.2 V and showed a peak at +0.6 V (Fig. S1B, red line). Compared to the carbon-based materials and gold, much lower potentials can be applied to measure H2O2 when using Co(II)-phthalocyanine, obtaining higher current intensities. The cyclic voltammetry of Prussian Blue/carbon electrodes showed well- defined oxidation and reduction peaks, with a midpoint redox potential of +0.12 V and a peak- to-peak separation of 100 mV (Fig. S1C, black line). The addition of H2O2 resulted in the chemical oxidation of the immobilised mediator (from Fe2+ to Fe3+) and the subsequent increase in the reduction current (Fig. S1C, red line). Current intensities obtained were comparable to those attained using Co(II)-phthalocyanine/carbon electrodes.

The detection of H2O2 on different HRP-modified electrodes was then investigated for the subsequent development of bi-enzymatic biosensors. In this case, the transduction strategy was based on the enzymatic reaction between HRP and its substrate, H 2O2, and the subsequent mediated bioelectrocatalysis. First trials were performed using the redox mediator

MPMS in solution and HRP adsorbed on screen-printed carbon electrodes. Before H 2O2 addition, MPMS showed an oxidation and a reduction peak, with a midpoint redox potential of -0.15 V and a peak-to-peak separation of 60 mV (Fig. S2A, black line). After H 2O2 addition, an increase in the reduction peak current and a decrease in the oxidation peak current were observed (Fig. S2A, red line). This effect is certainly due to the bioelectrocatalysis, and not to the direct H2O2 reduction on the electrode, since no response was observed in the controls without HRP. Otherwise, the bioelectrocatalytic currents from HRP adsorbed on Prussian Blue/carbon electrodes were also recorded. Results did not shown significant differences compared to the configurations without HRP (results not shown).

The last configuration incorporated Os-wired HRP. The electrical wiring of HRP with Os- polyvinylpyridine (Os-PVP) redox polymer favours the electron transfer from the redox centre of the enzyme to its periphery and, if it is immobilised, to the electrode [1]. When adsorbing the wired enzyme on the electrode, the osmium showed well-defined oxidation and reduction peaks, with a midpoint redox potential of +0.23 V and a peak-to-peak separation of 20 mV (Fig. S1E, black line). Since osmium complexes have particularly high self-exchange rates and, additionally, PVP polymer relays the electrons via the osmium centres to the electrode [2], the lower current intensity of the redox peaks compared to those of MPMS in solution (Fig. S2B, black line) were attributed to a lower amount of immobilised Os redox mediator. In the presence of H202, the Os oxidation peak completely disappeared and a bioelectrocatalytic reduction current was observed (Fig. S2B, red line). Although the current intensity of the reduction peak increased, it did not reached a steady-state plateau. The use of a twice higher amount of Os-wired HRP did not substantially increase the reduction current neither resulted in a steady-state plateau.

At this stage, three electrode supports were selected for the subsequent detection of BAs based on the attained current intensities: Co(II)-phthalocyanine/carbon and Prussian Blue/carbon electrodes for the development of mono-enzymatic biosensors, and Os-wired HRP-modified carbon electrodes for the development of a bi-enzymatic biosensor. Single- walled carbon nanotubes/carbon, graphene/carbon and gold electrodes showed H2O2 oxidation currents but at potentials too high for the subsequent development of biosensors. On the contrary, the use of Co(II)-phthalocyanine and Prussian Blue redox mediators resulted in well-defined redox peaks of higher current intensities and at lower potentials.

Each configuration presents advantages and limitations. Whereas in the mono-enzymatic approaches, Co(II)-phthalocyanine/carbon and Prussian Blue/carbon electrodes are commercially available and ready to use, the bi-enzymatic approaches require the immobilisation of HRP, step that increases the experimental procedure time. Moreover, the HRP immobilisation may increase the variability among electrodes due to possible enzyme leaking. Nevertheless, the use of a bi-enzymatic approach may increase the sensitivity and selectivity, and decrease the limit of detection (LOD) [3]. In fact, the current intensities attained when using HRP were slightly higher than in the mono-enzymatic approaches with immobilised mediators. Regarding the choice of redox mediator in the bi-enzymatic approach, immobilised Os was preferred to free diffusing MPMS because HRP wiring directly connects the enzyme with the electrode, which may also increase the sensitivity and decrease the LOD [4].

A 4 µ

I 2 0 -2 -0,1 0,1 0,3 0,5 0,7 0,9 E / V

15 12 B 9 A µ

I 3 0 -3 -0,1 0,1 0,3 0,5 0,7 0,9 E / V

6 3 C 0

A -3 µ

/ -6 I -9 -12 -15 -0,4 -0,2 0 0,2 0,4 0,6 E / V

Fig. S1 Cyclic voltammograms performed in 0.1 M potassium phosphate buffer, 0.1 M KCl, pH 7.2 at 5 mV/s for: (A) carbon (black), single-walled carbon nanotubes (green), graphene/carbon (blue) and gold (red) electrodes after the addition of 10 mM H 2O2 (cyclic voltammograms before H2O2 addition were always lower than carbon electrodes after H2O2 addition and are not shown for clarity); (B) Co(II)-phthalocyanine/carbon electrodes before

(black) and after (red) the addition of 10 mM H2O2; (C) Prussian Blue/carbon electrodes before

(black) and after (red) the addition of 10 mM H2O2

20 5 10 A 0 B 0 -5 A A µ µ

-10 -10 / /

I I -20 -15 -30 -20 -40 -25 -0,5 -0,3 -0,1 0,1 -0,2 0 0,2 0,4 0,6 E / V E / V

Fig. S2 Cyclic voltammograms performed in 0.1 M potassium phosphate buffer, 0.1 M KCl, pH 7.2 at 5 mV/s for: (A) HRP-modified carbon electrodes by adsorption in the presence of N- methylphenazonium methyl sulphate (MPMS) in solution before (black) and after (red) the addition of 10 mM H2O2; and (B) Os-wired HRP-modified carbon electrodes by adsorption before (black) and after (red) the addition of 10 mM H2O2 100 %

80 / e

n 4 °C o

p 4 °C + 10% Glyc s 40 e

R -20 °C 20 -20 °C + 10% Glyc 0 0 15 30 45 60 75 90 Storage time / days

Fig. S3 Storage stability of DAO-MB conjugates

References [1] Heller A (1992) Electrical connection of enzyme redox centers to electrodes. J Phys Chem 96: 3579-87. [2] Ohara TJ (1995) Osmium bipyridyl redox polymers used in enzyme electrodes. Platinum Metals Rev 39: 54-62. [3] Gu M, Wang J, Tu Y, Di J (2010) Fabrication of reagentless glucose biosensors: A comparison of mono-enzyme GOD and bienzyme GOD-HRP systems. Sensor Actuat B-Chem 148: 486-491. [4] Vreeke M, Maidan R, Heller A (1992) Hydrogen peroxide and β-nicotinamide adenine dinucleotide sensing amperometric electrodes based on electrical connection of horseradish peroxidase redox centers to electrodes through a three-dimensional electron relaying polymer network. Anal Chem 64: 3084-3090.