Mapping the antioxidant activity of apple peels with soft

probe scanning electrochemical microscopy

Supporting information

Tzu-En Lin,a Andreas Lesch,*,a Chi-Lin Li,b and Hubert H. Girault*,a

a École Polytechnique Fédérale de Lausanne, EPFL Valais Wallis, Laboratoire d’Electrochimie Physique et Analytique, rue de l'Industrie 17, Case Postale 440, CH-

1951 Sion, Switzerland b Department of Chemistry, National Taiwan University, 1, Section 4, Roosevelt Road,

Taipei 10617, Taiwan

SI-1. Apple sample preparation SI-2. Microscopic pictures of soft probeSI-3. Approach curve on rough surfacesSI-4. Cyclic voltammetry (CV) in antioxidant containing solutionsSI-5. Lenticels on apple peelsSI-6. Soft-Probe-SECM of an apple peel with lenticel area in phosphate bufferSI-7. Investigation of porosity in apple peelsSI-8. Investigation of apple peel compression during soft probe approachingSI-9. Photograph of apple peel treated with hydrogen peroxideSI-10. Soft-Probe-SECM on apple fruit cross section

SI-1. Apple sample preparation

Figure S1. Preparation steps of an apple peel sample: 1. The peel was cut with a peeler; 2.The pulp was

removed; 3. The apple peel was cut into a rectangular piece of about 0.5 cm 2 size. The apple peel

sample was fixed on a glass slide with a frame made of Parafilm and then a Teflon cup was mounted on

the top forming the electrochemical cell.

Fig. S1 displays the procedure of apple peel sample preparation. The apple was purchased from a local market and first cleaned with water. A small piece of peel was sliced by using a peeler (step 1). The fruit part was immediately and gently removed by using a spoon (step 2). Afterwards, the peel was chopped into a small square of about 0.5 cm2 size (step 3). Then the surface of the peel was treated carefully with isopropanol to remove the hydrophobic epicuticular wax layer from the cuticle. Rapidly, the peel was fixed on a glass slide by placing a frame made of Parafilm and a Teflon cup on the top building the electrochemical cell that was immediately filled with electrolyte solution.

SI-2. Microscopic pictures of soft probe

Figure S2. a) Optical microscopic picture of a cross-section of a soft probe showing the active carbon

paste electrode area. b) Optical microscopic picture of the top view of the soft probe. The probe width

is around 100 μm. The carbon paste electrode track can be roughly identified through the layer of

Parylene C. With depth of the picture, the probe resolution is reduced.

The soft probe was manufactured by performing the following steps: 1. UV laser ablation to fabricate a microchannel into a 100 µm thin polyethylene terephthalate (PET) sheet, 2. filling of the microchannel with a carbon paste followed by a curing process at 80 °C, 3. covering and sealing of the conductive carbon track with a Parylene C coating, 4. shaping of the probe body, exposure of the active electrode area (cross-section) and preparation of the electrical connection pad. The probe was cut prior to each experiment in order to expose a fresh electrode surface. For a detailed description and characterization of the soft probes the reader is directed to previous publications [1,2].

Fig. S2a shows the cross section of the soft probe which reveals the active electrode area of the probe. The carbon paste was manually filled into the microchannels so that the exposed area can slightly vary also depending on the accuracy of cutting. Fig. S2b displays the optical microscopic image of the tip at the soft probe. The dimension of the tip is around 100 µm.

SI-3. Approach curve on rough surfaces

Figure S3. a) Apple topography recorded with a laser scanning microscope. b) Apple topography

recorded with a mechanical profilometer. c) Schematic representation of a soft probe approaching a

rough sample surface.

Fig. S3a shows the topography of an exemplary area (280 µm × 210 µm) of an apple peel sample recorded by using a Keyence VK 8700 laser scanning microscope. The height difference between the highest and lowest points is around 10 µm. Furthermore, irregular hills and valleys on the surface of the apple peel can be seen clearly. The macroscopic topography of the apple peel was investigated by using a mechanical Dektak XT profilometer (Bruker). One topographic x- and one y-line scan

are shown in Fig. S3b. As it can be seen clearly, the height differences can be as large as ~200 µm over a lateral distance of several millimeters. It can be expected that the diffusion of the redox mediator towards a soft probe, which is approaching such an irregular surface, blocks the diffusion in a different way as it is the case on a flat surface (Fig. S3c). This might result in a faster decay of the diffusion limited current at the microelectrode than it can be expected from a flat glass substrate.

SI-4. Cyclic voltammetry (CV) in antioxidant containing solutions

Figure S4. a) CVs of various concentrations of GSH in 10 mM PB. b) CVs of GSH in the presence of

FcMeOH. c) CVs of quercetin in 10 mM PB. d) CVs of quercetin in the presence of FcMeOH.

Experimental conditions: 2 mM FcMeOH in PB (pH 6, 10 mM). Scan rate = 10 mV/s. WE = Soft

stylus probe. RE = Ag/AgCl. CE = Pt.

The CVs of GSH and quercetin in absence and presence of FcMeOH are presented in Fig. S4. In the solutions, which did not contain FcMeOH (Fig. S4a and c), the current signals were very low. However, in the presence 2 mM FcMeOH, the current signals increase with AO concentration indicating the recycling of FcMeOH by AOs (Fig. S4b and d).

SI-5. Lenticels on apple peels

Figure S5. a) Photograph of an apple with yellow spots on its peel. b) Schematic representation of a

lenticel on the apple peel. These lenticels are centered in yellow spots forming a lenticel area.

Fig. S5 contains a photograph of an apple peel and the schematic representation of the cross-section of a lenticel. Lenticels are special types of cells that are blister-like, lenticular breaks in the apple surface. These structures provide the option for gas exchanges by "breathe and transpiration". Lenticels can also be the portals of russet and disease.

SI-6. Soft-Probe-SECM of an apple peel with lenticel area in phosphate buffer

Figure S6. a) Soft-Probe-SECM feedback mode image of a lenticel area. Experimental conditions: 10

mM phosphate buffer (pH = 6.0), ET = 0.4 V, hP = –200 µm, step size = 25 µm, translation speed = 50

µm/s. b) Photograph of the apple peel sample containing the scanned as marked by the black rectangle.

Soft-Probe-SECM of the AO activity on the apple peel without redox mediator and hence in pure phosphate buffer was performed. The recorded currents over the peel were homogeneous and notably low (i.e., few pA, Fig. S6a). This confirms that no direct electrochemical detection of AOs at the microelectrode took place. The recorded peak in the middle of the lenticel area might be the result from AOs or other redox active species diffused out from the fruit part through the crack of lenticel. However, the recorded current is still in the lower pA-range.

SI-7. Investigation of porosity in apple peels

Figure S7. Soft-Probe-SECM feedback mode images of a red apple peel with lenticel area recorded in

the 1.) pure PB (a and b), FcMeOH solution (c and d) and pure PB (e and f). For each measurement the

SECM image is shown as a map (left) and 2D plot (right). Experimental solutions: a) and c): 10 mM

phosphate buffer (pH = 6.0). b) 2 mM FcMeOH containing 10 mM phosphate buffer (pH = 6.0). Other

experimental conditions: ET = 0.4 V, hP = –200 µm, step size = 25 µm, translation speed = 50 µm/s.

The volume of all the experimental solutions was 3 mL.

Soft-Probe-SECM of the apple peel in pure phosphate buffer was performed (Fig. S7a and b). The recorded current was very low, indicating no direct signals from the AOs. Afterwards, FcMeOH solution was used and the same area was imaged again, but with more line scans. The lenticel area with lower recorded currents can be seen on the right (Fig. S7 c and d). Afterwards, the FcMeOH solution was removed and the sample was washed twice with phosphate buffer. The same area was immediately scanned again in pure PB. (Fig. S7e and f).

In the first SECM image, performed in phosphate buffer (Fig. S7a and b), the recorded currents were very low as expected. These data represent the baseline for the third SECM measurement in this experiment. In Fig. S6c and d, the lenticel area can be seen clearly on the right confirming the results in the main manuscript. The

recorded currents in Fig. S7e and f are significantly larger than in the first redox mediator-free experiment. This shows that FcMeOH had accumulated in the apple peel and diffused slowly out from apple peel surface. During the first three line scans the currents were even higher. This experiment indicates the permeability of the redox mediator through the apple peel plays a role. Moreover, the recorded currents over lenticel area and red apple peel are very similar suggesting a similar permeability of the redox mediator in both areas.

SI-8. Investigation of apple peel compression during soft probe approaching

Figure S8. Snapshots of recorded videos of a soft probe sliding along a vertical apple peel cross-

section forward during approaching (upper panels) and during line scans (lower panels).

The full videos are available online. The upper panels of Fig. S8 show the probe sliding in contact mode during an approach curve on apple. A very little compression can be recognized due to a slight pressure that was not observed during line scan sliding (lower panels) where no decompression of the apple peel was recognized.

SI-9. Photograph of apple peel treated with hydrogen peroxide

Figure S9. Photograph of the investigated apple peel after treatment with H2O2 (60%). The area

scanned by Soft-Probe-SECM is indicated by the black rectangle.

Fig. S9 shows the photograph of the apple peel after H2O2 treatment. The H2O2

artificially degraded the AO defense system including some red pigments.

SI-10. Soft-Probe-SECM on apple fruit cross section

Figure S10. Schematic representation of a soft probe passing the gap between apple and wax.

Fig. S10 explains the behavior of soft probe when passing a gap between apple cross-section and wax in Fig. 5a in the manuscript. The probe slid well on wax and apple (steps 1 and 4). However, when passing the gap, the probe flips from the wax to the apple (steps 2 and 3).

References[1] Lin, T. E.; Bondarenko, A.; Lesch, A.; Pick, H.; Cortés‐Salazar, F.; Girault, H. H., Angew. Chem. Int. Ed. 2016, 55, 3813-3816.[2] Lesch, A.; Vaske, B.; Meiners, F.; Momotenko, D.; Cortés‐Salazar, F.; Girault, H. H.; Wittstock, G., Angew. Chem. Int. Ed. 2012, 51, 10413-10416.