Fabrication of a New, Low-Cost, and Environment-Friendly Laccase-Based Biosensor by Electrospray Immobilization with Unprecedented Reuse and Storage Performances

The fabrication of enzyme-based biosensors has received much attention for their selectivity and sensitivity. In particular, laccase-based biosensors have attracted a lot of interest for their capacity to detect highly toxic molecules in the environment, becoming essential tools in the fields of white biotechnology and green chemistry. The manufacturing of a new, metal-free, laccase-based biosensor with unprecedented reuse and storage capabilities has been achieved in this work through the application of the electrospray deposition (ESD) methodology as the enzyme immobilization technique. Electrospray ionization (ESI) has been used for ambient soft-landing of laccase enzymes on a carbon substrate, employing sustainable chemistry. This study shows how the ESD technique can be successfully exploited for the fabrication of a new promising environment-friendly electrochemical amperometric laccase-based biosensor, with storage capability up to two months without any particular care and reuse performance up to 63 measurements on the same electrode just prepared and 20 measurements on the one-year-old electrode subjected to redeposition. The laccase-based biosensor has been tested for catechol detection in the linear range 2–100 μM, with a limit of detection of 1.7 μM, without interference from chrome, cadmium, arsenic, and zinc and without any memory effects.

S2 voltage increases, the electric field becomes stronger and elongates the liquid meniscus into a cone whose apex extends into a jet (inset in Figure 1 main text). The onset voltage for the cone-jet mode of electrospray is given by the (1) where r c is the radius of the needle, γ is surface tension of the solution, D is the distance between the needle and the counter electrode, and ε 0 is the permittivity of vacuum. The angle of 49° guarantees the equilibrium between an external electric field and a conical fluid surface, according to the model developed by Taylor [2]. In our set-up, the γ and D parameters, as well as the operating conditions with the focusing electrode, were optimized for the best performance in terms of spray stability and spot size of the deposit (Φ deposit ). In detail, at r c = 100 μm, the surface tension was optimized by testing different solutions described in Table S2 and the optimal distance D was found to be 14 mm. In order to deposit on a C-SPE working electrode area of about 4 mm diameter, the voltages of the needle and the focusing cone placed at a distance of 5 mm (Table S1) with respect to the SPE were set to 4.92 and 2 kV, respectively. Table S1.
Geometrical sizes of the cones and diameter of the deposited film for each cone at d=5mm. The geometric h, Φ1 and Φ2 parameters are described in Figure 1 main text.
S3 two different solvents as methanol and ethanol in aqueous solution or in acid aqueous solution (0,01 % formic acid), that usually helps in the formation of a good nebulisation of the spray during the ESD process, has been tested (Choice of the Solvent). Furthermore the variation of the activity caused by the pH of solution (Choice of pH) and the electrospray ionization process itself (Effect of Electrospray Ionization Process in the main text) have been analysed.
Finally the amount of laccase deposited on C-SPE can vary with the deposition time. By changing the focusing cone we can control the deposition area and find a good condition for the minimum deposited quantity of laccase which gives a detectable amperometric signal (Choice of deposition time and focusing cone).

Choice of the solvent: the study of the solvent effect
The effect of the solvent on the enzyme activity was studied by spectrophotometric measurements of four aqueous solutions containing different percentage of methanol, ethanol and formic acids. The absorbance measurements of syringaldazine (syr) at 530 nm showed that the best solution to be sprayed in order to preserve the enzyme activity was an acqueous one with the 20% of methanol. A control was run in parallel in the absence of organic solvent. The four solutions named A, B, C and D (Table S2)  taken. The activity of the enzyme has been measured by spectrophotometric analysis (tA, tB, tC and tD in Table S2) of the syringaldazine absorbance at 530 nm and 25°C. Syringaldazine is oxidised by laccase to the corresponding quinone producing water from oxygen molecules in solution; quinone production is monitored by light adsorption measurements at 530 nm. The buffer used is a 0.1 M pH 4.5 citric acid/sodium citrate solution (see section 2.2). The list of the prepared solutions is reported in Table S2. S4 Table S2.
List of working solutions tested to study the effect of different solvents on laccase activity. The solutions A, B, C and D correspond to aqueous solutions with different percentages of organic solvent at laccase concentration of 2μg/μl. The tests tA, tB, tC and tD refer to the mixtures used in the cuvette for spectrophotometric analysis containing the respective solutions. The 'Blank' is the control one.  Figure S1. These results are in agreement with the ones by Leonowicz and Gzywnowicz [5] for laccase from Trametes Versicolor, who demonstrated no variation in syringaldazine absorption up to 25% of ethanol concentration in solution. They observed a first decrease in laccase activity in solutions containing an amount between 27.5% and 80% of ethanol or 50% of methanol. In particular they found a loss of activity of 50% with a 50% concentration of methanol in the reaction mixture. This is consistent with our results of a 17% loss of activity for a solution with a 20% methanol S5 concentration obtained by comparing the slopes of curves A and Blank in the steady state region [6]. On the basis of these results and having observed a greater stability of the electrospray process with solution A, methanol has been chosen as a co-solvent of water for all the other tests.

Choice of the pH: the study of optimal pH before and after ESD
The crucial role of pH on enzymatic activity is well-known. The optimal pH value is characteristic of each enzyme and it depends on the chemical environment, temperature and enzyme stability in acid and alkaline neighbourhood.
According to literature [7] the most commonly used buffer for laccase Trametes Versicolor of 0.5 U/mg is a citric acid/sodium citrate 0.1 M solution. The optimal pH value was searched within the range 3.5 -6 using spectrophotometric measurements of the syringaldazine absorbance at 530 nm before deposition and by amperometric measurements of the current produced by the red-ox reaction of the catechol catalyzed by the electrosprayed laccase on C-SPE after the deposition. The laccase sensing mechanism for catechol detection is based on the electrocatalysis of catechol oxidation to its corresponding 1,2 benzoquinone, which is coupled with the electrocatalytic reduction of dioxygen to water on the working electrode surface. Before ESD, in a systematic exploration, six different solutions of citric acid/sodium citrate 0.1 M were prepared at pH values between 3.5 and 6 (see Figure S2a). The absorbance curves were measured by repeating test tBlank in Table S2 with the six buffer solutions to establish the optimal pH for laccase. Three spectrophotometric measurements have been performed for each pH value and their average is shown in Figure S2a. Then, three depositions of 30 minutes each were performed on three C-SPEs with the set-up shown in Figure 1. of the manuscript. The so modified electrodes were tested by amperometric measurements, to analyse the activity of the enzyme after the ESD, using the same buffer solutions (citric acid/sodium citrate at 0.1 M) at room temperature.
Before ESD Figure S2. Absorbance values for the syringaldazine oxidation catalyzed by laccase, at different pH values (a). Determination of the initial rate of syringaldazine oxidation reaction at pH=4.5 (b).

S6
In Figure S2b the slope of the absorbance A versus time at the beginning of the reaction represents the initial rate v 0 of the reaction for pH of 4.5.  Table S3.
The results clearly show that the optimal pH value for the enzymatic reaction in solution is 4.5.

S7 After ESD
Since it is possible that, during the electrospray process, the enzyme can suffers: (i) inactivation inside the ES capillary due to the electrochemical reactions, (ii) inactivation as a result of reaction with corona products in the gas phase, (iii) inactivation as a result of impact with the target electrode [9], and therefore be deposited in conformations different from those in solution, the study of the activity versus the pH was also carried out by amperometric measurements on laccase deposited for 30 minutes on C-SPE at catechol concentration of 30μM ( Figure S3b). The data in Figure S3b do not show significant differences and therefore the use of the optimal pH of the native enzyme has been preferred.

Effect of Electrospray Ionization process
The results of the siringaldazyne assay performed on test solution tA and on laccase dissolved after deposition (without cone) of solution A are shown in Figure S4 where the absorbance curves measured in the two cases are compared. The change of slope of the two curves in the steady state region shows that laccase activity after ESD immobilization is equal to 70% of that from starting enzymatic solution. Figure S4. Comparison of the absorbance measured at 530 nm for the solution A (blue curve) and for the laccase dissolved after deposition by ESD (red curve). The linear fits (black lines) in the steady state (grey box) are also shown.

Choice of deposition time and focusing cone
To quantify the fraction of material intercepted by the cone, the amount of deposited enzyme has been evaluated by substituting the target with the resonator of a quartz crystal microbalance (QCM).
Our custom QCM is composed by two gold electrodes with a diameter of 6 mm obtained by vapour deposition on a quartz crystal disk of 14 mm diameter [10,11] and a resonance frequency f0 = 10 MHz. The variation of the resonance frequency is proportional to the amount of deposited material [12]. However, it also depends on the viscoelastic properties of the deposited material, the adhesion on the gold material and among different layers. As such, the response frequency versus mass had to be calibrated specifically for laccase.
To calibrate the QCM response, controlled amounts of laccase were deposited by dropcasting on the resonator. Then the QCM has been used to measure the amount of the deposited laccase for each focusing electrode (Table S1). Three independent depositions for each cone were done. Then, in order to test the linearity in the ESD rate the amount of the deposited laccase was measured versus the deposition time, with three measurements at each time.
Considering that the concentration of the sprayed solution is 2 μg/μl and the spraying rate is 1 μl/min, the expected 'nominal' amount of deposited material can be easily calculated and compared to the measured one through the procedure reported in the section devoted to the study of the deposition time in the manuscript. The fraction of deposited material, depending on the focusing electrode varies, from 45 to 21 % of the sprayed material.
To evaluate this result one can compare these quantities with the ones calculated for an hypothetical direct deposition on a mask with a hole of diameter ϕ deposit equal to the one of the spot obtained with a focusing electrode (see Table S1). The amount of deposited material can be calculated by the following equation (5): where Vol is the total volume of solution sprayed, c is enzyme concentration, and ϕ 0 = 15 mm is the diameter of the deposited film at 14 mm distance from the spray needle without any focusing electrodes. In Table S4 the amount of enzyme deposited in 20 min by the use of conical electrodes ( Figure S5) or in the hypothesis of using a mask to achieve the same spot size (eq. 5) are reported. The comparison clearly shows a gain in the amount of deposited material of a factor between 4 and 6 thanks to the focusing electrode.
Amount (μg) of deposited enzyme after 20 min at flow rate of 1 μl/min and a concentration of 2 μg/μl by using focusing conical electrodes or in the hypothesis to use a mask (eq.5). The percentage of the deposited enzyme with respect to the nominal amount (40 μg) is reported in bracket. In the calculation, possible focusing effects introduced by the material of the mask and the ions deposited on it have not been considered [13]. Figure S5. Amount of deposited laccase on the QCM electrode using the conical electrodes labelled Ci (i=1,5) in Table S1 to focus the spray. The spray solution A in Table S2 has been used. The voltage settings and geometrical parameters for the deposition are the ones in Figure 1 in the main text.