378 Acta Chim. Slov. 2022, 69, 378–384 Štukovnik et al.: The Use of Yeast Saccharomyces Cerevisiae ... DOI: 10.17344/acsi.2021.7301 Scientific paper The Use of Yeast Saccharomyces Cerevisiae as a Biorecognition element in the Development of a Model Impedimetric Biosensor for Caffeine Detection Zala Štukovnik,1 Regina Fuchs Godec1 and Urban Bren1,2,* 1 University in Maribor, Faculty for Chemistry and Chemical Engineering, Smetanova 17, 2000 Maribor, Slovenia 2 University of Primorska, Faculty of Mathematics, Natural sciences and Information Technologies, Glagoljaška 8, 6000 Koper, Slovenia * Corresponding author: E-mail: urban.bren@um.si Received: 11-26-2021 Abstract In the present study, an electrochemical-impedimetric biosensor using Saccharomyces cerevisiae as an effective biorecog- nition element was designed to detect caffeine. The presented biosensor consists of a previously developed stainless steel electrochemical cell constructed as a three-electrode system in the RCW side-by-side configuration. The electrochemical stability of the sensing electrode was evaluated by measuring the open circuit potential (OCP), and electrochemical im- pedance spectroscopy (EIS) was applied to determine the impedimetric response of the biosensor with Saccharomyces cerevisiae cells attached to the working electrode (WE) in the absence (0.9% NaCl) and presence (10 mg/mL in 0.9% NaCl) of caffeine. Moreover, the limit of detection (LOD) was determined. In this way, a new approach in biosensor de- velopment has been established, which involves assembling a low-cost and disposable electrochemical system to detect alkaloids such as caffeine. The developed biosensor represents a good candidate for detecting caffeine in beverages, foods, and drugs with the merits of time-saving, robustness, low cost, and low detection limit. Keywords: Impedimetric biosensor, Saccharomyces cerevisiae, electrochemical impedance spectroscopy, caffeine 1. Introduction The demand for biosensors has increased significant- ly in the recent years due to the need for specific sensors that can provide fast and reliable measurements in various research areas. The development of biosensors is of interest for different applications ranging from biochemical profil- ing of normal and pathologic cells, over clinical diagnos- tics and drug discovery to more straightforward analyses such as fermentation, process monitoring, environmental testing, and food and beverages quality control.1–3 Detec- tion of alkaloids such as caffeine has attracted abundant attention due to their extensive occurrence in beverages and drugs.4 However, conventional detection methods for caffeine (high-performance liquid chromatography-mass spectrometry (HPLC-MS), thin-layer chromatography (TLC), and immunoassay) have several drawbacks, in- cluding expensive equipment as well as complex and labo- rious sample preparation.5 Biosensors, as analytical devices, convert a biological response into an electrical signal and provide us with the information on the concentration of the target analyte.6,7 Biosensors may present the best candidates for detecting caffeine with the merits of high sensitivity and specifici- ty, convenience, time-saving, low cost, and low detection limit.8 Biosensors can be based on animal tissues, bacteria, or eukaryotic microorganisms such as yeasts.9 Although yeasts are highly resistant to adverse environmental con- ditions, they can sense and respond to a variety of stimuli and, unlike several alternative biological components, do not require sophisticated sterile techniques or complex media.10,11 Yeast Saccharomyces cerevisiae represents a sin- gle-cell eukaryotic organism used primarily in the food in- dustry to produce bakery products and alcoholic beverag- es.9,12 It is chemoorganotrophic and anaerobic organism classified in the kingdom of Fungi, phylum Ascomycota, class Saccharomycetes, order Saccharomycetales, and family Saccharomycetaceae.13 Saccharomyces cerevisiae can exist in two different forms, the haploid or the diploid form.13,14 A yeast cell possesses the typical characteristics of a eu- karyotic cell and characteristic organelles such as vacuoles 379Acta Chim. Slov. 2022, 69, 378–384 Štukovnik et al.: The Use of Yeast Saccharomyces Cerevisiae ... and lipid droplets.12 It is usually spherical to slightly spher- ical and occasionally ellipsoidal to cylindrical.15 Caffeine (1,3,7-trimethylxanthine), with the chemi- cal molecular formula of C8H10N4O2, has been used for thousands of years and represents one of the most widely consumed food ingredient throughout the world.1,16 It is found in common beverages such as coffee, tea, and soft drinks, as well as in products containing cocoa or choc- olate, and in a variety of medications and dietary supple- ments.17,18 Due to the high consumption of caffeinated foods, beverages, and medicines worldwide, caffeine is also considered to be the most representative pharmaceu- tically active pollutant with regard to its abundance in the environment.19 Based on the data reviewed, it is concluded that in the healthy adult population, daily caffeine intake at a dose exceeding 400 mg is associated with adverse effects such as general toxicity, cardiovascular effects, effects on bone status and calcium balance, changes in adult behav- ior, increased cancer incidence and effects on male fertil- ity.20 The caffeine content in coffee products ranges from 0.27 to 1.85 mg/mL, in tea from 0.11 to 0.23 mg/mL, in energy drinks from 0.30 to 0.37 mg/mL, and in soft drinks such as regular cola from 0.10 to 0.13 mg/mL.21–23 Given these values, the biosensor may be sufficiently sensitive and robust enough to cover the range of caffeine concen- trations present in beverages. Figure 1: Chemical structure of the caffeine molecule For the detection of different caffeine concentra- tions, the electrochemical impedance spectroscopy (EIS) method was applied. EIS is widely used in the production and optimization of biosensors as this method allows for the characterization of the biological component attached to the sensor and of the analyte present in the sample.24,25 Because biosensors produce a rapid response, they can be applied to monitor molecular events in real-time.26 The EIS method was used to measure the frequency response of the electric current, which provides data on the adhe- sion layer of Saccharomyces cerevisiae on the electrode surfaces. The aim of this study was to develop a model elec- trochemical impedimetric biosensor for the detection of caffeine using Saccharomyces cerevisiae as an effective bi- orecognition element with many advantageous properties such as cell robustness, ease of maintenance, and cell pro- duction rate. 2. Experimental The developed biosensor consists of a stainless steel electrochemical cell (Figure 2) constructed as a three-elec- trode system in the RCW-side by side configuration, in- cluding the working electrode (WE) with yeast cells on the surface, the reference electrode (RE), and the counter electrode (CE). Such electrochemical cell was previously developed and tested.10 In assembling the electrochemical cell, stainless steel type SS316 (manufacturer TBJ Indus- tries, Germany) was used. The electrodes were manufac- tured with a dimension of electrode 20 mm × 5 mm, where the active component was applied to the 5 mm × 5 mm. The electrodes were insulated on the fixation side, and the system was sealed with glass. Saccharomyces cerevisiae was applied to the working electrode using a technique involving a mold made with a 3D printer, which ensured that the layer thickness (0.10 mm) was similar for all measurements. 3.8 g Saccharomy- ces cerevisiae was mixed with 1 mL 0.9% NaCl to ensure that the mixture was viscous. The mixture was applied on the stainless-steel electrode inserted in the mold. The pro- cess of coating the working electrode (WE) was taken at 25 °C and took approximately 30 seconds. Figure 2: Electrochemical cell in the RCW-side-by-side configura- tion and the Saccharomyces cerevisiae cells attached to the working electrode (WE). Two solutions were prepared for the measurements, the 0.9% NaCl solution (Sigma Aldrich, CAS: 7647-14-5, M: 58.44 g/mol) and 10 mg/mL caffeine (Sigma Aldrich, 380 Acta Chim. Slov. 2022, 69, 378–384 Štukovnik et al.: The Use of Yeast Saccharomyces Cerevisiae ... CAS: 58-08-2, M: 194.19 g/mol) in 0.9% NaCl solution. The electrochemical cell with Saccharomyces cerevisiae on the working electrode was connected to the Multi Palms- ens4 potentiostat. Initially, 1 mL of the 0.9% NaCl solution was injected into the system, and the open circuit potential (OCP) and EIS measurements were performed. Then, the excess saline (0.9% NaCl) was drained. Afterward, 1 mL of the 10 mg/mL caffeine solution was injected into the system, and OCP and EIS measurements were repeated. The electrochemical characterization of the working electrode was evaluated by measuring the OCP to assess the stability of the electrode. The duration of the measure- ment was 600 seconds since living yeast cells were utilized. The EIS method was applied to evaluate the biosen- sor with yeast on the stainless-steel surface. Impedance spectra were obtained at a steady open circuit potential (OCP) in the frequency range from 100 kHz to 10 mHz with 10 points per decade and a 20 mV amplitude of the excitation signal. The potential amplitude was chosen at 20 mV since the lower amplitudes have not provided the stable signal due to the yeast layer on the stainless-steel electrode The EIS measurement‘s expected duration was 2 minutes and 15 seconds, although this time was often ex- tended up to 3 minutes. EIS was used to obtain data on the processes on the surface of the electrode and the applied layers, and the Bode and Nyquist plots were interpreted as the results. Moreover, the limit of detection (LOD) was deter- mined based on the impedance drop with the increasing concentration of caffeine in 0.9% NaCl. For the measure- ment, eight different caffeine concentrations in saline (0.0 mg/mL, 0.05 mg/mL, 0.01 mg/mL, 0.1 mg/mL, 0.25 mg/ mL, 0.5 mg/mL, 1.0 mg/mL, and 5.0 mg/mL) were pre- pared and 1mL of each sample was injected into the sys- tem separately. The blank solution consisted of 0,9% NaCl solution (saline). Three measurements were taken for each concentration, where the mean value (MV), standard de- viation (SD), precision, and accuracy of the measurements were calculated. The measurements were taken using iden- tical parameters as in the measurements mentioned before, and the data were obtained at the frequency 125 mHz. 3. Results and Discussion 3. 1. Open Circuit Potential (OCP) Measurements The electrochemical characterization of the sensing electrode was evaluated by measuring the OCP to assess the stability of the electrode. The OCP provides valuable insight into the thermodynamic stability of the electrode material involved in the electrochemical response.27 The results are represented in Figure 1S in the Supple- mentary information. The data provided from the meas- urements indicate that the system was thermodynamically stable. When measuring with Saccharomyces cerevisiae on the stainless steel surface of the working electrode (WE) with the addition of 0.9% NaCl and the addition of 10 mg/ ml caffeine in 0.9% NaCl, a slight difference is observed at OCP in each measurement. The slight change in OCP is due to the living cells on the surface, which react to envi- ronmental conditions. 3. 2. Electrochemical Impedance Spectroscopy (EIS) EIS represents a non-destructive method that can be used to quantify specific parameters and simultaneously monitor multiple electrochemical processes.28 The meas- urements are explained with the real (electrical resistance) and imaginary (capacitance) components of the imped- ance response of an electrochemical system.10 In the Nyquist diagram (Figure 3), the decrease in the resistance (real component, x-axis) and the decrease in the capacitance (imaginary component, y-axis) when 10 mg/mL caffeine in 0.9% NaCl was added to the system compared to the blank solution (0.9% NaCl) is seen. The results indicate that the electrode surface was released due to the detachment of yeast cells from the electrode surface. Figure 3: Nyquist diagram of the EIS measurement of the biosensor containing Saccharomyces cerevisiae on the WE, with the addition of 0,9% NaCl (blue) and 10 mg Caffeine in 1 mL 0.9% NaCl (red). Bode plots consist of two spectra simultaneously, the impedance spectrum and the phase spectrum, in which the dependence of impedance (Z) and the dependence of phase angle on the frequency is shown. In the impedance spectrum, the activity at the working electrode is deter- mined from the slopes of the line, and in the phase spec- trum, the activity is determined from a phase angle. Based on the impedance spectrum of the Bode dia- gram (Figure 4), the solution resistance (Rs) was determined with a slope of approximately 0 (high-frequency range), the capacitance of the electrical double layer (Cdl) was deter- 381Acta Chim. Slov. 2022, 69, 378–384 Štukovnik et al.: The Use of Yeast Saccharomyces Cerevisiae ... mined with a slope of approximately –0.8, which occurs at the phase boundary between the electrode and the electro- lyte (middle-frequency range), the charge transfer resistance was determined with a slope of approximately 0 (Rct) which occurs due to the electrochemical reaction or due to the charge transfer between the electrolyte and the metal (mid- dle- frequency range), as well as the diffusion with a slope approximately –0.5 (low-frequency range) was determined. In the phase spectrum of the Bode diagram (Figure 5), the resistance is described as the negative phase at ap- proximately 0°, the non-ideal capacitance with the nega- tive phase at approximately 55°, the diffusion at the nega- tive phase at approximately 45°. Figure 4: Bode diagram of the EIS measurement of the biosensor containing Saccharomyces cerevisiae on the WE, with the addition of 0,9% NaCl (blue) and 10 mg Caffeine in 1 mL 0.9% NaCl (red). The Bode diagram includes an impedance diagram described with squares and a phase diagram described with triangles, where I Z I represents an impedance and -Phase presents a negative phase shift. The equivalent electrical circuits (EEC) of the stain- less steel electrochemical cell without and with the yeast on the working electrode (WE) are shown in Figure 5. The EEC of the electrochemical cell without yeast cells attached to the stainless steel electrode is depicted in Figure 5A. The equivalent circuit consists of the solution resistance (Rs), the capacitance of the electrical double lay- er (Cdl), the charge transfer resistance (Rct), and the War- burg impedance (Wo). The EEC of the electrochemical cell with the yeast cells attached to the working electrode is shown in the Figure 5B and consists of the solution resistance (Rs), the yeast layer capacitance (Cy), the yeast layer resistance (Ry), the capaci- tance of the electrical double layer (Cdl), the charge transfer resistance (Rct), and the Warburg open diffusion (Wo). The comparison of the values of the parameters where the 0.9% NaCl and the 10 mg/mL caffeine in 0.9% NaCl were separately added to the system is provided in Table 1. When caffeine was added to the system, the  Sac- charomyces cerevisiae  cells detached from the stainless steel surface, and consequently, the electrode surface was released. Consequently, the resistance of the system dropped, and the capacitance and the impedance of dif- fusion increased. The χ2 of the measurement where 0.9% NaCl was added to the system was 2.94 × 10–3, and for the measurement where the 10 mg/mL caffeine in 0.9% NaCl was added 1.48 × 10–3. Table 1: Comparison of the EEC parameters when the 0.9% NaCl and the 10 mg/mL caffeine in 0.9% NaCl were added to the system. Parameters 0.9% NaCl 10 mg/mL Caffeine Rs (kOhm/cm2) 3.07 0.69 Ry (kOhm/cm2) 55.65 1.08 Rct (kOhm/cm2) 103.35 78.97 Cy (µF/cm2) 21.17 2237.20 Cdl (µF/cm2) 77.26 208.72 Wor (kOhm/cm2) 8.42 12.68 Woc (Ohm/cm2) 0.09 1.21 Figure 5: The equivalent electrical circuit (EEC) of the electrochemical cell without the yeast attached to the working electrode (Figure 5A) and with the electrochemical cell with yeast attached to the working electrode (Figure 5B). Figure 5A consists of the solution resistance (Rs), the capacitance of the electrical double layer (Cdl), the charge transfer resistance (Rct), and the Warburg impedance (Wo). Figure 5B consists of the solution resistance (Rs), the yeast layer capacitance (Cy), the yeast layer resistance (Ry), the capacitance of the electrical double layer (Cdl), the charge transfer resistance (Rct), and the Warburg open diffusion (Wo). 382 Acta Chim. Slov. 2022, 69, 378–384 Štukovnik et al.: The Use of Yeast Saccharomyces Cerevisiae ... 3. 3. Limit of Detection (LOD) and Limit of Quantification (LOQ) In Table 2, the decrease in impedance (|Z|) with the increasing concentration of caffeine in saline (c) is re- ported. Compared to the blank solution, the decrease in impedance is observed with the addition of 0.1 mg/mL of caffeine in 0.9% NaCl. The data were obtained at a fre- quency of 125 mHz. Three measurements were taken for each concentration, where the mean value (MV), standard deviation (SD), precision, and accuracy of the measure- ments were calculated. Table 2: The decrease in impedance with increasing concentration of caffeine in 0.9% NaCl C logC MV log|Z| SD Precision Accuracy (mg/mL) (mg/mL) (kOhm (kOhm (%) (%) /cm2) /cm2) 0.00 / 237.472 0.539 99.503 99.681 0.01 –2.00 231.935 2.895 97.051 98.318 0.05 –1.30 221.839 2.998 96.827 98.166 0.10 –1.00 106.922 1.929 96.161 97.515 0.25 –0.61 98.402 0.555 98.627 99.310 0.50 –0.30 94.551 0.272 99.312 99.616 1.00 0.00 88.514 0.583 97.949 98.859 5.00 0.70 75.801 0.332 98.175 99.010 The impedance decrease with increasing concen- trations is also represented as a box plot (Figure 6). The data were obtained at a frequency of 125 mHz. The box plot shows the mean values as a dot, the upper whiskers represent the maximum, the lower whiskers represent the minimum, and the box represents the interquartile range. The distinct decrease in the impedance is observed at 0.1 mg/mL concentration. The system‘s linearity was obtained in the concentra- tion range from 0.1 mg/mL to 5 mg/mL with R2 of 0.997 (Figure 7). The concentrations were calculated to logarith- mic values (Table 2) to obtain linear regression since the impedance values read from the Bode plot were logarith- mic. Based on the 3-Sigma criteria, the LOD was deter- mined at 0.728 mg/mL, and based on the 10-sigma criteria, the LOQ was determined at a concentration of 0.382 mg/ mL. It was observed that the impedance decreased with the increasing concentration of the caffeine in the solution. Thus, it can be concluded that the biosensor can sense the presence of caffeine in the solution. Figure 7: Calibration curve between the impedance (log|Z|) and the caffeine concentrations (logc) In conclusion, the Nyquist plot and the Bode plot of the investigated biosensor show that adding 10 mg/mL of caffeine in saline decreased the resistance and increased the capacitance, indicating that the electrode surface is released. Due to the Saccharomyces cerevisiae detachment from the stainless steel surface, the parameters of the EEC changed: the system‘s resistance decreased, and the capaci- tance increased. Consequently, it can be indeed concluded that caffeine can cause the desorption and death of Saccha- romyces cerevisiae cells. Some other research was done in the field of electro- chemical biosensors and caffeine detection. An ampero- metric biosensor for the determination of caffeine in solu- tions was developed, where whole cells of Pseudomonas alcaligenes were utilized. The biosensor system was able to detect caffeine in solution over a concentration range of 0.1 to 1 mg/mL.1 In comparison, the linearity range with our biosensor was in the range of 0.1 to 5 mg/mL. A biosensor based on the inhibition of alkaline phosphatase (ALP) en- zyme was developed for caffeine determination, where caf- feine concentration can be determined accurately between 0.1 and 10 μM and the LOD of the biosensor was 0.08 μM. Figure 6: Box plot representing the impedance (log|Z|) decrease with the increasing concentrations (c) of caffeine 383Acta Chim. Slov. 2022, 69, 378–384 Štukovnik et al.: The Use of Yeast Saccharomyces Cerevisiae ... This biosensor, compared to ours, had a lower LOD and linearity range as the enzymes were used as a biorecogni- tion element. Also, an electrochemical impedance aptasen- sor based on a porous organic framework supported silver nanoparticles for ultrasensitively detecting theophylline, with the LOD of 0.191 pg/mL (1.06 pmol/L) in a wide con- centration range of 5.0 × 10–4 to 5.0 ng/mL (2.78 × 10–3 to 27.8 nmol/L) was developed.29 4. Conclusions A new approach in biosensor development has been established, which involves assembling a low-cost and disposable electrochemical system for the detection of alkaloids such as caffeine. The caffeine detection with the presented method avoids an excessive use of solvents, requires only a small amount of analyte, and does not re- quire lengthy preparation. It was observed that the impedance decreased with the increasing concentration of the caffeine in the solution. It can be concluded that the developed biosensor is ro- bust enough to detect the various caffeine concentrations. Based on the linear calibration curve of the impedance de- crease with the increasing caffeine concentration, the LOD was determined at 0.728 mg/mL, and the LOQ was deter- mined at 0.382 mg/mL. Therefore, it can be concluded that yeasts, although very resistant to adverse environmental conditions, can sense and respond to caffeine as stimuli. Biosensors have the potential to represent the best candidate for caffeine detection with the merits of time-saving, robustness, low cost, and low detection limit. 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Predstavljen biosenzor je sestavljen iz predhodno razvite elektrokemijske celice, narejene iz nerjavnega jekla v RCW konfiguraciji. Elektrokemijska stabilnost delovne elektrode je bila ocenjena s potencialom odprtega tokokroga (OCP). Elektrokemijska impedančna spektroskopija (EIS) je bila uporabljena za spremljanje imped- imetričnega odziva biosenzorja s celicami Saccharomyces cerevisiae na površini delovne elektrode (WE) pri odsotnosti (0.9% NaCl) in prisotnosti (10 mg/mL v 0.9% NaCl) kofeina. Določena je bila tudi meja zaznavnosti (LOD). Razvit je bil nov pristop v razvoju biosenzorjev, ki vključuje sestavo ekonomično dostopnega biosenzorja, namenjenega enkratni up- orabi za detekcijo alkaloidov kot je kofein. Razvit biosenzor je dober kandidat za detekcijo kofeina v pijači, hrani ter zdra- vilih, saj omogoča hitro detekcijo, z nizko mejo zaznavnosti ter z nizko mejo določljivosti, hkrati pa je tudi ekonomičen.