Amperometric Miniaturised Portable Enzymatic Nanobiosensor for the Ultrasensitive Analysis of a Prostate Cancer Biomarker

Screen-printing technology is a game changer in many fields including electrochemical biosensing. Two-dimensional nanomaterial MXene Ti3C2Tx was integrated as a nanoplatform to immobilise enzyme sarcosine oxidase (SOx) onto the interface of screen-printed carbon electrodes (SPCEs). A miniaturised, portable, and cost-effective nanobiosensor was constructed using chitosan as a biocompatible glue for the ultrasensitive detection of prostate cancer biomarker sarcosine. The fabricated device was characterised with energy-dispersive X-ray spectroscopy (EDX), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Sarcosine was detected indirectly via the amperometric detection of H2O2 formed during enzymatic reaction. The nanobiosensor could detect sarcosine down to 7.0 nM with a maximal peak current output at 4.10 ± 0.35 × 10−5 A using only 100 µL of a sample per measurement. The assay run in 100 μL of an electrolyte showed the first linear calibration curve in a concentration window of up to 5 μM with a slope of 2.86 μA·μM−1, and the second linear calibration curve in the range of 5–50 μM with a slope of 0.32 ± 0.01 μA·μM−1 (R2 = 0.992). The device provided a high recovery index of 92.5% when measuring an analyte spiked into artificial urine, and could be used for detection of sarcosine in urine for at least a period of 5 weeks after the preparation.


Introduction
Prostate cancer (PCa) is one of the leading global causes of cancer death and the most frequent type of cancer diagnosed for men in 112 countries. Around 650,000 new patients are diagnosed with prostate cancer each year, and the incidence continues to rise [1,2]. Among PCa risk factors, the following are at the top of the list: age, race, genetic predispositions, and family history. PCa is also linked with other factors, including eating habits (a high consumption of saturated fat of animal origin and red meat, and a low intake of fruits, vegetables, vitamins, and coffee), being overweight, physical inactivity, inflammation, hyperglycaemia, infections, and exposure to hazardous chemicals and radiation [3,4]. Male androgenic hormones also play key roles in the development and progression of PCa [5].
Most PCa types are slow-growing and often asymptomatic, or their symptoms are similar to those of benign prostate hyperplasia (BPH) and are, hence, underestimated [6,7]. Since PCa is often asymptomatic in its earlier stages, it is very difficult to diagnose it before it reaches advanced stages. Late stages of PCa are associated with frequent night urination (nocturia), pain and difficulties during urination (dysuria), and the presence of blood in urine (haematuria) [8,9]. PCa may also cause problems with sexual function and performance [10].
PCa is screened via the analysis of the serological level of the prostate-specific antigen (PSA). Initially, the US Food and Drug Administration (FDA) agency approved PSA tests urination (nocturia), pain and difficulties during urination (dysuria), and the presenc blood in urine (haematuria) [8,9]. PCa may also cause problems with sexual function performance [10].
PCa is screened via the analysis of the serological level of the prostate-specific ant (PSA). Initially, the US Food and Drug Administration (FDA) agency approved PSA together with a digital rectal examination (DRE) for PCa screening in 1994 [11]. A low l of PSA is present in healthy men, and an increased level is not only associated with P but also with prostate enlargement, inflammation, and even cycling [7]. Hence, PSA is a reliable PCa biomarker, with high false-negative and false-positive results due to low specificity and sensitivity of the assay [7,8].
Since PSA is not able to provide reliable information as a biomarker for the diag tics and screening of PCa, more specific biomarkers need to be studied and valida Numerous types of biomolecules have been proposed as PCa biomarkers, including tabolites (i.e., sarcosine), proteins (i.e., prostate-specific antigen), RNAs (i.e., transm brane protease serine 2-ETS-related gene fusion), and glycans [12][13][14][15].
Sarcosine is an intermediate metabolite involved in glycine synthesis and degr tion. The first paper showing a link between changed sarcosine levels and cancer was p lished by A. Sreekumar and co-workers in 2009 [16]. Since this publication, there h been numerous studies that aimed to examine the association between sarcosine and [17]. Elevated levels of sarcosine associated with prostate cancer can be found in pros tissue, blood, and urine [18]. Significantly elevated levels of sarcosine are present in u and urine is the biofluid of choice since the analysis of biomarkers in urine is consid noninvasive [19].
The level of sarcosine in urine can vary from 20 nM to 5 µM [20]. The role of sarco in the onset and development of the disease is the stimulation of the growth of malig and metastatic cells [21]. Various methods are available for the determination of sarco [18,[22][23][24][25] that exhibit several drawbacks that can be addressed with biosensing dev that are simple, fast, inexpensive, and reliable. Electrochemical techniques coupled w SPCEs can offer additional benefits to users, including portability and miniaturisation 28]. The integration of such devices for "cancer-on-a-chip" assay platforms is an a tional advantage for their use in many applied areas [29,30].
We have been witnessing a revolution in the integration of nanomaterials to de biosensors over the past few decades. MXenes, discovered in 2011, are one of the lar families of two-dimensional (2D) materials for a diverse range of applications, includ biosensing due to their high electrical conductivity, large active area, hydrophilic nat easily tuneable structure, excellent thermal stability, and large interlayer spacing [31-In this work, MXene Ti3C2Tx was applied to the integration of sarcosine oxidase (S onto SPCEs to fabricate a miniaturised, portable, cost-effective nanobiosensor for the trasensitive analysis of sarcosine as a PCa biomarker ( Figure 1). The Materials and Methods section is provided in the supporting information file.

Basic CV Studies in a Plain Buffer
Initial electrochemical experiments were performed by running CVs in a plain 0.1 M PB pH 7.4 in potential windows from 0.1 to −1.0 V (a cathodic window) and from 0.0 to 1.0 V (an anodic window) with a scan rate of 0.1 V·s −1 using modified SPCEs. The following electrodes were examined: SPCE, MXene/SPCE, MXene-chitosan/SPCE, and SOx/MXenechitosan/SPCE ( Figure 2). droplet in the potential window from +0.1 V to −1.3 V vs. silver RE at a scan rate of 0.1 V·s −1 using 10 (blank) or 3 (sarcosine) scans.
The Materials and Methods section is provided in the supporting information file.

Basic CV Studies in a Plain Buffer
Initial electrochemical experiments were performed by running CVs in a plain 0.1 M PB pH 7.4 in potential windows from 0.1 to −1.0 V (a cathodic window) and from 0.0 to 1.0 V (an anodic window) with a scan rate of 0.1 V·s −1 using modified SPCEs. The following electrodes were examined: SPCE, MXene/SPCE, MXene-chitosan/SPCE, and SOx/MXenechitosan/SPCE ( Figure 2). As shown in the Figure 2a, the capacitive current increased in the following order: unmodified SPCE, MXene/SPCE, MXene-chitosan/SPCE, and SOx/MXene-chitosan/SPCE. For MXene-modified SPCE, a higher current was observed than that of pristine SPCE because MXene/SPCE, due to the porous MXene structure, has a greater electrochemically active surface area when compared to the unmodified surface. These findings are in good agreement with already published results [35]. In the cases of MXenechitosan/SPCE and SOx/MXene-chitosan/SPCE, the capacitive current was significantly higher than the unmodified SPCE and MXene/SPCE surface, suggesting the presence of enhanced MXene loading onto the electrode surface when present within the nanocomposite compared to the electrode modified only by MXene.
On the other hand, cyclic voltammetry in an anodic potential window (0.0 to 1.0 V) revealed that the MXene present on the MXene/SPCE surface underwent irreversible anodic oxidation with an oxidation peak at 0.457 V recorded in the first CV scan (9.67 ± 0.27) × 10 −7 A (Figure 2b). The experiment revealed that the beneficial redox behaviour of MXene substantially dropped in the second scan, and a further decrease in anodic current with the increasing number of scans was observed. The exposure of MXene to an anodic potential in the aqueous solution oxidised the nanomaterial, forming a TiO2 layer or TiO2 domains with the subsequent TiO2 dissolution by Fions, rendering the resulting nanomaterial less electrochemically active compared to the pristine MXene [36]. Similar results could be observed in the cases of the MXene-chitosan/SPCE and SOx/MXene-chitosan/SPCE surfaces. The prepared devices could, thus, be applied to electrochemical reactions in a cathodic potential window. As shown in the Figure 2a, the capacitive current increased in the following order: unmodified SPCE, MXene/SPCE, MXene-chitosan/SPCE, and SOx/MXene-chitosan/SPCE. For MXene-modified SPCE, a higher current was observed than that of pristine SPCE because MXene/SPCE, due to the porous MXene structure, has a greater electrochemically active surface area when compared to the unmodified surface. These findings are in good agreement with already published results [35]. In the cases of MXene-chitosan/SPCE and SOx/MXene-chitosan/SPCE, the capacitive current was significantly higher than the unmodified SPCE and MXene/SPCE surface, suggesting the presence of enhanced MXene loading onto the electrode surface when present within the nanocomposite compared to the electrode modified only by MXene.
On the other hand, cyclic voltammetry in an anodic potential window (0.0 to 1.0 V) revealed that the MXene present on the MXene/SPCE surface underwent irreversible anodic oxidation with an oxidation peak at 0.457 V recorded in the first CV scan (9.67 ± 0.27) × 10 −7 A (Figure 2b). The experiment revealed that the beneficial redox behaviour of MXene substantially dropped in the second scan, and a further decrease in anodic current with the increasing number of scans was observed. The exposure of MXene to an anodic potential in the aqueous solution oxidised the nanomaterial, forming a TiO 2 layer or TiO 2 domains with the subsequent TiO 2 dissolution by Fions, rendering the resulting nanomaterial less electrochemically active compared to the pristine MXene [36]. Similar results could be observed in the cases of the MXene-chitosan/SPCE and SOx/MXenechitosan/SPCE surfaces. The prepared devices could, thus, be applied to electrochemical reactions in a cathodic potential window.

EIS Measurements Using a Ferricyanide/Ferrocyanide Redox Couple
EIS is a powerful characterisation technique to probe the interfacial properties of individual unmodified and modified SPCEs. EIS measurements were performed to observe the characteristics of individual modified SPCEs, and investigate the short-term stability of the prepared MXene/SPCE and MXene-chitosan/SPCE surfaces. Figure 3 shows the impedance spectra of the prepared modified electrodes.

EIS Measurements Using a Ferricyanide/Ferrocyanide Redox Couple
EIS is a powerful characterisation technique to probe the interfacial properties of individual unmodified and modified SPCEs. EIS measurements were performed to observe the characteristics of individual modified SPCEs, and investigate the short-term stability of the prepared MXene/SPCE and MXene-chitosan/SPCE surfaces. Figure 3 shows the impedance spectra of the prepared modified electrodes. A Nyquist plot shows two main features: a semicircular part with a diameter representing charge transfer resistance Rct and a linear part representing the diffusional properties of the interface [37].
EIS using a ferricyanide/ferrocyanide redox couple revealed that the Rct of MXenemodified SPCE was approximately 1.9 times larger than that of bare SPCE (Table 1), indicating that a layer of negatively charged nanomaterial had formed on the surface, which hindered the electron transfer between [Fe(CN)6] 3−/4− and the electrode surface (Table 1). Though MXene could hinder the electron transfer of a negatively charged soluble redox probe pair, it is an excellent candidate with high metallic conductivity and great filmforming ability for the design of electrochemical biosensors. In contrast, the MXene-chitosan/SPCE and SOx/MXene-chitosan/SPCE surfaces exhibited significantly lower Rct compared to those of the unmodified and MXene/SPCE. The Rct value changed because of the electrostatic interactions between the positively charged chitosan-containing surface and [Fe(CN)6] 3−/4− ions.

SPCE Modification Charge Transfer Resistance (Rct) Values (Ω)
MXenes are promising nanoplatforms for the construction of biosensing devices due to their excellent physicochemical properties. A natural biopolymer, chitosan, is an excellent counterpart to MXene since it is a hydrophilic hydrogel that can form adhesive A Nyquist plot shows two main features: a semicircular part with a diameter representing charge transfer resistance R ct and a linear part representing the diffusional properties of the interface [37].
EIS using a ferricyanide/ferrocyanide redox couple revealed that the R ct of MXenemodified SPCE was approximately 1.9 times larger than that of bare SPCE (Table 1), indicating that a layer of negatively charged nanomaterial had formed on the surface, which hindered the electron transfer between [Fe(CN) 6 ] 3−/4− and the electrode surface (Table 1). Though MXene could hinder the electron transfer of a negatively charged soluble redox probe pair, it is an excellent candidate with high metallic conductivity and great film-forming ability for the design of electrochemical biosensors. In contrast, the MXenechitosan/SPCE and SOx/MXene-chitosan/SPCE surfaces exhibited significantly lower R ct compared to those of the unmodified and MXene/SPCE. The R ct value changed because of the electrostatic interactions between the positively charged chitosan-containing surface and [Fe(CN) 6 ] 3−/4− ions. MXenes are promising nanoplatforms for the construction of biosensing devices due to their excellent physicochemical properties. A natural biopolymer, chitosan, is an excellent counterpart to MXene since it is a hydrophilic hydrogel that can form adhesive membranes/films with biocompatible properties [38,39]. Thus, the combination of chitosan and nanomaterials such as MXene is an efficient strategy for the design of mechanically robust biosensing devices [40,41].
In order to investigate the short-term stability of the prepared MXene/SPCE and MXene-chitosan/SPCE surfaces, and the stabilisation of nanomaterial with a chitosan matrix, seven subsequent EIS measurements were run in 0.1 M PB containing 5 mM membranes/films with biocompatible properties [38,39]. Thus, the combination of chitosan and nanomaterials such as MXene is an efficient strategy for the design of mechanically robust biosensing devices [40,41].
In order to investigate the short-term stability of the prepared MXene/SPCE and MXene-chitosan/SPCE surfaces, and the stabilisation of nanomaterial with a chitosan matrix, seven subsequent EIS measurements were run in 0. 1   The obtained results confirmed the stabilisation effect of the chitosan matrix on biosensor performance. In the case of the MXene/SPCE surface, a more significant change in Rct value was observed compared to that on the MXene-chitosan/SPCE surface. The greatest decrease in Rct value was recorded during the first four EIS measurements (Figure 4a). When the assay was performed in an electrochemical cell, a much quicker change (decrease) in Rct was observed, i.e., 84.5% of the initial value on the MXene/SPCE interface ( Figure 4b  The obtained results confirmed the stabilisation effect of the chitosan matrix on biosensor performance. In the case of the MXene/SPCE surface, a more significant change in R ct value was observed compared to that on the MXene-chitosan/SPCE surface. The greatest decrease in R ct value was recorded during the first four EIS measurements (Figure 4a). When the assay was performed in an electrochemical cell, a much quicker change (decrease) in R ct was observed, i.e., 84.5% of the initial value on the MXene/SPCE interface (Figure 4b) compared to the assay performed on MXene-chitosan/SPCE (Figure 4c), i.e., 93.0% of the initial value (Figure 4d) within 22 min, indicating that chitosan had a positive effect on the stability of the MXene-modified interface. The exponential fitting of the operational stability curve performed on an electrochemical cell revealed that the signal would be stable at the R ct value of 341 Ω (i.e., 77.6% of the initial value).

Detection of Sarcosine Using SOx/MXene-Chitosan/SPCE Biosensor
CV measurements with SOx/MXene-chitosan/SPCE electrodes for detection of sarcosine were performed in a cathodic potential window. SOx enzyme was characterised by maximal stability at pH value of 7.4 [42]. The bioelectrochemical activity of the biosensor was evaluated by constructing the corresponding calibration curves when 10 mM sarcosine stock solution was added into the cell during the measurement. The final sarcosine concentration was in the range of 2.5-50 µM. Well-defined irreversible redox peaks were observed on the SOx/MXene-chitosan/SPCE electrode in the presence of sarcosine. The cathodic peak of H 2 O 2 reduction increased with increasing biomarker concentration ( Figure 5, inset). The plot of the peak current in the first CV scan vs. sarcosine concentration was applied to calibrate the device by subtracting a blank signal. In this work, blank was considered the fifth CV scan obtained in the absence of sarcosine ( Figure 5). cosine were performed in a cathodic potential window. SOx enzyme was characterised by maximal stability at pH value of 7.4 [42]. The bioelectrochemical activity of the biosensor was evaluated by constructing the corresponding calibration curves when 10 mM sarcosine stock solution was added into the cell during the measurement. The final sarcosine concentration was in the range of 2.5-50 µM. Well-defined irreversible redox peaks were observed on the SOx/MXene-chitosan/SPCE electrode in the presence of sarcosine. The cathodic peak of H2O2 reduction increased with increasing biomarker concentration (Figure 5, inset). The plot of the peak current in the first CV scan vs. sarcosine concentration was applied to calibrate the device by subtracting a blank signal. In this work, blank was considered the fifth CV scan obtained in the absence of sarcosine ( Figure 5). In comparison with our previous study, several differences could be observed when working with SOx/MXene-chitosan/SPCE biosensor compared to SOx/MXene-chitosan/GCE device [43]. Significant differences in the values of the current responses were recorded. In the case of SPCE-based detection platform, they were about 19 times higher (1.21 × 10 −5 A in the presence of 2.5 µM sarcosine) in comparison with those of GCE-based biosensing (6.38 × 10 −7 A in the presence of 2.5 µM sarcosine). Changes in the current responses could be attributed to the properties of the carbon working-electrode material. The fitting of the calibration curve revealed two different ranges in which the calibration curve was linear for both types of measurements ( Figure 5).
The assay in the electrochemical cell exhibited a first linear calibration curve in a range up to 10 µM with a slope of 1.19 ± 0.25 µA·µM −1 (R 2 = 0.910), and a second linear calibration curve in the range of 10-50 µM with a slope of 0.38 ± 0.03 µA·µM −1 (R 2 = 0.982) (Figure 5a). Additionally, the prepared SOx/MXene-chitosan/SPCE biosensor exhibited a satisfactory LOD value of 7.0 nM. Hence, the LOD was successfully reduced by 2.6 times [43]. In comparison with the SOx/MXene-chitosan/GCE device, a noticeable shift in peak potential values with respect to Ag/AgCl/KCl (3 M) RE was recorded. The peak potential shifted to more positive values with the increasing concentration of sarcosine. The electrochemical reduction in H2O2 by the device started at −0.2 V, with the peak maximum present at approx. −0.6 V ( Figure 5, inset).
The proposed biosensing detection platform was subsequently further optimised. For this purpose, electrochemical measurements focusing on sample volume reduction were performed. For each measurement, drops with a volume of 100 µL were applied In comparison with our previous study, several differences could be observed when working with SOx/MXene-chitosan/SPCE biosensor compared to SOx/MXene-chitosan/GCE device [43]. Significant differences in the values of the current responses were recorded. In the case of SPCE-based detection platform, they were about 19 times higher (1.21 × 10 −5 A in the presence of 2.5 µM sarcosine) in comparison with those of GCE-based biosensing (6.38 × 10 −7 A in the presence of 2.5 µM sarcosine). Changes in the current responses could be attributed to the properties of the carbon working-electrode material. The fitting of the calibration curve revealed two different ranges in which the calibration curve was linear for both types of measurements ( Figure 5).
The assay in the electrochemical cell exhibited a first linear calibration curve in a range up to 10 µM with a slope of 1.19 ± 0.25 µA·µM −1 (R 2 = 0.910), and a second linear calibration curve in the range of 10-50 µM with a slope of 0.38 ± 0.03 µA·µM −1 (R 2 = 0.982) (Figure 5a). Additionally, the prepared SOx/MXene-chitosan/SPCE biosensor exhibited a satisfactory LOD value of 7.0 nM. Hence, the LOD was successfully reduced by 2.6 times [43]. In comparison with the SOx/MXene-chitosan/GCE device, a noticeable shift in peak potential values with respect to Ag/AgCl/KCl (3 M) RE was recorded. The peak potential shifted to more positive values with the increasing concentration of sarcosine. The electrochemical reduction in H 2 O 2 by the device started at −0.2 V, with the peak maximum present at approx. −0.6 V ( Figure 5, inset).
The proposed biosensing detection platform was subsequently further optimised. For this purpose, electrochemical measurements focusing on sample volume reduction were performed. For each measurement, drops with a volume of 100 µL were applied onto the surface of the SPCE's working electrode, since SPCEs are suitable for either working with microvolumes or dipping them into solutions. In this case, the electrochemical cell consisted of a modified carbon working electrode, carbon auxiliary electrode, and silver RE (Figure 5b). The assay run in 100 µL of an electrolyte showed a first linear calibration curve in the concentration window up to 5 µM with a slope of 2.86 µA·µM −1 and a second linear calibration curve in the range of 5-50 µM with a slope of 0.32 ± 0.01 µA·µM −1 (R 2 = 0.992) (Figure 5b). In comparison with the previous measurement (Figure 5a), a shift in the reduction process towards more negative potentials was observed. The electrochemical reduction in H 2 O 2 started at −0.5 V with the peak maximum present at −0.9 V. Such an electrochemical behaviour can be assigned to the characteristics of the silver reference electrode used in three-electrode system. Moreover, similar findings were observed in the peak potential shift towards more positive values with an increasing concentration of sarcosine when compared to that in Figure 5a. Additionally, the prepared SOx/MXene-chitosan/SPCE biosensor exhibited a satisfactory LOD value of 10.4 nM. A slight increase in the LOD value in the case of the detection of sarcosine in the drop using the SOx/MXene-chitosan/SPCE device was caused by the increased intensity of noise during the electrochemical measurements. The intensified noise could be attributed to the nature of the used SPCE devices.
The reproducibility of the biosensor device's fabrication, examined as the sensitivity of the devices, was within 10 % (n = 5) when measured in an electrochemical cell, and within 3 % (n = 4) when measured in 100 µL of an electrolyte. Such observations indicate that it is much better to use the biosensor device for measurements in 100 µL of an electrolyte with the bionanocomposite at the bottom in the measurement setup. Part of the bionanocomposite in the electrochemical cell was most likely dissolving over time.
Scanning electron microscopy was used to visualise both pristine MXene (Figure 6, left) and the Ti 3 C 2 T X MXene@chitosan composite (Figure 6, right).

Clinical Application of SOx/MXene-Chitosan/SPCE Biosensor
The practical application of the biosensor was evaluated by analysing the analyte in real samples. For that purpose, the SOx/MXene-chitosan/SPCE device was applied to analyse sarcosine (from 0.1 to 1.0 µM) spiked into 10× diluted artificial urine using sarcosine as the PCa biomarker [20]. Calibration curves were obtained from measurements in 0.1 M PB pH 7.4 and 10× diluted artificial urine together with the CVs of the prepared SOx/MXene-chitosan/SPCE biosensor. The value of the obtained recovery index was 92.5% (data not shown). The recovery index was calculated via the standard addition of 1 µM sarcosine into artificial urine, and the current response obtained in urine was compared to the current response obtained in plain PB. This means that, under such conditions, there were no significant interferences affecting the biosensor s performance. The results prove that the as-fabricated biosensor is a reliable tool for sarcosine detection in urine samples. Obviously, analysis with real human urine is needed to reach more general conclusions about the performance of the prepared MXene-based sarcosine biosensor in the analysis of real urine samples.

Long-Term Stability of the SOx/MXene-Chitosan/SPCE Device
To investigate the long-term stability of the prepared SOx/MXene-chitosan/SPCE device, CV measurements in the presence of 30 µM sarcosine in the potential window from 0.1 to −1.0 V and 0.1 to −1.3 V in the cell and in a 100 µL droplet, respectively, were performed ( Figure 7). All prepared devices were stored in the refrigerator throughout the experiment.

Clinical Application of SOx/MXene-Chitosan/SPCE Biosensor
The practical application of the biosensor was evaluated by analysing the analyte in real samples. For that purpose, the SOx/MXene-chitosan/SPCE device was applied to analyse sarcosine (from 0.1 to 1.0 µM) spiked into 10× diluted artificial urine using sarcosine as the PCa biomarker [20]. Calibration curves were obtained from measurements in 0.1 M PB pH 7.4 and 10× diluted artificial urine together with the CVs of the prepared SOx/MXene-chitosan/SPCE biosensor. The value of the obtained recovery index was 92.5% (data not shown). The recovery index was calculated via the standard addition of 1 µM sarcosine into artificial urine, and the current response obtained in urine was compared to the current response obtained in plain PB. This means that, under such conditions, there were no significant interferences affecting the biosensor's performance. The results prove that the as-fabricated biosensor is a reliable tool for sarcosine detection in urine samples. Obviously, analysis with real human urine is needed to reach more general conclusions about the performance of the prepared MXene-based sarcosine biosensor in the analysis of real urine samples.

Long-Term Stability of the SOx/MXene-Chitosan/SPCE Device
To investigate the long-term stability of the prepared SOx/MXene-chitosan/SPCE device, CV measurements in the presence of 30 µM sarcosine in the potential window from 0.1 to −1.0 V and 0.1 to −1.3 V in the cell and in a 100 µL droplet, respectively, were performed ( Figure 7). All prepared devices were stored in the refrigerator throughout the experiment.
In the case of the performed measurements in the cell (Figure 7a), the average current responses of the prepared SOx/MXene-chitosan/SPCE device decreased from 7.05 ± 0.51 × 10 −5 A on day 0 to 5.94 ± 0.65 × 10 −5 A on Day 35 after biosensor preparation. Thus, a 15.7% drop of the SOx/MXene-chitosan/SPCE current response was observed for measurements in the cell within 35 days. For the measurements performed in the 100 µL droplet (Figure 7b), a 6.8% decrease in the SOx/MXene-chitosan/SPCE current response was observed on Day 35 after fabrication. In this case, the average current responses of the prepared SOx/MXene-chitosan/SPCE device decreased from 6.12 ± 0.77 × 10 −5 A on Day 0 to 5.71 ± 0.01 × 10 −5 A on Day 35. Despite a slight decrease in the current response, the as-fabricated SOx/MXene-chitosan/SPCE device was able to detect sarcosine even after a period of 5 weeks. In summary, the obtained data confirm that the as-fabricated device could be used for analysis for at least 5 weeks after its preparation. In the case of the performed measurements in the cell (Figure 7a), the average current responses of the prepared SOx/MXene-chitosan/SPCE device decreased from 7.05 ± 0.51 × 10 −5 A on day 0 to 5.94 ± 0.65 × 10 −5 A on Day 35 after biosensor preparation. Thus, a 15.7% drop of the SOx/MXene-chitosan/SPCE current response was observed for measurements in the cell within 35 days. For the measurements performed in the 100 µL droplet (Figure 7b), a 6.8% decrease in the SOx/MXene-chitosan/SPCE current response was observed on Day 35 after fabrication. In this case, the average current responses of the prepared SOx/MXene-chitosan/SPCE device decreased from 6.12 ± 0.77 × 10 −5 A on Day 0 to 5.71 ± 0.01 × 10 −5 A on Day 35. Despite a slight decrease in the current response, the asfabricated SOx/MXene-chitosan/SPCE device was able to detect sarcosine even after a period of 5 weeks. In summary, the obtained data confirm that the as-fabricated device could be used for analysis for at least 5 weeks after its preparation.

Conclusions
The goal of this paper was to design an enzymatic nanobiosensor for the detection of PCa biomarkers using disposable SPCEs and modern 2D nanomaterial MXene in order to prepare a sensitive, reliable, portable and miniaturised, stable, cost-effective, and quick detection platform based on previously obtained results and findings. By dipping SPCEs into the solution, the prepared SOx/MXene-chitosan/SPCE biosensor exhibited a satisfactory LOD value of 7.0 nM. Hence, the LOD was successfully reduced 2.6 times when compared to our previous paper. The proposed detection platform was subsequently further optimised with a focus on a sample volume reduction of up to 100 µL, exhibiting a satisfactory LOD value of 10.4 nM. To investigate the long-term stability of the prepared SOx/MXene-chitosan/SPCE device, CV measurements were performed in the presence of 30 µM in both the cell and a 100 µL droplet. For the measurements performed in the 100 µL droplet, only a 6.8% decrease in the SOx/MXene-chitosan/SPCE current response could be observed during the first 35 days after fabrication. Thus, the obtained data confirm that the as-fabricated device could be used for the detection of sarcosine in urine for a period of at least 5 weeks after its preparation. Moreover, to evaluate the potential application of the biosensor for the analysis of real samples, the as-fabricated device was successfully applied for the determination of sarcosine spiked into 10× diluted artificial urine with the 92.5% recovery index value. Such results prove that the as-fabricated biosensor is a reliable tool for sarcosine detection in urine samples. The portable SOx/MXenechitosan/SPCE device is one of the most sensitive electrochemical biosensor devices for

Conclusions
The goal of this paper was to design an enzymatic nanobiosensor for the detection of PCa biomarkers using disposable SPCEs and modern 2D nanomaterial MXene in order to prepare a sensitive, reliable, portable and miniaturised, stable, cost-effective, and quick detection platform based on previously obtained results and findings. By dipping SPCEs into the solution, the prepared SOx/MXene-chitosan/SPCE biosensor exhibited a satisfactory LOD value of 7.0 nM. Hence, the LOD was successfully reduced 2.6 times when compared to our previous paper. The proposed detection platform was subsequently further optimised with a focus on a sample volume reduction of up to 100 µL, exhibiting a satisfactory LOD value of 10.4 nM. To investigate the long-term stability of the prepared SOx/MXene-chitosan/SPCE device, CV measurements were performed in the presence of 30 µM in both the cell and a 100 µL droplet. For the measurements performed in the 100 µL droplet, only a 6.8% decrease in the SOx/MXene-chitosan/SPCE current response could be observed during the first 35 days after fabrication. Thus, the obtained data confirm that the as-fabricated device could be used for the detection of sarcosine in urine for a period of at least 5 weeks after its preparation. Moreover, to evaluate the potential application of the biosensor for the analysis of real samples, the as-fabricated device was successfully applied for the determination of sarcosine spiked into 10× diluted artificial urine with the 92.5% recovery index value. Such results prove that the as-fabricated biosensor is a reliable tool for sarcosine detection in urine samples. The portable SOx/MXene-chitosan/SPCE device is one of the most sensitive electrochemical biosensor devices for the analysis of sarcosine when classified by LOD. Additionally, the as-fabricated MXene-based biosensor is one of the most sensitive MXene-based devices.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jfb14030161/s1, Figure S1: Enlarged graph shown in Figure 5A in the inset; Figure S2: Enlarged graph shown in Figure 5B