An Amperometric Acetylcholine Biosensor Based on Co-Immobilization of Enzyme Nanoparticles onto Nanocomposite

An electrochemical biosensor was fabricated using nanoparticles of acetylcholinesterase (AChE) and choline oxidase (ChO)/Pt nanoparticles (PtNPs)/porous graphene oxide nanosheet (GONS) composite. A pencil graphite electrode (PGE) was used for the electrodeposition of nanocomposite and the determination of acetylcholine (ACh), a neurotransmitter. Various techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectra and cyclic voltammetry (CV) were used for characterization. This biosensor (AChENPs-ChONPs/GONS/PtNPs/PGE) indicated a very short response time (3 s), a lower limit of detection (0.001 µM), good linearity (0.001–200 µM), longer storage stability (6 months) and better reproducibility. The percent analytical recoveries of added acetylcholine in serum (5.0 and 10 µM) were found to be 97.6 ± 0.7 and 96.5 ± 0.3 for the present biosensor. The coefficients of variation were obtained to be 8% and 3.25%, correspondingly. The biosensor was applied to measure the ACh amount in the serum of healthy individuals and patients with Alzheimer’s disease. The number of interferents had no effect on the biosensor at their physiological concentrations.


Introduction
Neurotransmitters are substances that the body utilizes to communicate between cells. In the case of the human body, acetylcholine (ACh) is one of the most common neurotransmitters. It is secreted by cholinergic neurons. Choline (Ch) and its coenzyme A ester (acetyl CoA) are converted to ACh (CH 3 COOCH 2 CH 2 N + (CH 3 ) 3 by an enzyme known as acetylcholine transferase. ACh, scientifically referred to as 2-acetoxy-N, N, N-trimethylethanaminium, is an important neurotransmitter in which both the central and peripheral nervous systems (PNS) are involved [1]. Henry Hallett Dale first discovered it in 1914, and Otto Loewi subsequently verified its existence in 1922. Nearly one-hundredth of the world's population is affected by neurological illnesses, according to the World Health Organization (WHO) [2]. Medications that interfere with ACh, a neurotransmitter critical to muscular activity, may induce varying degrees of movement disturbances or even paralysis. Functions that are impacted by ACh are cognition, memory, sleep, focus, and learning capacity.
ACh levels in neurological disorders can be determined by using a variety of procedures, together with an improved Elman colorimetric test for cholinesterase activity estimation [3], capillary electrophoresis, capillary zone electrophoresis, ion-sensitive field effect transistors (ISFETs), high-performance liquid chromatography (HPLC) [4,5], mass spectrometry [6], hydrophilic interaction chromatography [7], etc. However, the majority of these methods are complex, costly and time-consuming, with less sensitivity and selectivity. For the clinical diagnosis of acetylcholine, biosensing methods are more efficient, accurate, time-saving, specific, rapid and portable.

Synthesis of Platinum Nanoparticles (PtNPs)
The preparation of PtNPs was performed as described by Thirumurugan et al. [32], with minor modifications. To synthesize PtNPs, 10 mL of leaf extract of citrus lemon was added to 190 mL of 1 mM aqueous chloroplatinic acid extract (H 2 PtCl 6 .6H 2 0) under continuous stirring. After complete addition of the leaf extract, the mixture was kept for incubation at 30 • C for 24 h. The color of the solution changed from light yellow to dark brown, which revealed the synthesis of PtNPs. The colored solution was centrifuged for 30 min, followed by redispersion in DW to remove further unwanted biological impurities.

Preparation of Porous Graphene Oxide Nanosheets (GONS)
Firstly, the improved Hummer method [33] was used for the preparation of graphene oxide (GO) as follows: 0.5 g of pencil graphite powder, 0.50 g of sodium nitrate and 25 mL of sulfuric acid (H 2 SO 4 98%) were mixed in a reaction flask of 500 mL under continuous stirring for 15 min in a water bath with ice at a temperature between 0 and 5 • C. Then, 4.0 g of potassium permanganate was added slowly to the above solution within 15 min at 20 • C. Following that, DW (20 mL) was added slowly under stirring at 40 • C for 90 min in the water bath. A brown-colored suspension was formed, which was treated with 30% H 2 O 2 solution (6 mL). A 5% amount of hydrochloric acid (HCl) was used to wash the GO suspension and was monitored by DW for the removal of excess manganese (Mn) until it reached neutrality. An oven was used to dry the purified GO at 60 • C for 24 h. To prepare the graphene oxide nanosheet (GONS), GO (0.1 g) and broccoli juice (240 µL) were added to a beaker (250 mL), producing a homogeneous dispersion. This dispersion was stirred for 24 h. After stirring, it was sonicated for 60 min and, lastly, dried at 60 • C for 24 h.

Preparation of AChENPs/ChONPs
For the preparation of AChENPs/ChONPs, the desolvation method was used by using ethanol [34]. Firstly, the enzyme (acetylcholinesterase/choline oxidase separately) was dissolved in DW (2 mg/mL). Then, 4 mL of absolute ethanol was added dropwise (0.1 mL/min) under continuous stirring (500 rpm). To confirm the complete cross-linking of the respective enzyme nanoparticles (ENPs), 1.8 mL of glutaraldehyde solution (2.5%) was added to the solution under the same stirring settings at 4 • C for 24 h. The differential centrifugation technique was used for the precipitation of ENPs from two suspensions at 12,000× g (min, 4 • C). After that, a 0.12 g amount of cysteamine di-hydrochloride was added to the ENPs under continuous stirring (5-6 h), which provides an amino group to the ENPs. These functionalized ENPs were separated from the enzyme solution by centrifugation at 1200 rpm for 10 min and stored at 4 • C until use. The size and shape of the functionalized AChENPs/ChONPs were studied by TEM from AIRF commercially.

Preparation of GNs/PtNPs/PG Electrode
Firstly, the surface of the PG electrode (2 cm × 5 mm) was manually polished with alumina slurry by using a polishing cloth. It was monitored by systematic washing with DW. The cleaned electrode was dipped in 4 mL of piranha solution (3H 2 SO 4 :1H 2 O 2 ) for 10 min and then washed with DW. A PtNPs/GONS hybrid was prepared by taking both in an equal ratio (2 mg/2 mL) under continuous stirring for 12 h, followed by centrifugation at 1200 rpm to separate the nanocomposite (PtNPs/GONS). Cyclic voltammetry in a potentiostat/galvanostat was used for the electrodeposition of PtNPs/GONS on the surface of the PGE by immersing it in 23 mL of 2.5 mM K 3 Fe(CN) 6 /K 4 Fe(CN) 6 (1:1) and 2 mL of PtNPs/GONS solution with 40 continuous polymerization circles (−1.1 to 0.1 V) at a scan rate of 20 mV/s. The subsequent PtNPs/GONS-modified PGE was washed thoroughly with DW to get rid of boundless matter ( Figure 1).

Co-Immobilization of AChENPs/ChONPs onto PtNPs/GONS-Modified PG Electrode
The GONS/PtNPs/PGE was immersed into 4 mL of AChENPs + ChONPs suspension for 24 h at 4 • C. The resulting AChENPs/ChONPs/GONS/PtNPs/PG electrode was washed 3-4 times with DW to remove boundless AChENPs and ChONPs. The AChENPs/ChONPs/GONS/PtNPs/PGE was used as a working electrode, as shown in Figure 2, and stored at 4 • C while not in practice. The working electrode was characterized by means of SEM and FTIR before and after immobilization of AChENPs and ChONPs.
Biosensors 2023, 13, x FOR PEER REVIEW rate of 20 mV/s. The subsequent PtNPs/GONS-modified PGE was washed thoro with DW to get rid of boundless matter ( Figure 1).

Co-Immobilization of AChENPs/ChONPs onto PtNPs/GONS-Modified PG Electrode
The GONS/PtNPs/PGE was immersed into 4 mL of AChENPs + ChONPs suspe for 24 h at 4 °C. The resulting AChENPs/ChONPs/GONS/PtNPs/PG electrode was w 3-4 times with DW to remove boundless AChENPs and ChONPs. AChENPs/ChONPs/GONS/PtNPs/PGE was used as a working electrode, as shown i ure 2, and stored at 4 °C while not in practice. The working electrode was characte by means of SEM and FTIR before and after immobilization of AChENPs and ChON

Construction of Amperometric Acetylcholine Biosensor
An amperometric acetylcholine biosensor was constructed using AChENPs/ChONPs/GONS/PtNPs/PGE (working electrode), Pt wire (counter electrode) and Ag/AgCl (reference electrode) saturated 3.5 M KCl. All measurements were performed on an electrochemical analyzer (AutoLab PGSTAT 30) at room temperature. The cyclic voltammetry experiments were performed in 25 mL of 2.5 mMK3Fe(CN)6/K4Fe(CN)6 (1:1) with 200 µL of AChCl (0.05 M), and a voltage range between −0.1 and +0.1 V at a scan rate of 20 mV/s was applied. Hence, the working of this biosensor is based on the follow-

Construction of Amperometric Acetylcholine Biosensor
An amperometric acetylcholine biosensor was constructed using AChENPs/ChONPs/ GONS/PtNPs/PGE (working electrode), Pt wire (counter electrode) and Ag/AgCl (reference electrode) saturated 3.5 M KCl. All measurements were performed on an electrochemical analyzer (AutoLab PGSTAT 30) at room temperature. The cyclic voltammetry experiments were performed in 25 mL of 2.5 mMK 3 Fe(CN) 6 /K 4 Fe(CN) 6 (1:1) with 200 µL of AChCl (0.05 M), and a voltage range between −0.1 and +0.1 V at a scan rate of 20 mV/s was applied. Hence, the working of this biosensor is based on the following reaction: nanoparticles; PGE = Pencil graphite electrode).

Construction of Amperometric Acetylcholine Biosensor
An amperometric acetylcholine biosensor was constructed using AChENPs/ChONPs/GONS/PtNPs/PGE (working electrode), Pt wire (counter electrode) and Ag/AgCl (reference electrode) saturated 3.5 M KCl. All measurements were performed on an electrochemical analyzer (AutoLab PGSTAT 30) at room temperature. The cyclic voltammetry experiments were performed in 25 mL of 2.5 mMK3Fe(CN)6/K4Fe(CN)6 (1:1) with 200 µL of AChCl (0.05 M), and a voltage range between −0.1 and +0.1 V at a scan rate of 20 mV/s was applied. Hence, the working of this biosensor is based on the following reaction: Acetylcholine + H2O Acetylcholine esterase Choline + Acetate The current measurement or flow of electrons in the galvanostat remains directly related to the acetylcholine concentration.

Optimization of Acetylcholine Biosensor
The effects of various analytical parameters such as pH, incubation temperature, substrate (acetylcholine) concentration and reaction period were considered for the optimization of the AChENPs/ChONPs/GONS/PtNP-modified PGE-based nanosensor. For the determination of optimum pH, phosphate buffers (0.1 M) with pH between 5.0 and 10 at a break of 0.5 were prepared. Similarly, the incubation temperature range (20-50 °C) at an interval of 5 °C in a temperature-controlled water bath and at a time duration of 2-90 s was measured for the detection of the most favorable temperature and incubation time, respectively. The acetylcholine effect on the biosensor response was studied by changing the concentration of acetylcholine in the range of 0.001-200 µM.
The current measurement or flow of electrons in the galvanostat remains directly related to the acetylcholine concentration.

Optimization of Acetylcholine Biosensor
The effects of various analytical parameters such as pH, incubation temperature, substrate (acetylcholine) concentration and reaction period were considered for the optimization of the AChENPs/ChONPs/GONS/PtNP-modified PGE-based nanosensor. For the determination of optimum pH, phosphate buffers (0.1 M) with pH between 5.0 and 10 at a break of 0.5 were prepared. Similarly, the incubation temperature range (20-50 • C) at an interval of 5 • C in a temperature-controlled water bath and at a time duration of 2-90 s was measured for the detection of the most favorable temperature and incubation time, respectively. The acetylcholine effect on the biosensor response was studied by changing the concentration of acetylcholine in the range of 0.001-200 µM.

Evaluation of Acetylcholine Biosensor
The acetylcholine biosensor was evaluated by measuring its lower detection limit (LOD), % analytical recovery of added acetylcholine in serum, within and between batch coefficients of variation (precision) and correlation with the standard method.

Transmission Electron Microscope (TEM) Study
The TEM study revealed that the mean size of the PtNPs is 20 nm, and they are predominantly spherical in shape, while some of the nanoparticles have a dodecahedron shape, as shown in Figure 3A. The TEM image shown in Figure 3A confirms thin, porous graphene sheets with a wrinkled-like material and crumpled ends ( Figure 3B). The average size of AChENPs and ChONPs was in the range of 1-100 nm in diameter, as studied by taking their images in TEM, as shown in Figure 3C,D. The diameter of further ENPs was also described similarly [35].

X-ray Diffraction (XRD) Study
The XRD study revealed a sharp diffraction peak at 2θ = 25 • , which indicated that the porous graphene nanosheets existed individually with a highly disordered nature ( Figure 4).

Fourier-Transform Infrared (FTIR) Study
The FTIR analysis of PtNPs showed a broad band at 3341 cm −1 , which was owed to the N-H enlarging vibration of the -NH 2 group. The value at the 2332 cm −1 wavelength corresponds to C-N stretching, and the strong intense peak value at 1634 cm −1 may be due to the presence of -C=C stretching. The peaks at 1173 cm −1 were due to C-O bonding, as shown in Figure 5A. The spectrum of porous graphene nanosheets is represented by the peaks at 809 cm −1 , 1636 cm −1 , 2344 cm −1 and 3309 cm −1 , resulting from C-H bonding, C=C conjugation, C-N band and N-H extending vibrations. The lack of bands corresponding to the oxygen-functional groups in the porous graphene oxide nanosheet spectrum further confirms its formation ( Figure 5B). The bare PG electrode showed peaks at 1530 cm   The XRD study revealed a sharp diffraction peak at 2θ = 25°, which indicated that the porous graphene nanosheets existed individually with a highly disordered nature ( Figure  4).

Fourier-Transform Infrared (FTIR) Study
The FTIR analysis of PtNPs showed a broad band at 3341 cm −1 , which was owed to the N-H enlarging vibration of the -NH 2 group . The value at the 2332 cm −1 wavelength corresponds to C-N stretching, and the strong intense peak value at 1634 cm −1 may be due to the presence of -C=C stretching. The peaks at 1173 cm −1 were due to C-O bonding, as shown in Figure 5A

Scanning Electron Microscope (SEM) Study
The bare electrode studied by SEM shows no deposition ( Figure 6A) onto PGE, whereas the AChENPs/ChONPs/GONS/PtNP-modified PGE depicts the deposition of nanoparticles, as it confirmed the co-immobilization of AChENPs and ChONPs ( Figure 6B). Some earlier studies reported the co-immobilization of ENPs onto the PGE [33].

Fourier-Transform Infrared (FTIR) Study
The FTIR analysis of PtNPs showed a broad band at 3341 cm −1 , which was owed to the N-H enlarging vibration of the -NH 2 group . The value at the 2332 cm −1 wavelength corresponds to C-N stretching, and the strong intense peak value at 1634 cm −1 may be due to the presence of -C=C stretching. The peaks at 1173 cm −1 were due to C-O bonding, as shown in Figure 5A

Optimization of Acetylcholine Biosensor
The present acetylcholine biosensor showed an optimum current at pH 7.5 ( Figure  7A) and an incubation temperature of 35 °C ( Figure 7B). All these optimization studies were carried out in 0.1 M sodium PB in the pH range of 5.0-10. The current response de-

Optimization of Acetylcholine Biosensor
The present acetylcholine biosensor showed an optimum current at pH 7.5 ( Figure 7A) and an incubation temperature of 35 • C ( Figure 7B). All these optimization studies were carried out in 0.1 M sodium PB in the pH range of 5.0-10. The current response decreases at various pH and temperatures due to the slight shifting of redox peaks. Therefore, acetylcholine determination was performed in 0.1 M sodium phosphate buffer (pH 7.5; tempera-ture, 35 • C; and maximum response potential, +0.1 V). The biosensor showed an optimum response within 4 s, which is similar to biosensors based on AChE-ChO/GrNP/PtNPs/ITOcoated glass plates [16]. The optimum pH of the present biosensor was similar to that of earlier research based on AChE-ChO/AuNPs-GO/indium tin oxide (ITO)-coated glass plates [15]; closer to the optimum pH 7.4 of AChE-ChO/metallic organic framework (MOF) and AChE-ChO/PtNPs/AuE-based biosensors [14]; higher than biosensors based on AChE-ChO/Fe 2 O 3 NPs/rGO/PEDOT/fluorine-doped tin oxide (FTO) electrodes [17], AChE-ChO/MWCNT-manganese oxide MnO 2 /rGO/gold electrodes (AuEs) [13] and AChE-ChO/CDs-APTES/CPEs [19]; but lower than AChE-ChO/quantum dots (QDs)/reduced graphene oxide (rGO)/GCE biosensors [10] and biofunctional AMs-AChE-ChO/graphenegold nanoparticles-chitosan (GR-AuNPs-CS)/GCE [9] and AChE-ChO/polypyrrole-polyvinyl sulfonate (PPy-PVS)/platinum (Pt) electrodes [12]. The optimum temperature of the present nanosensor (35 • C) is similar to biosensors based on AChE-ChO/GrNPs/PtNPs/ITOcoated glass plates [16]. The performance of the present biosensor increased with an increase in incubation temperature, obtaining the most favorable value of 35 • C, and then further declined with a decline in temperature. The present AChENPs/ChONPs/GNs/PtNPmodified pencil graphite electrode indicated a linear increase in the current response with the rise of the acetylcholine concentration ( Figure 7C).

Study of Scan Rate
The biosensor (AChENPs/ChONPs/GONS/PtNPs/PGE) showed that with varying scan rates (20, 40, 60, 80, and 100 mV s -1 ), the current response also increased accordingly, which also confirms the stability in the performance of the present biosensor ( Figure 8).

Study of Scan Rate
The biosensor (AChENPs/ChONPs/GONS/PtNPs/PGE) showed that with varying scan rates (20,40,60,80, and 100 mV s -1 ), the current response also increased accordingly, which also confirms the stability in the performance of the present biosensor ( Figure 8).

Analytical Recovery
The percent analytical recoveries of added acetylcholine in serum (5.0 and 10 µM) were found to be 97.6 ± 0.7 and 96.5 ± 0.3 for the present biosensor, showing the accuracy of this biosensor.

Reproducibility
The coefficients of variation (within and between batches) obtained were 2.8% and 3.25%, correspondingly, indicating the reliability of the present biosensor. These results also revealed the improved analytic performance of the present biosensor in terms of better repeatability and reproducibility, which can be attributed to the excellent immobilization of AChENPs/ChONPs onto the GONS/PtNPs/PG electrode.

Analytical Recovery
The percent analytical recoveries of added acetylcholine in serum (5.0 and 10 were found to be 97.6 ± 0.7 and 96.5 ± 0.3 for the present biosensor, showing the acc of this biosensor.

Reproducibility
The coefficients of variation (within and between batches) obtained were 2.8% 3.25%, correspondingly, indicating the reliability of the present biosensor. These r also revealed the improved analytic performance of the present biosensor in terms o ter repeatability and reproducibility, which can be attributed to the excellent immob tion of AChENPs/ChONPs onto the GONS/PtNPs/PG electrode.

Application of Acetylcholine Biosensor
The present biosensor measured the level of the acetylcholine (ACh) concentrat the serum of healthy individuals (n = 20), ranging from 9.0 to 12.0 nM within the n

Correlation of Acetylcholine Biosensor
The levels of ACh in serum as determined by the present biosensor were compared with those by the standard colorimetric method. The calculated correlation coefficient (R 2 = 0.989) showed a good correlation between the existing sensor with earlier methods, as shown in Figure 9.

Interferents
The interferents study was performed amperometrically using glucose, ascorbic acid, uric acid, urea, sodium chloride, potassium chloride and L-cysteine at their physiological concentrations in 25 mL of PBS (pH 7.5). In all assays, the concentration of ACh (0.1 mM) was kept constant. The value of the current did not particularly change in the presence of these interferents. Therefore, it was concluded that the interferants impart minimal impact on the analysis of ACh by the present biosensor ( Figure 10).

Interferents
The interferents study was performed amperometrically using glucose, ascorbic acid, uric acid, urea, sodium chloride, potassium chloride and L-cysteine at their physiological concentrations in 25 mL of PBS (pH 7.5). In all assays, the concentration of ACh (0.1 mM) was kept constant. The value of the current did not particularly change in the presence of these interferents. Therefore, it was concluded that the interferants impart minimal impact on the analysis of ACh by the present biosensor ( Figure 10).

Storage Stability and Reusability of AChENPs/ChONPs/GONS/PtNPs/PGE
The present nanosensor investigated the response of the acetylcholine concentration in 0.1 M PBS for a time period of 6 months. During this time, the original performance of the nanosensor decreased by 35% upon continuous use 250 times ( Figure 11). Therefore, the storage stability of the present biosensor was higher than earlier biosensors based on AChE-ChO/GrNP/PtNPs/ITO (4 months) [15] AChE-ChO/MWCNT-MnO2/rGO/Au (3 months) [13] and AChE-ChO/ePAMAM-Sal/CPE (1 month) [19]. Various analytical parameters of the present ACh biosensor were compared with those of earlier biosensors and are summarized in Table 2.

Storage Stability and Reusability of AChENPs/ChONPs/GONS/PtNPs/PGE
The present nanosensor investigated the response of the acetylcholine concentration in 0.1 M PBS for a time period of 6 months. During this time, the original performance of the nanosensor decreased by 35% upon continuous use 250 times ( Figure 11). Therefore, the storage stability of the present biosensor was higher than earlier biosensors based on AChE-ChO/GrNP/PtNPs/ITO (4 months) [15] AChE-ChO/MWCNT-MnO 2 /rGO/Au (3 months) [13] and AChE-ChO/ePAMAM-Sal/CPE (1 month) [19]. Various analytical parameters of the present ACh biosensor were compared with those of earlier biosensors and are summarized in Table 2.

Storage Stability and Reusability of AChENPs/ChONPs/GONS/PtNPs/PGE
The present nanosensor investigated the response of the acetylcholine concentration in 0.1 M PBS for a time period of 6 months. During this time, the original performance of the nanosensor decreased by 35% upon continuous use 250 times ( Figure 11). Therefore, the storage stability of the present biosensor was higher than earlier biosensors based on AChE-ChO/GrNP/PtNPs/ITO (4 months) [15] AChE-ChO/MWCNT-MnO2/rGO/Au (3 months) [13] and AChE-ChO/ePAMAM-Sal/CPE (1 month) [19]. Various analytical parameters of the present ACh biosensor were compared with those of earlier biosensors and are summarized in Table 2.

Conclusions
An enhanced biosensor was constructed for the determination of acetylcholine in the serum samples of healthy patients and Alzheimer's patients. The present ENP-based biosensor demonstrated better analytical performance, such as a lower detection limit (0.001 µM), shorter reaction time (3 s), good linearity (0.001-200 µM), longer storage stability (6 months) and better reproducibility, compared to earlier reports. Before this, an ENP-based biosensor for acetylcholine detection had not been prepared. Hence, this nanocomposite (GONS/PtNPs/PGE) also improved the performance of the present biosensor compared to the direct immobilization of native enzymes on the electrode surface. Therefore, it can also be used for the improvement of other biosensors.