Electroanalytical Overview: The Determination of Levodopa (L-DOPA)

L-DOPA (levodopa) is a therapeutic agent which is the most effective medication for treating Parkinson’s disease, but it needs dose optimization, and therefore its analytical determination is required. Laboratory analytical instruments can be routinely used to measure L-DOPA but are not always available in clinical settings and traditional research laboratories, and they also have slow result delivery times and high costs. The use of electroanalytical sensing overcomes these problems providing a highly sensitivity, low-cost, and readily portable solution. Consequently, we overview the electroanalytical determination of L-DOPA reported throughout the literature summarizing the endeavors toward sensing L-DOPA, and we offer insights into future research opportunities.


■ INTRODUCTION: L-DOPA
L-DOPA is a naturally occurring isomer of the amino acid 3,4dihydroxyphenylalanine which was first isolated in 1913 from a seedling of Vicia faba; Hornykiewicz provides a thorough historical review on L-DOPA. 1 L-DOPA is a therapeutic agent, and the precursor to dopamine, and clinicians use L-DOPA as a dopamine replacement agent for the treatment of Parkinson's disease and dopamine-responsive dystonia. 2 Parkinson's disease is reported to be the second most common neurodegenerative disorder after Alzheimer's disease. It is reported that more than 10 million people globally are living with Parkinson's disease and more than 1.2 million are within Europe; this is forecast to double by 2030. 3 L-DOPA is administered with carbidopa for the treatment of Parkinson's disease where carbidopa prevents the conversion of L-DOPA into dopamine outside the brain, permitting more L-DOPA to reach the brain. 4 Owing to the short half-life of L-DOPA, ∼90 min, this provides a narrow therapeutic window which can contribute to patients experiencing nonmotor fluctuations and motor symptoms. 5,6 L-DOPA needs to be sustained at the therapeutic level to avoid low and high doses to circumvent Parkinsonism, anxiety and orthostatic hypotension, dyskinesias, and psychosis. 5 Overall, there is a clinical need to measure L-DOPA.
Traditionally analytical based laboratory equipment have reported including high-performance liquid chromatography, 7 gas chromatography/mass spectrometry, 8 and liquid chromatography−electrospray ionization to name a few. 9 Although such methods are the most sensitive, akin the "gold standard", they usually require small sample volume and are not always available in clinical settings, treatment environments, and traditional research laboratories. 7 Another issue is that such laboratory methods are not a viable methodology for the timely adjustment of L-DOPA dosage due to their slow delivery of the results and high costs.
An alternative approach is the use of electrochemical based sensing strategies which offer rapid, robust, portable electroanalytical solutions. 10 Electroanalytical approaches exhibit high selectivity and sensitivity when careful attention is given to the electrode materials, sizes, their modification, and the chosen electrochemical technique toward the target analyte. 11 In this review, we have overviewed the recent advances in the electroanalytical sensing of L-DOPA, and future challenges and opportunities are emphasized. ■ ELECTROANALYTICAL APPROACHES Table 1 presents an overview of the electroanalytical detection of L-DOPA; we will consider key highlights below. Blandini et al. 12 applied the use of high-performance liquid chromatog- raphy (HPLC) using electrochemical (coulometric) detection for the quantification of L-DOPA and its metabolite 3-Omethyldopa (3-OMD) within plasma. The authors reported that the limit of detection (LOD) for L-DOPA and 3-OMD was 2 and 6 ng/109 platelets, respectively; this was extended to a population of patients with Parkinson's disease under treatment with L-DOPA. 12 Rizzo et al. 13 reported the extension of this approach which they applied to the measurement of the total and the nonprotein-bound fraction of L-DOPA in plasma which exhibited sample runs of less than 5 min. Galal and co-workers 14 reported sensing of norepinephrine, L-DOPA, epinephrine, and dopamine using HPLC with amperometric detection. The authors reported that the conducting polymer poly(3-methylthiophene) gave detection limits as low as 10 −8 to 10 −9 M which were superior to those using platinum or glassy carbon electrodes, 10 −6 to 10 −8 M. 14 This was attributed to the intrinsic catalytic property of the polymer electrode surface toward the redox behavior of the compounds studied. 14 Dutton et al. 15 employed ion-pair reverse-phase HPLC with an electrochemical detector for the quantification of L-DOPA within urine and plasma. This approach was applied to therapeutic monitoring of elderly patients with established Parkinson's disease being treated with L-DOPA. This experimental setup has been applied using HPLC−amperometric detection using a carbon fiber (14 μm diameter) microelectrode flow cell. 16 Upon inspection of Table 1, we can see that various researchers have explored bare (solid) electrodes for the sensing of L-DOPA such as boron-doped diamond, 17 which was applied in extracts from the seeds of velvet bean (Mucuna prurita Hook or Mucuna pruriens), carbon discs, which were evaluated in the presence of benserazide and (R,S)-2-amino-3hydroxypropanohydrazide, 18 or carbon paste electrodes. 19 People have adapted their electroanalytical approach where the majority have explored modified electrodes, 20 such as modification with carbon nanotubes (multiwalled/singlewalled). 21−41 This approach is due to carbon nanotubes (CNTs) arising as a novel nanomaterial following their discovery by Ijima, 42 where their major role in electrocatalysis underpinned many different applications in sensors. For example, Yan and coworkers 28 explored a single-walled CNT modified glassy carbon (GC) electrode for L-DOPA detection, and they reported that the "modified electrode exhibited good promotion of the electrochemical reaction of L-DOPA and greatly increased the standard heterogeneous rate constant". 28 The authors demonstrated that the sensing of L-DOPA was possible over the range of 0.5 to 20 μM with a LOD reported to be 0.3 μM. That said, there is no evidence of CNTs being electrocatalytic which is attributed to a decrease in the peak-topeak separation at the CNT modified electrode over that observed at the bare/supporting electrode. It is noted that such comparisons are valid if both the bare and modified electrodes have similar mass transport regimes since the peak potential reflects a point of balance between the electrode kinetics and the rate of mass transport; 43 Compton et al. provide a thorough overview of the use of CNTs as "electrocatalysts" which is a must read. 43 Researchers have changed their approach to the use of graphene (reduced graphene oxide) modified electrodes for the sensing of L-DOPA. 44−50 Graphene is a 2D nanoscale single-atom-thick, sp 2 -bonded material which was rediscovered in 2004 by Novoselov and Geim 51 and, of course, has attracted extensive attention from electrochemists. For example, Arvand and Ghodsi 52 reported the development of a graphene modified GC electrode toward the sensing of L-DOPA which gave rise to a linear range of 0.04 to 79 μM and a LOD of 22 nM. This was successfully applied to sensing within a mouse brain extract and a pharmaceutical. The reason for the use of graphene was that "better performance of graphene/GC electrode may be due to the nanometer dimensions of the graphene, the electronic structure, and the topological defects present on the graphene surfaces". 52 Indeed, if correctly fabricated, that is not being pristine but having edge plane defect sites, this is highly likely. 53,54 The electrochemical sensing of L-DOPA is well established; for example, the electrochemical oxidation of L-DOPA is exemplified within Figure 1A which shows the electrochemical behavior recorded on a graphene nanosheet modified glassy carbon, depicting the first and 20th voltammetric scans which provide insights into the mechanism of L-DOPA. 48 In the case of the first scan, an electrochemical peak is observed E a1 = +0.34 V, which is the oxidation of L-DOPA to an open-chained quinone, as shown within Figure 1A. In the case of the 20th scan, a new redox couple becomes evident (E a2 = +0.09 V, E c2 = −0.12 V) which is indicative of a slow chemical follow up reaction after the oxidation of L-DOPA; this has been seen with the use of bare glassy carbon electrodes. 55 Note that at a neutral pH, sufficient unprotonated quinones are available to favor the cyclization reaction while the second redox couple corresponds to the oxidation of the cyclized product, cyclodopa to dopachrome ( Figure 1B). 48 Other work utilizes a composite modified electrode, common throughout the literature in electroanalytical sensing, 56 comprising many items to help promote the sensing of L-DOPA. For example, Đurđićand co-workers 36 demonstrated the development of a sensor based on carboxylated single-walled carbon nanotubes (SWCNT-COOH) which have been modified with SiO 2 coated Nd 2 O 3 nanoparticles for the sensing of L-DOPA.
Note that SWCNTs have unique physical and chemical properties, such as electrical and thermal conductivity, mechanical strength, and flexibility, and they are ultralight weight; some of these aspects make them useful as the supporting material for metal nanoparticles, but they have suffered in the past due to metallic impurities. 57 The authors carboxylate the SWCNTs by using a mixture of sulfuric and nitric acids which were subjected to 2 h of ultrasonication and then fluxed for 12 h. Following this, the suspension was cooled and centrifuged and washed with distilled water and then dried for 6 h in a vacuum oven. In terms of SiO 2 coated Nd 2 O 3 nanoparticles, the procedure utilized cyclohexane solution containing polyoxyethylene (10) tridecyl ether, which was used to form reverse micelles (solution 1). Another solution containing a suspension of Nd 2 O 3 nanopowder in ultrapure water (solution 2) alongside tetraethyl orthosilicate was diluted in NH 3(aq) (solution 3). Solution 2 was added into a preheated to 50°C solution 1, while solution 3 was added to this mixture, after which the formed suspension was exposed to hydrolysis (50°C, 1 h). 36 Subsequently, the precipitate, comprising SiO 2 coated Nd 2 O 3 nanoparticles, was centrifuged, washed three times with propanol, and dried (80°C, 24 h). Next, the calcination of the dried precipitate was performed within an airflow dryer at 400°C for 5 h. The final nanocomposite was prepared by placing SWCNT-COOH and Nd 2 O 3 −SiO 2 nanoparticles in DMF. After 6 h of ultrasonication, DMF was evaporated, and the nanocomposite was dried at 70°C within a vacuum oven. Figure 2A shows a transmission electron microscopy (TEM) image taken of the Nd 2 O 3 , and a scanning electron microscopy (SEM) image of the SWCNT-COOH@Nd 2 O 3 −SiO 2 is shown in Figure 2B. The former demonstrates that the Nd 2 O 3 nanoparticles are spherically shaped and well-dispersed with a reported average particle size of D TEM = 94 ± 20 nm. Figure  2C displays the X-ray powder diffraction (XRD) spectra for the SWCNT-COOH@Nd 2 O 3 −SiO 2 , which shows that the Nd 2 O 3 (black line) is a combination of Nd 2 O 3 and the Nd(OH) 3 reflections, while the red line reveals a broad peak at 22.5°2θ which can be allocated to amorphous nanosilica. The other two patterns, green and blue lines, are a combination of the previous, which could be assigned to the amorphous nature of SWCNT-COOH@Nd 2 O 3 −SiO 2 . As shown within Figure 2D, the sensing performance of the SWCNT-COOH@Nd 2 O 3 − SiO 2 toward L-DOPA exhibited a linear range from 2 to 52 μM with a LOD reported to be 0.7 μM. The sensor was evaluated within a pharmaceutical product, where the results agreed well with the declared dose, and in human urine samples, which exhibited good recoveries over the range of 94−102%.
Naghian and co-workers 45 reported a metal−organic framework (MOF) that was mixed with reduced graphene oxide to produce an electrochemical sensor that has a high surface area, coupled with improved electrode transfer kinetics. The sensor was able to show a linear range of 0.1 to 85 μM with a LOD of 0.02 μM. The sensors exhibited no interference from such competitive analytes when applied to measuring L-DOPA in spiked human urine and within a pharmaceutical tablet showing recovery rates of 94.0−102.0%, with the RSD value for each sample being less than 3.0%.
Renganathan and co-workers 47 reported the novel fabrication of nitrogen-doped graphene supported with nickel oxide (N-GE/NiO) nanocomposite which was immobilized upon a GC electrode ( Figure 3A). This approach required the fabrication of graphene oxide prepared by the modified Hummers method, where nickel oxide was mixed and kept under continuous stirring for 1 h at a room temperature, after which urea was added as the reducing agent. 47 This mixture was placed into a Teflon-lined autoclave with a stainless-steel shell (180°C for 2 h), after which, the precipitate was washed and then dried overnight. Last, the final composite was achieved by calcination in a flowing argon atmosphere by heating at a rate of 10°C min −1 to 400°C at 2 h. The composite was immobilized upon a glassy carbon electrode. As shown in Figure 3B, the N-GE/NiO exhibited an optimized voltammetric response which was attributed by the authors to the higher electron transfer capacity compared to a bare GC electrode, NiO, and graphene oxide. 47 The electroanalytical response demonstrated a linear range of 0.03 to 386.8 μM with a LOD of 17 nM. The authors were able to show a novel application and measured L-DOPA in sweet potato juice, Another notable approach is that reported by Bergamini and co-workers, who have described the first use of gold screenprinted electrodes (SPEs) for the measurement of L-DOPA. 58 SPEs are highly useful because differing from classic electrode platforms they can be used as single-shot, disposable, reproducible, and ready to use electrodes. On the other hand, classic (solid) electrodes such as glassy carbon, edge plane and basal plane pyrolytic graphite, or highly ordered pyrolytic graphite need to be rigorously polished and cleaned before undertaking every measurement and require the presence of external reference electrode (RE) and counter electrode (CE). SPEs offer a disposable, reproducible, and lowcost electrode which is easily modified for electrochemical sensor platforms. 59,60 Though SPEs have inherent advantages, there are limited reports of their use toward the sensing of L-DOPA. 32,46,58,59,61,62 The authors of ref 58 demonstrated that the sensor was able to measure L-DOPA within 1 to 660 μM with the LOD reported to be 0.99 μM. The method was successfully applied to the determination of L-DOPA in two commercial dosage forms without any pretreatment, which resulted in 104% and 108% recoveries of L-DOPA. Work around this theme reported the first disposable electrochemical biosensor for L-DOPA determination in undiluted serum samples. This approach utilized an outer cross-linked layer containing tyrosinase on the top of a carbon nanotube (CNT) modified SPE/polythionine film. This sensor was able to measure L-DOPA over the range of 0.8 to 22 μM with the LOD reported to be 2.5 μM; the use of a reagent layer enhances sensitivity and stability, decreasing the detection limit of L-DOPA in undiluted serum samples. 32 Shoja and co-workers 22 have reported a biosensor for the measurement of L-DOPA using a horseradish peroxidase/ organic nucleophilic-functionalized carbon nanotube composite; see Figure 4A. This biosensor was made via physically immobilizing horseradish peroxidase (HRP) as a catalyst through a sol−gel approach upon the surface of a GC electrode, which was already modified with p-phenylenediamine (pPDA) as an organic nucleophile chemically bonded with functionalized (carboxylic groups) multiwalled carbon nanotubes (MWCNTs). As shown in Figure 4B, typical cyclic voltammograms are recorded in the presence of L-DOPA where there is no redox peak at the bare GC electrode but a smaller, broader redox peak around +0.19 V for anodic peak and −0.09 V for cathodic peak was observed using the MWCNT-pPDA/GCE. Also shown is the response of sol−gel/ HRP/MWCNT-pPDA/GCE, which exhibits an anodic peak at +0.19 V and a cathodic peak at +0.08 V. The authors 22 reported that the high electrocatalytic activity for oxidation of L-DOPA is related to the simultaneous presence of HRP as electrocatalyst mediator and MWCNT-pPDA as a nucleophilic composite, which is coupled with a high surface area and good conductivity from the presence of MWCNTs. Figure 4C shows the electrochemical mechanism of the oxidation of L-DOPA, but in the presence of the pPDA, the scheme shows that in the first step, L-DOPA is oxidized to L-DOPA quinone by HRP which then undergoes an 1,4-Michael addition with pPDA as a nucleophile leading to a respective aromatic amino acid in the second step. This aromatic amino acid, which is formed in the second step, is an electroactive intermediate and is subsequently oxidized to a respective quinone by HRP in the third step. 22 The authors were able to show that their composite achieved the determination of L-DOPA from 0.1 μM to 1.9 μM, with a low LOD of 40 nM, but only in model solutions; despite this the authors noted that their sensor has advantages such as rapid response, high stability, and reproducibility.
Pinho et al. 49 reported the use of a 3D gold nanoelectrode ensemble (GNEE) in a flow-injection analysis system for the sensing of L-DOPA. A schematic representation of how the biosensor is prepared is shown in Figure 5A, which comprises of 4 steps involving the electroless gold deposition in polycarbonate membranes, followed by partial etching exposing gold nanoarrays. Figure 5B shows an SEM image of the 3D GNEE which shows that the gold nanowires have an average diameter of 50 nm and a length of 180 (±20) nm which also shows the absence of voids on surface, which suggest that this method produces 3D GNEEs with protruding gold wires. 49 The 3D GNEE was explored toward the sensing of L-DOPA using a flow-injection analysis system, as shown in Figure 5C where a linear current response for L-DOPA between 10 nM and 10 mM was achieved. The LOD was found to be 1 nM with a resultant % RSD of 7.23% (n = 5). No significant interference from ascorbic acid, glucose, and urea on the measurement L-DOPA was observed. This approach was explored with human urine and had a recovery of 96%.
Another approach is the design and development of Molecular Imprinted Polymers (MIPs). 38,50,63 MIPs are synthetic receptors that can form high affinity binding sites corresponding to the specific analyte of interest 64 and can utilize the shape, size, and functionality to produce sensitive and selective recognition of target analytes. 65 Despite the advantages, there are few literature reports utilizing MIPs. Lin and co-workers have developed a selective sensor utilizing MIPs, based on a composite of graphene and chitosan. 50 Figure 6A shows how the graphene−MIP sensor was fabricated. The electrodeposition on a GC was achieved via holding the potential of −1.1 V for 150 s within a dispersion containing graphene, chitosan, and L-DOPA. The graphene− MIP/GCE was obtained after removing the template molecule from the composite by applying a potential of +0.6 V for 20 min within a new solution (0.1 M KCl solution containing 100 μL ethanol). A nonimprinted polymer sensor was prepared in the same way except that the template molecule was absent in the electrodeposition step. The sensor was evaluated for the sensing of L-DOPA which, as shown in Figure 6B, shows a linear response from 0.4 μM to 100 μM and a reported LOD of 12 nM. Figure 6C shows the specific recognition toward L-DOPA using the graphene−MIP/GCE sensor in the presence of D-tyrosine, L-tyrosine, L-tryptophan, and dopamine. The current in the presence of L-DOPA is the highest of all and changes significantly, which indicates that the sensor had a good specific recognition toward L-DOPA. The sensor was last evaluated for the measurement of L-DOPA within a pharmaceutical tablet and human blood serum. The authors conclude that their graphene−MIP/GC sensor exhibited a high sensitivity, low detection limit, good selectivity, and stability. In another example, following on from Lin and coworkers, 50 Sooraj et al. 38 developed copper nanoparticles (CuNPs) grafted with a MIP on MWCNTs. This was supported upon a GC electrode and was specific and selective giving the fabricated sensor a response to L-DOPA and not structurally related compounds such as dopamine, uric acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid. This sensor was able to demonstrate a linear range of 0.01−1 μM and a LOD of 7.2 nM. This was extended to the measurement of L-DOPA within noninfected and infected human urine and pharmaceutical samples with a recovery between 98.3 and 102.4%.
As mentioned above, L-DOPA is administered with carbidopa for the treatment of Parkinson's disease which prevents the conversion of L-DOPA into dopamine outside the brain, permitting more L-DOPA to reach the brain. 4 L-DOPA is almost always given in combination with the drug carbidopa, which reduces or prevents the nausea that L-DOPA alone can cause. Reports of the simultaneous determination of L-DOPA in the presence of carbidopa are rather limited, 48,66,67 despite   the need for both in pharmaceuticals and human samples; this is an area that needs progressing.
Wang and co-workers 5 reported for the first time a fantastic microneedle sensing platform for continuous minimally invasive electrochemical sensing of L-DOPA. The motivation was an urgent need for a reliable sensing device to provide timely individualized feedback on the proper L-DOPA dosing regimen in a decentralized and rapid fashion. 5 Figure 7A shows an overview of their direct (nonenzymatic) L-DOPA sensing which was carried out using square-wave voltammetry and a microneedle, while a tyrosinase (TYR)-based biocatalytic detection was performed at a neighboring enzyme-paste microneedle electrode using chronoamperometric measurements of the corresponding dopaquinone product with the third microneedle completing the circuit as the reference electrode. The three-working electrode microneedle array was fabricated using carbon paste (CP) that was packed into the hollow microneedles. Figure 7B shows the square-wave voltammetry and chronoamperometry experiments that were performed simultaneously on three CP-modified microneedle electrodes ( Figure 7B(A,B)). The square-wave voltammetry and the chronoamperometric response of both microneedle sensors were recorded over the range of 50 to 250 L-DOPA to artificial interstitial fluid. As shown in Figure 7B(C,E), the sensing of L-DOPA is achieved for artificial interstitial fluid through mouse skin, and the response of the sensor is shown for skin-mimicking phantom gel ( Figure 7B(F,H)). Future work needs to focus on antibiofouling protective coatings and clinical testing and validation in Parkinson's disease patients.
More recently, additively manufactured electrodes (AMEs) have been utilized for the production of electroanalytical platforms. 68 In this way, AMEs of varying shapes and sizes 69 can be 3D-printed and utilized in various systems, even with the electrodes printed within the cell walls. 68,70 Whittingham et al. 71 are the only report to date of an AME for the detection of L-DOPA, using it to show the proficiency of their 3D-printed rotating disc electrode and experimental setup. They report the use of additive manufacturing (AM) to produce both the rotator housing and disc electrode for their setup and compare this to a commercially purchased system with a GC rotating electrode. They show that L-DOPA could be detected with a LOD of 0.23 μM using an experimental set up of less than 2% and electrode of less than 0.05% material cost of the comparative commercial options. It should be noted that to produce the best AM electrochemical platforms, the connection length of AMEs should be kept to as short a distance as possible. 72 Currently, these AMEs must be used as single-shot electrodes, similar to SPEs, because of solution ingress into the electrodes, 73 among other issues. However, due to the flexibility, low-cost, and rapid prototyping capabilities of AM (in addition to the recent reports of the use of recycled conductive feedstocks 74 ), we expect many further reports in this field over the coming years.

■ SUMMARY AND OUTLOOK
In our review, we have overviewed the electroanalytical sensing of L-DOPA, which provides credible approaches with high sensitivity and selectivity over common analytes that have the potential to be low-cost and portable. This is crucial due to the desire for rapid monitoring of L-DOPA dosages for the optimal treatment of Parkinson's disease. One area that needs progressing is the measurement of L-DOPA and carbidopa within pharmaceuticals and human samples since these are both given to patients since it prevents the nausea that L-DOPA alone can cause. The measurement of L-DOPA in the literature is rather limited to pharmaceuticals and blood (serum) and urine, and only a handful have measured L-DOPA within food. Note that it is reported that the global demand for L-DOPA has an annual market value of USD 2.64 billion in 2020. 75 For example, it is well-known that relatively high L-DOPA concentrations occur in natural sources, such as Mucuna and fava beans, which have been measured using LC-MS, 76 5 ] 6+ ; SWV, square-wave voltammetry; SPE, screen-printed electrodes; WO 3 NP, tungsten oxide nanoparticle; ZnONF, zinc oxide nanofiber; ZnO NR, zinc oxide nanorod; 3D HGB, 3-dimensional hollow graphene ball; 35DT, 3,5-diamino-1,2,4-triazole; 3,4′-AAZ, 3-(4′-amino-3′hydroxy-biphenyl-4-yl)-acrylic acid