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Electrochemical detection of phosphate ion in body fluids with a magnesium phosphate modified electrode

An electrochemical sensor for phosphate detection in body fluids is developed based on the hydration transition of magnesium hydrogen phosphate (newberyite, MgHPO 4 ·3H 2 O). The sensor was fabricated through incubation of a multi-walled carbon nanotubes/Nafion (MWCNTs/Nafion) modified glassy carbon electrode (GCE) in magnesium phosphate solution, where MgHPO 4 ·3H 2 O was self-assembled on the electrode surface (denoted as MgPs/MWCNTs/Nafion). An electrooxidation peak at 1.0 V vs Ag/AgCl was observed when the as-prepared electrode was subjected to a differential pulse voltammetry (DPV) scan in presence of phosphate in acetate solution. When the DPV scan was performed in 0.4-1.3 V vs Ag/AgCl, a linear relationship was observed between the peak height and the phosphate concentration in the range from 0.01 μM to 25 μM in the presence of 0.1 mM Mg 2+ in the acetate solution with a limit of detection 32 nM. And the sensor was successfully applied for the phosphate detection in the human urine and saliva with recoveries at 94.7%-104.4% and 96%-103.3% respectively.


Introduction
The electrochemical detection of phosphate ions in body fluids attracted lots of recent interests due to its real time, in situ detection, portable instrument and low cost of analysis. 1,2 The electrochemical inertness of phosphate ion, however, has imposed a great challenge to direct method of detection, hence many indirect strategies were proposed for phosphate sensing. Ion selective electrodes, 3 enzyme based electrochemical biosensors, 4,5 amperometric sensors [6][7][8] and voltammetry sensors [9][10][11] were developed in recent years. In the last case, some direct strategies for phosphate detection under auxiliary steady atmosphere supply were also developed. For instance, the reaction of phosphate with molybdate was utilized to realize the detection of phosphate. 12 Based on the catalytic hydrogen waves, molybdenum phosphide (MoP) modified electrode was fabricated for the detection of phosphate in blood. These methods have shown the feasibility of phosphate detection with electrochemical sensors.
However, some intrinsic disadvantages were also witnessed. For example, the detections are often influenced by the dissolved oxygen and their limit of detection is still not high enough for body fluidic analysis. 13 In our recent work, an electrochemical sensor for the detection of phosphate in environmental samples was developed based on an intriguing redox behavior observed from calcium phosphate (CaPs) modified electrode. 14 It was understood that phosphate could tune the generation of coordinated water in the calcium phosphates, which is responsible for the appearance of the oxidation peak at relatively low voltage (1.0 V vs Ag/AgCl ). Calcium ion was found to be able to promote the electrochemical oxidation peak, hence can enhance the sensitivity of detection. However, it was also noted that Ca 2+ can easily precipitate with phosphate ion at increased concentration, so the promotion effect of Ca 2+ is restricted to some extent. To overcome such limitation, 4 magnesium ion (Mg 2+ ) is considered in present work. Mg 2+ has greater charge density than that of Ca 2+ ion, 15 hence it could have a stronger enhancement effect to the hydrolysis of the coordinated H2O. This assumption has been evidenced by the fact that Mg 2+ -coordinated H2O has stronger acidity (pKa = 11.4) than that of Ca 2+ -coordinated H2O (pKa = 12.67). 16 The free energy required to deprotonate a coordinated H2O in presence of Mg 2+ ion is only 40% of that required to deprotonate a free H2O. 17 These observations indicate that Mg 2+ ion can promote the ionization of water, i.e., under the modulation of Mg 2+ , the proton of coordinated H2O is prone to be donated to phosphate.
The ionization of the coordinated H2O would facilitate the oxygen release during the subsequent voltage scan. Furthermore, the relatively high solubility of magnesium phosphates (MgPs) should have less likelihood to form precipitation in bulk solution, hence the MgPs production is more localized in the electrode surface. 18-21 Therefore, it is expected that the sensitivity of MgPs based phosphate sensor could be further enhanced in the presence of Mg 2+ .
In our previous work, Nafion and MWCNTs composite was used for the assembly of CaPs on the electrode surface, where singlet oxygen production was evidenced by the accompanied electrochemiluminescence. 22 Nafion has excellent ion exchange property 23,24 and MWCNTs can effectively improve the conductivity of modified electrode, so the composite is often used for electrode modification to expand surface area and to improve the electrochemical performance. 25,26 Therefore, the composite of Nafion and MWCNTs was adopted in present work to modify the glassy carbon electrode (GCE) and the coated layer acted as the substrate for the assembly of MgPs.
The electrochemical sensor, denoted as MgPs/MWCNTs/Nafion, was obtained by sequentially incubating the Nafion/MWCNTs electrode in Mg 2+ and phosphate solution.
The MgPs assembled on the electrode surface were comprehensively studied before and 5 after voltage application. And the oxidation of coordinated H2O originated from hydration of MgPs and the effect of Mg 2+ on the electrochemical oxidation of coordinated H2O were comprehensively investigated. Finally, the as-prepared sensor (MgPs/MWCNTs/Nafion) was validated by successful determination of phosphate contents in urine and saliva samples.
All solutions were prepared with ultra-pure water (18.2 MΩ· cm), which was obtained from Milli-Q Plus water purification system (Millipore, USA).

Characterization
Scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) images were obtained on a SUPRA 55 field emission scanning electron microscope was added in the acetate buffer for phosphate detection and the pH of the solution was adjusted to 9.0 with 0.1 M NaOH. All tests were conducted at room temperature (25°C).

Preparation of MgPs/MWCNTs/Nafion modified electrode
The route for MgPs/MWCNTs/Nafion preparation is shown in Scheme S1. Before the electrode modification, the glassy carbon electrode was subsequently polished with 0.

Determination of the phosphate concentration in urine and saliva
The MgPs/MWCNTs/Nafion electrode was applied to determine the phosphate concentration in human urine and saliva. Before the electrochemical measurement, urine and saliva samples were respectively diluted 4000 times and 1500 times with ultrapure water so that the phosphate concentrations fall in the linear range of calibration. In order to validate the results obtained from the MgPs/MWCNTs/Nafion sensor, the ICP-MS analysis of phosphate was also conducted on the same samples.

Preparation and characterization of MgPs/MWCNTs/Nafion
As described in the experiment section, the MgPs/MWCNTs/Nafion was prepared by subsequently inserting the MWCNTs/Nafion electrode in Mg 2+ and phosphate solution to assemble MgPs on the electrode surface. The morphology and structure of MgPs after 5 days incubation were characterized. The SEM image (Fig. 1a) shows that the MgPs formed on the electrode are uniformly distributed particles, which are similar to the morphology reported for MgHPO4· 3H2O. 27,28  in the assembled products. 31,32 The result agrees with that obtained from the XRD spectra (Fig. 1f), where the (022), (040) planes as well as others planes are also observed in the standard card of newberyite (PDF No. 97-003-1281). The element ratio of Mg and P was measured to be approximately 1.0 (Fig. S1), which also agrees to the chemical stoichiometric ratio of MgHPO4· 3H2O. In conclusion, the newberyite (MgHPO4· 3H2O) was assembled on the surface of MWCNTs/Nafion successfully, and its prominent hydration feature would provide a crucial fundamental for its subsequent electrochemical investigation.

Electrochemical investigation based on the hydration process of MgPs/MWCNTs/Nafion
As a powerful tool to reveal the electrochemical behavior of electrical active material, DPV is widely used in electrochemical studies as well as in phosphate detection. 33,34 For example, the catalytic hydrogen waves were observed when the molybdenum phosphide modified electrode was subjected to a DPV scan, which was applied for the phosphate detection in blood samples. 13 Fig. 2a shows the typical DPV curves obtained from MWCNTs/Nafion electrode (solid line) and MgPs/MWCNTs/Nafion electrode (dashed line) in phosphate buffer solution. As can be seen that only the MgPs modified electrode shows the typical oxidation wave, which is similar with that of CaPs modified electrode 9 reported in our previous work. 14 The transition processes may be described by following equation 1 to equation 4. In the first step, MgPs interact with water to produce coordinated H2O due to the hydration effect of Mg 2+ in MgPs (equation 1). Upon the addition of phosphate in the equilibrium system, the coordinated OHis generated sequentially due to the deprotonation effect of phosphate (equation 2 and equation 3).
The in-situ formed coordinated OHis believed to be more easily oxidized when compared with their free anion counterparts. 35 Hence upon voltage application, the oxygen is released by the oxidation of coordinated OH -(equation 4).

MgPs + H2O
MgPs-H2O MgPs-H2O + PO4 3-HPO4 2-+ MgPs-OH - MgPs-H2O + HPO4 2-H2PO4 -+ MgPs-OH - 2MgPs-OH -+ 2OH --4e → 2MgPs + 2H2O + O2 (4) As described in the introduction part, Mg 2+ should have stronger hydrolysis effect than Ca 2+ due to the smaller ion radius (Table S1). The Mg 2+ exhibit stronger coordinated ability to H2O, and the pKa of corresponding coordinated H2O is about 11.4, which is greater than that of the coordinated H2O in CaPs (pKa = 12.67). Thus compared with CaPs/MWCNTs/Nafion electrode, a more enhanced deprotonation would be proceeded, leading to a pronounced oxidation wave in the MgPs/MWCNTs/Nafion system (Fig. 2b) could form an octahedral complex Mg(H2O)6 2+ with water, the 6 tightly coordinated water molecules composes first layer around the central atom. 36 Then a second layer is formed, which is composed of 12 less firmly associated H2O solvation shell. [38][39][40] can activate the coordination H2O in the inner layer of Mg(H2O)6 2+ , so the first layer of coordination H2O is more prone to deprotonation. 17,40 The generated protons are more likely transferred to the free water near the electrode surface to from hydronium. In the presence of phosphate (HPO4 2and PO4 3-), the excess hydrodium can be rapidly delocalized, thereby promoting the reaction and generating more OHon the MgPs/MWCNTs/Nafion, which is subsequently oxidized at a voltage of 1.0 V vs Ag/AgCl during the voltage scan.

Electrochemical conditions optimization for phosphate determination
To accomplish the phosphate detection based on the hydration process on the MgPs/MWCNTs/Nafion, the crucial parameters that affect the sensitivity was initially investigated. Fig. 3

Electrochemical determination of phosphate with MgPs/MWCNTs/Nafion
A sensitive electrochemical response to phosphate can be obtained with the MgPs/MWCNTs/Nafion electrode in pH 9 acetate solution containing 0.1 mM Mg 2+ . Fig. 4 shows that a good linear relationship between the peak currents and the concentrations of phosphate from 0.01 μM to 25 μM was observed, the calibration curve can be described by the regression equation, y = 13.42-1.331x (R 2 = 0.995) (Fig. 4b).
The limit of detection is estimated to be 32 nM based on the conventional 3σ criterion.
It is well known that the concentration of phosphate is in the range of 15-40 mM in human urine, and in the range of 5-14 mM in saliva, 41,42 hence the MgPs/MWCNTs/Nafion electrode could be a promising sensor for the determination of phosphate concentration in urine and saliva. In Table S2, compared with other phosphate detection methods reported in the literature, our electrode not only exhibits a lower detection limit, but also a wider detection range. It can be seen that MgPs/MWCNTs/Nafion exhibits good sensitivity with the help of Mg 2+ . We have also studied phosphate solutions in the concentration range 0.01 μM to 25 μM in the 1.0 mM acetate solution 0.1 mM Mg 2+ , and the addition phosphates should not result in the pH shift ( Fig S2).
Selectivity is also one important factor for the practical application of a sensor. Some potential interferences in body fluid, including SO4 2-, NO3 -, CO3 2-, Cl -, Na + , K + and Ca 2+ have been tested. As shown in Fig. 5 lower than that of phosphate in urea and saliva, their interferences would be negligible.
The above results prove that the MgPs/MWCNTs/Nafion electrode has good selectivity for the detection of phosphate.

The analytical feature of MgPs/MWCNTs/Nafion in biological fluids
The phosphate in body fluid plays an essential role, hence the detection of its concentration in urine and saliva is significant for health survey and clinical diagnosis. 1,43,44  human saliva and urine proves that the as-prepared phosphate sensor also has excellent sensitivity, good selectivity and stability. This work not only constructed a feasible electrochemical platform for the phosphate detection in biological system, but also provides new insight into the transition process of MgPs in the solid/water interface.