heavy metal detox

Amino Acid based SWASV

This post describes the development of an alanine coated glassy carbon electrode that can detects heavy metals Cu, Zn, Hg, and Pb, Kokab et al 2019. Heavy metals in a fluid are coated on this electrode in cathode mode. Heavy metal cations in detox solutions will theoretically electroplate on the cathode. Then the cathode becomes the anode. Square waves are used to strip the anode of plated metals into their cation form.Could this technique One Day be used to detect heavy metals in body fluids after detox? treatments?

Kokab T, Shah A, Iftikhar FJ, Nisar J, Akhter MS, Khan SB. Amino Acid-Fabricated Glassy Carbon Electrode for Efficient Simultaneous Sensing of Zinc(II), Cadmium(II), Copper(II), and Mercury(II) Ions. ACS Omega. 2019 Dec 9;4(26):22057-22068. PMC free article

Making the electrode

For electrode fabrication, a bare glassy carbon electrode

  1. was gently rub on 6 and 1 μm alumina slurries having a nylon buffing pad repeatedly to achieve a smooth shiny surface.
  2. Then, the polished electrode was thoroughly washed with doubly distilled water.
  3. To obtain reproducible surface conditions before modification, physical pretreatment was followed by electrochemical pretreatment by passing the electrode surface through several polarization cycles of −1.4 to +0.9 V in buffer media at 100 mVs–1 until reproducible cyclic voltammogram was accomplished.
  4. Then, the clean activated electrode surface was covalently modified via electrochemical-aided grafting of the known concentration of amino acids on the carbon surface.
  5. To obtain modified electrode having a stable monolayer with a broad potential range, bare GCE was scanned four times between 0 to +1.4 V with a scan rate of 10 mV/s by a cyclic voltammetric technique in the solution of the selected amino acid in acetonitrile (ACN) having 0.1 M NBu4BF4 under inert atmosphere.
  6. At a sufficiently positive potential, controlled electrolysis of amino acid solution causes electrooxidation of its amino group and produces a corresponding cation radical that forms carbon–nitrogen covalent linkage at the electrode surface.
  7. Thus, individual amino acids are grafted onto GCE through its N-terminus and allow the binding of analytes from their carboxylic acid terminus during the complexation process as shown in the modification
  8. The modified electrode was cautiously cleaned with ethanol and doubly distilled water to remove any physiosorbed, unreacted, and loosely bound amino acid molecules.
  9. All cyclic voltammograms with AA/GCE as WE in 0.1 M KCl in a potential range between −1.5 to +0.8 V, that is, working potential window for metal ions, revealed no redox peak for amino acids.
  10. The prepared AA/modified electrodes are ready to be used and can be stored in PBS (phosphate buffer) of pH 6.0 at 4 °C.
  11. For simultaneous detection of toxic metal ions, the modified electrode was then subjected to square-wave anodic stripping voltammetry (SWASV), which involves a deposition step where a predefined deposition potential of −1.3 V for a 140 s deposition time is applied to electroplate metal ions on the electrode surface. While in the stripping step, the electro-reduced metal ions are oxidized back into the solution during anodic stripping with a potential scan ranging from −1.3 to 0.8 V.
The scheme. What is nice about this one is that there are no aromatic amino acids prone to oxidation

Figure 1

The Warburg element is a resistance associated with charge transfer. The constant phase element (CPE) is essentially a double layer of charge that functions as an imperfect capacitator. The 3D structures of the four amino acids tested in this system are shown. Charges have been added. The Fe atom is surrounded by cyanide anions that might impede its access to the negatively charged cathode. The carboxyl groups (red) of he amino acids have the potential to displace the CN- counter ions.

A charge transfer cartoon and data

Any time we have a charge surface, we collect a cloud of solutes with the opposite charge. Cations migrate to the cathode, with a negative charge. Our Fe atom (rust color) has to shed its cyanide counter ions (blue and gree with three parallel lines) to approach the surface of the cathode.

Figure 1. Comparative (A) cyclic voltammograms obtained from bare
GCE and alanine, threonine, glutamic acid, and lysine amino acidmodified GCE in a medium containing 5 mM K3[Fe(CN)6] solution and a 0.1 M KCl electrolyte. (B) Nyquist plots using electrochemical impedance spectroscopic data with applied frequency ranges varying from 100 kHz to 0.1 Hz. (Inset) Randles equivalent circuit model forthe system under study showing resistors, capacitor, and Warburg
impedance elements.

Not shown in this cartoon are solution counter ions like Cl, K+, and CN.

Table of circuit parameters

Note that the charge transfer resistance is much lower in the Ala coated glassy carbon electrode (GCE) is much less than in the parent GCE. The resistance coming from the electrodes remain pretty constant regardless of the coating.

electrodesR ct (kΩ)Re (Ω)CPE (μF)Jo (μA/cm2)
bare GCE6.41 ± 0.057201.7 ± 1.745.52 ± 0.544.010.79
Lys/GCE3.34 ± 0.026199.5 ± 1.682.57 ± 0.127.690.81
Glu/GCE2.62 ± 0.025198.2 ± 1.582.53 ± 0.109.800.83
Thr/GCE1.77 ± 0.017186 ± 1.412.51 ± 0.1414.500.86
Ala/GCE0.50 ± 0.007179.8 ± 1.631.43 ± 0.0451.400.90
Table 1. Randles Circuit Parameters Calculated for Bare and Amino Acid-Modified GCEs from Electrochemical Impedance Spectroscopy (EIS). The exchange current density can be calculated by the equation Jo = RT nFRct where R, n, F, T, and Rct are the gas constant, number of electrons involved in the electrode reaction (here n = 1 for [Fe(CN)]3-/4- redox reaction), Faraday’s constant, temperature (here T = 298 K),
and charge-transfer constant, respectively

Figure 2. SWASV obtained from unmodified and alanine, threonine,
glutamic acid, and lysine amino acid-modified GC electrodes for the
detection of 10 μM Zn2+, 7.5 μM Cd2+, 5 μM Cu2+, and 7.5 μM Hg2+
in BRB of pH = 4 as striping solvent, keeping a scan rate of 100 mV/s,
a deposition potential of -1.3 V, and a deposition time of 140 s.

Figure 2 Optimal alanine in electrode coating solution

Kokab at this point had decided that alanine was the the best amino acid for ferrocyanide reduction and the best overall matrix for detection of their four heavy metals of interest. What are the optimal concentrations in electrode coating? Too much could be a barrier for the heavy meal. Too little just does not overcome the counterion impedance.

Figure 3. (A) Alanine concentration effect on the SWASV response of 100 μM Zn2+, 75 μM Cd2+, 50 μM Cu2+, and 75 μM Hg2+ ions in BRB of pH = 4, keeping scan rate = 100 mV/s, deposition time = 5 s,
deposition potential = -1.3 V by electrochemical assisted modification of GC electrode surface with different concentrations of alanine solution. (B) Plot of Ip vs alanine (modifier) concentration. The center panel is a magnification of the Cd2+ peak in panel A.

These data are really nice in that reiterate the importance of the surface concentration in either easing heavy metals to the cathode that becomes the anode…

Figure 4 The best supporting/stripping electrolytes

The”thought” diagram of figure 1 suggests heavy metal ion counter ion may affect how well the heavy metal can approach the cathode (soon to be anode) surface. In a theoretical sort of way, we want the pH to promote the charged state of the alpha carbon carboxylate group on analine. Theoretically, we’d like a loading buffer anion to have less affinity for the heavy metal than the carboxylate group of alanine. Going in the other direction, when the cathode becomes the anode and the goal is to strip it of heavy metals, perhaps we want a buffer that provides the heavy metal a counter ion shell that will facilitate exit from the anode.

Figure 4. (A) Effect of various stripping media (supporting electrolytes) such as BRB (pH = 4), phosphate buffer (pH = 7), acetate buffer (pH = 4.8), 0.1 M HCl, 0.1 M NaOH, 0.1 M KCl, 0.1 M H2SO4, and 0.1 M H3BO3 on the SWASV peak currents of 100 μM Zn2+, 75 μM Cd2+, 50 μM Cu2+, and 75 μM Hg2+ ions using 65 μM Ala/GCE, a deposition potential of -1.3 V, and an accumulation time of 5 s at a scan rate of 100 mV/s. (B) The plots of SWASV anodic peak currents Ip as a function of pH of BRB (3-9) obtained from 65
μM Ala/GCE at an accumulation time of 5 s, a deposition potential of -1.3 V with a scan rate of 100 mV/s.

Note the not so good performance of borate pH 4. This is the buffer system that Kemio uses for loading and/or stripping. The pH 7 phosphate buffer is of interest for those interested in plasma concentration of heavy metals. The pKa of the carboxylate groups was mentioned. At very low pH, these groups are fully protonated that reduces their oxidation signal. Oxidation would be the removal of an electron yielding the cation.

However, at very low pH protonation of carboxylate ion results, removing the ability to bind heavy metals. Higher pH promote the formation of metal hydroxides such as
Zn(OH)2, Cu(OH)2, Hg(OH)2, and Cd(OH)2, that forms a solid coat.

Figure 5 Deposition conditions

What is the optimal voltage needed to deposit heavy metal cations on the cathode that will become our anode? Too much might cause formation of Group 1 and 2 element metals that are extremely reactive with water. Too little will not enable full electroplating of the new anode with the desired metals.

Figure 5. (A) Plot of Ip vs Ed shows the influence of accumulation potentials on the oxidative peak currents of 100 μM Zn2+, 75 μM Cd2+, 50 μM Cu2+, and 75 μM Hg2+ ions in BRB of pH = 4, a scan rate of 100 mV/s, and a deposition time of 5 s at 65 μM Ala/GCE.(B-I) Effect of deposition times on the stripping current responses of 100 μM Zn2+, 75 μM Cd2+, 50 μM Cu2+, and 75 μM Hg2+ ions in BRB of pH = 4 with a deposition potential of -1.3 V, and a scan rate of 100 mV/s at 65 μM Ala/GCE. (B-II) Plot between Ip vs td at
different deposition times in BRB of pH = 4

Panel 5A suggests multi inorganic acid buffer BRB is very good at the deposition of -1.3 V. Panel 5B-I looks at depositions time at defined concentrations of the heavy metals. Panel 5B-II is those data plotted in a different manner.The deposition time of 150 seconds seems to be reasonable

Figure 6 The linear range

Those performing detox would want to know if a device like this one could detect lower amounts of heavy metals. The linear range does seem to be populated by the lower concentrations. The body fluids for the detox could be urine, sweat, or water from a sonic food bath.

Figure 6. (A) SWASV recorded at Ala/GCE by varying concentrations of Zn2+, Cd2+, Cu2+, and Hg2+ ions in BRB (pH = 4), a scan rate of 100 mV/s, a deposition potential of -1.3 V, and a deposition time of 140 s. The investigated concentration ranges arementioned above each peak. (B) Corresponding calibration plots with
linear equation and correlation values from data obtained from selected portion of plot-A SWASV showing linearity of Zn2+, Cd2+, Cu2+, and Hg2+ ion concentrations with Ip obtained under chosen optimized conditions for Ala/GCE for each metal ions

Figure 7 Interfering agents

The good news is that agents used for detoxification like EDTA will not reduce expected concentration more than 20%. SDS is a detergent found in self care products that might come into play with sonic foot bath detox protocol.

Figure 7. (A) Voltammograms of metal analytes performed with an alanine-modified electrode in the presence of 2 mM of one of the interfering agents, i.e., K+, Na+, As3+, Ag+, Cs+, Ca2+, Sr2+, Co2+, Pb2+,
Cl-, EDTA, citric acid, glucose, SDS, CTAB, 2-amino-4- nitrophenol, and 3-chloro-5-nitrophenol in cell having 10 μM Zn2+, 7.5 μM Cd2+, 5 μM Cu2+, and 7.5 μM Hg2+ ions in BRB of pH 4 under chosenoptimized conditions. (B) Corresponding bar graphs showing adsorptive stripping peak current Ip of SWASV affected by 2 mM concentrations of various ions and organic interfering agents.

It is disappointing that Kokab and coauthors did not include Pb, As, and other heavy metals in their analysis. It is understandable that they did not examine agents used in sonic foot baths as interfering agents.

Figure 8 Reproducibility

Several elements of reproducibility that potential customhouses may want to know are

  1. How many times may I reuse this electrode? The Kemio is a single use electrode with limited analyte range.
  2. How reproducible are these electrodes? Will the lot number I ordered in October behave like the next lot I ordered in April of the next year?
Figure 8. Validation of the applied methodology by monitoring theSWASV peak current responses of 10 μM Zn2+, 7.5 μM Cd2+, 5 μM Cu2+, and 7.5 μM Hg2+ ion solutions under chosen optimized conditions; (A) showing repeatability of the designed Ala/GCE electrode at multiple scans (n = 4) and (B) SWASV showing reproducibility of multiple fabricated Ala/GCE electrodes (n = 4)

Future Prospects

Heavy metal detox programs are often dismissed as quackery because of lack of standards of the particular bodily fluid in which the toxin is being released. This system could be a game changer if it could be adapted for easy use in an alternative medicine clinic. Urine and sweat may be fairly uniform compared to solutions in sonic foot baths.

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