Real-Time Voltammetric Assay of Lead Ion in Biological Cell Systems

Trace lead detection for cyclic voltammetry (CV) and square-wave (SW) stripping voltammetry was performed using mercury immobilized onto a carbon nanotube electrode (HNPE). Using the characteristics of mercury and the catalytic carbon nanotube structure, a modified technique, the 0.45 µg/l detection limit of lead ion was attained. The developed method can be applied to pond water, fish tissue, plant tissue, and in vivo direct assay.


INTRODUCTION
In in vivo biological systems, absorbed lead is related to the medicinal risk facts of organic and neuro disease (Vagn et al., 2001). Trace Pb(lI) can be absorbed through the skin, contaminated food, cigarettes (Laura-Iynn et al., 2001) and water. Pb(lI) exposure remains significant for many populations worldwide despite the major restrictions on certain uses of lead (e.g., as gasoline additives) (Ellen et al., 2003). Trace detection methods are important in biological and environmental  monitoring controls (Joseph et al., 2001). For these reasons, some methods of detecting lead have been developed. These include the spectrophotometric graphite furnace atomic absorption spectrometry (Huge et al., 1995), microwave-enhanced anodic-stripping detection (Yu et al., 2001), retrospective analysis (Eric et al. , 1007), sono electroanalysis (Alastair et al. , 2000), sono ASV detection method (Richard et al., 1998) and others. However, these techniques demand specialized ionization energy systems and complicated voltametric modification techniques. The voltammetric utilizing devices are easy to use and inexpensive. Herein, various methods that have been developed include the nano hydroxamic acid carbon paste electrode (Tesfaye et al., 1999), boron doped diamond electrode (Andrew et al., 1999), and carbon paste polymer film electrodes (DomeÄnech et al. , 2000). Metal mercury (Hg) is also often used owing to its characteristic of forming amalgam. Methods include cathodic adsorptive-stripping voltammetry (Percio et al. , 2003), differential pulse adsorptive-stripping voltammetry using hanging mercury drop electrode (Bhim et al., 2003), mercury film deposition on glassy carbon electrodes (Sandra et al., 2004), and mercury film electrode (Jin et al., 1997) often used in detecting heavy metals. Also, the carbon nanotube catalytic (Joseph et al., 2004) structure is effective for bioassays . In this study, mercury and carbon nanotube paste were combined for lead detection. Optimized analytical result was attained to sensitive detection limits over other common methods, indicating that the method can be applied to plant and in vivo diagnostic analysis.

MATERIALS AND METHODS
Apparatus, reagents, HNPE preparation, and voltammetrie producer. Electrochemical Workstation (660A CH Instruments Inc., Cordova, TN) was used for setting up the electrochemical system. A three electrode cell system was used in the cyclic and squarewave stripping voltammetry. Analytical in vivo assay was performed using the plant cell (Eichhornia crassipes 150 gram) and living fish (250 gram, a carp). A needle type, counter and reference electrode were inserted in the muscle and cells using a 0.5 mm micro diameter hand drill under anesthesia, 10-20 mm deep into the tissue. All the electrodes were cemented with a tooth binder and connected to a 0.05 mm enamel coated copper wire with an electrochemical system. The modified HNPE working eleetrode (prepared using 40% metal Hg, 40% carbon nanotube, and 10% mineral oil mixed paste) and the PE (prepared using 90% earbon nanotube and 10% mineral oil mixed paste) were inserted in a 3 mm diameter glass tube. A referenee eleetrode was used on an Ag/AgCI (saturated KCI) or ehloride-eoated 0.5-mm diameter silver wiretype eleetrode, whereas a 0.5-mm diameter platinum wire was used for the auxiliary eleetrode. Eleetrolyte solutions were prepared using 18 M-Ohm-em· 1 doubledistilled water. For the blank eleetrolyte solution, a 0.1 M ammonium phosphate buffer solution was used. The optimum pH strength was adjusted by adding 0.1 M HCI or a 0.1 M NaOH standard. Under these eonditions, HNPE and PE were eompared, and their eoneentration effeets and optimum voltammetrie parameters were sought.

RESULTS AND DISCUSSION
Cyclic comparison. Fig. 1. shows the eomparison of the effeets of HNPE and PE. First, the various eleetrolyte solutions were tested, with the ammonium phosphate buffer solution yielding good results. Under the aforementioned eonditions, 1, 2, 3, 4, 5, 6, and 7 ppm Pb(lI) standards were put in the ammonium phosphate buffer solution. The peak eurrent of HNPE reaehed 0.7154, 0.6305, 1.514, 3.696, 5.98, 8.06, and 11.54 x 10. 1 A, respeetively. The peak eurrent of PE went up to 0.1021, 0.3356, 1.094, 1.933, 2.721, 3.661, and 6.219 x 10. 1 A, respeetively, and HNPE's peak became  narrower and more sensitive. This means that HNPE is more sensitive to Pb(lI) than PE. The oxidation peak current became more sensitive, instead of being reduced. As such, HNPE was chosen as the working electrode. Under anodic scan, the square wave oxidation stripping parameters were examined.
Working range, statisties and applieation. After obtaining the optimum conditions, the working ranges were examined. Fig. 3(A) shows the resulting ppm range. When 0.8, 1.6, 2.4, 3.2, 4, 4.8, 5.6, and 6.4 mg/I were spiked, the peak current reached 0. 7107, 2.506, 6.564, 10.85, 16.57, 20.12, and 25.47 x 10-5 A, respectively. The peak current appeared sharply at -0.2 V. The statistics of y = 4.679x -6.349 and R 2 = 0.956 herein can have environmental applications. Fig. 3(8), on the other hand, shows the result of the micro ranges. When 10,30,50,70,90,110,130,150,170,and 190 ppb were spiked,they reached 1.784,2.274,3.892,5.853,8.57,10.56,11.66,12.43,12.65,and 11.91 x 10. 1 A, respectively. The peak current also appeared sharply at -0.2 V. A linear curve was produced from the graph from a regression equation of y = 0.802x + 0.4663 (y: peak current in A; x: concentration in ppb) and a correlation of R 2 = 0.9486. This can be useful in biological assay and diagnostic in vive detection, among others. Moreover, the ppb range is more sensitive that other photometric and separation methods. Thus, it can be applied to biological cell systems such as water, fish, plant tissue, and potato cells. Fig. 4(A) shows the SW result of the analytical application of HNPE to pond water. Each peak yielded an electrolyte blank, 1-ml pond water, and 1-, 3-, and 5 f-lg/; Pb(lI) standards, respectively. In the 1-ml water sam pie, the SW reached 4.861 x 10-1 A, but at 1, 3, and 4.52 f--Lg/ml Pb(li) shows that it increased in terms of sensitivity scale. This means that HNPE can detect Pb within low working ranges. A more advanced application to a biological cell system was done. Fig. 4(B) shows the SW results of the analytical real-time application to fish tissue. A counter electrode (platin um), a reference electrode, and HNPE were inserted into a fish tissue, after which lead with HNPE was detected. The peak current appeared at -0.2 and -0.3 V Pb ion. Thus, Pb was found in the fish body. Fig. 4(C) shows the SW result of the analytical application of HNPE to plant tissue. Before the examination, the plant cell was contaminated using the 100 ppb Pb(li) spike in the plant water. Six hours later, three electrode systems were inserted into the plant cell tissue in a 5-mm dip. Under the cell tissue, the SW stripping voltammograms were scanned. The peak current also appeared at -0.2 and -0.3 V. This means that Pb(li) can be soaked in a plant. These developed techniques can be used for real-tissue assay and diagnostic analysis in in vive cell systems.

CONCLUSION
The optimal parameters of HNPE were found to be 0.04 V SW amplitude, 400 Hz frequency, -8 V initial potential, 0.02 V increment potential, 300 s accumulation time, and pH 4.0 strength. Under these conditions, HNPE was applied to the biological cell tissues of a fish, plant, and others. Pb(li) can also be soaked in plants via polluted water and detected direct. These results show that it is important to monitor lead, and that there is a need for a convenient lead detection method. The final method can easily detect even an infinitesimal trace range for in vive and vitro diagnostics.