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Technical Briefs |
1
Ecossensors Ltd., 74 Sunderland Rd., Sandy Bedfordshire SG19 1QY, UK and
2
Regional Lab. for Toxicol., City Hosp., Birmingham B18 7QH, UK,
3
present address;
a address for
correspondence: Inverness Medical Ltd., Beechwood Park North, Inverness IV2 3ED, UK, fax 44-1463-724601
In recent years concern over the adverse effects of low concentrations of lead on children has increased. In 1991, the CDC reduced the acceptable blood lead concentration from 250 µg/L to 100 µg/L and recommended screening of all American children <6 years old for lead poisoning (1). Graphite furnace atomic absorption spectroscopy is the most common method of measuring lead in blood, but the CDC has encouraged the development of other methods that could be used for mass population screening or near-patient testing. Such methods should be portable, cheap, and easier to use than graphite furnace atomic absorption spectroscopy as recommended by CDC Program Announcement 269, 1992.
The electrochemical technique of stripping voltametry at a mercury electrode has also been used for blood lead analysis. Commercially available instruments based on this method have been used widely but have insufficient accuracy and precision for measuring lead at low concentrations (2). Recently, improvements have been made in the electrochemical measurement of blood lead. Ostapczuk (3) and Jagner et al. (4) obtained good accuracy and precision at low concentrations by potentiometric stripping analysis. They used a mercury-coated graphite electrode that must be cleaned between each analysis, and their testing procedure required stirring of the acidified blood solution.
An approach to stripping analysis that simplifies the testing is the use of disposable electrodes, which can be used once and then thrown away. Microarray electrodes have properties that make them especially suitable for this application. They have high current densities in unstirred solution, have a high signal-to-background ratio, and are not affected by dissolved oxygen. Furthermore, they can be made cheaply by a combination of screen printing and laser photoablation (international patent WO 91/08474) (5). In sufficiently large numbers, e.g., 106 electrodes per year, they could be manufactured for ~$1 each. Recently Feldman et al. (6) described the use of disposable microelectrode arrays for measuring lead in blood. Their testing protocol required that the electrodes be preplated with mercury. They also found that the electrodes responded differently to different blood types, and they had to use standard additions to determine the lead concentration.
Here we report a simplified procedure for the use of disposable microelectrodes to measure lead in blood. Mercury is not preplated or added to the test solution but is contained in a precoated layer on the electrode and coplates with the lead. These devices can be precalibrated, removing the need to perform standard additions.
The disposable electrodes were similar to those described by Feldman et al. (6) but also incorporated a carbon counterelectrode in addition to the Ag/AgCl reference electrode and carbon microelectrode array. Each 2 mm x 3 mm array consists of 13 rows of 19 microelectrodes, each with a diameter of ~40 µm. The electrodes were cleaned by soaking for 30 min in 0.1 mol/L HCl. A reagent layer containing ~11 µg of mercury was ink-jetted over each array with the use of a Bio Dot Microdoser. The ink-jetting solution contained 12.5 g/L mercuric nitrate, 15.4 g/L hydroxyethylethylenediaminetriacetic acid (HEDTA) (Aldrich), 30.0 g/L carboxymethylcellulose (CMC) (Aqualon), 1.0 g/L hydroxyethylcellulose (HEC) (Fluka), and 50 mmol/L KCl. The CMC and HEC are film-formers, and the HEDTA is a chelating agent that stabilizes the mercury (international patent application PCT/GB96/00301).
The procedure for performing a blood lead test involved two steps, acidification and electrochemical measurement. The initial acidification step involved the addition of 200 µL of blood to 2.0 mL of 0.9 mol/L HCl in a 20-mL polystyrene vial. The vial was then mixed briefly by hand and put on a bottle roller for ~5 min to ensure complete release of the bound lead before the blood was tested. Earlier experiments showed that 1 min was sufficient for complete release, but results were unchanged for up to 45 min.
The electrochemical testing was carried out with an Autolab (EcoChemie) in the differential pulse mode. Acidified blood (75 µL) was pipetted to a disposable electrode, and deposition was effected by polarizing the electrode at -800 mV for 165 s. The stripping sequence used a pulse amplitude of 50 mV, step 5 mV, pulse width 3 ms, and trough width 120 ms. Lead-stripping peak heights were measured with use of the Autolab software.
The linear response of the electrodes was tested by the addition of various amounts of Pb(NO3)2 (Aldrich AA standard) to portions of a blood sample to give a range of lead concentrations up to 1000 µg/L. The exact lead concentration of each supplemented blood sample was measured by atomic absorption. Each blood was tested with 5 disposable electrodes. A graph of peak current against lead concentration was linear up to ~600 µg/L with a slope of 0.0070 µA per µg/L and an intercept of 0.212 µA. The intercept may result from the presence of lead in the mercury layer on the electrodes. This graph was used as a calibration curve for subsequent electrodes.
Blood samples were selected from those sent to the Regional Toxicology Laboratory for clinical investigation or occupational monitoring of lead exposure (7). In the majority of cases the specimens had been anticoagulated with potassium EDTA, although some pediatric samples had been collected into Li heparin. All the specimen collection tubes used were known to be free of lead contamination.
Blood lead was measured by electrothermal atomic absorption spectroscopy with the use of a Varian Spectra AA 44 spectrophotometer equipped with deuterium background correction and a GTA-96 graphite furnace (Varian UK). Blood was assayed directly after suitable dilution with an ammonium nitrate/ammonium phosphate modifier solution, with calibration against matrix-matched calibrators. Batch accuracy was monitored by the use of blood reference materials with accurately assigned values (7) (UK Supra Regional Assay Service Trace Elements Laboratories). Laboratory performance was assessed by participation in two national external quality-assessment schemes: NEQUAS lead and cadmium in blood (Wolfson EQA Laboratories) and TEQAS Trace Elements (Robens Institute, University of Surrey, Guildford).
The accuracy of the electrodes was determined by doing a correlation
study with 20 venous blood samples, the lead concentrations of which
had been determined as described above. Each blood was tested with 2
electrodes. The lead concentrations were calculated from the
lead-stripping peaks with the use of supplemented blood calibration.
The results of the correlation study (Fig. 1
) show an excellent agreement between the disposable electrodes
and the comparison method. All 40 measurements were within 20 µg/L of
the comparison method results. Of the 22 measurements on blood samples
with lead concentrations <200 µg/L, 21 of them were within 10 µg/L
of the comparison method result. The low intercept and slope close to 1
demonstrate that the supplemented blood calibration works well.
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The precision of the electrodes was tested with 3 venous blood samples with lead concentrations of 50, 110, and 250 µg/L. Each blood sample was acidified once, and the solution was tested with 10 electrodes. The observed CV was 11.9%, 7.2%, and 2.7%, respectively.
In this electrochemical method of measuring lead in blood by disposable electrodes, a minimum of sample preparation and the use of precalibrated electrodes should enable relatively unskilled people to use this method for screening purposes. Although this lead test was carried out with 200-µL venous samples, smaller volumes, which would enable fingerstick samples to be tested, could be used. The method is sufficiently accurate and precise to meet guidelines recommended by the CDC (Program Announcement 269, 1992). This technique might also have application in surveys of blood lead concentrations in lead-exposed remote populations in developing countries where laboratory facilities are not available.
Acknowledgments
We thank Manuel Alvarez-Icaza for helpful discussions and Enviromed PLC for financial assistance.
References
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