1. Field of the Invention
The invention relates to colloidal gold adsorbed enzyme biosensors sensitive to inhibition by metal ions in fluids and in particular to bioelectrodes capable of detecting lead at concentrations at least as low as 10 .mu.g/dl. Aspects of the invention include methods of detecting lead in various biological fluids and convenient devices incorporating the bioelectrode.
2. Description of Related Art
Certain metals have long been considered as hazardous to health or to pose an environmental threat. Some metal ions in water or other fluids are of particular interest because of toxicity to humans, cadmium, chromium, nickel, lead, thallium, zinc and arsenic being particular examples The effects of these metals may not be acute; indeed chronic toxicity is of particular concern because the metals accumulate in tissues over a period of long-term exposure. The result, especially in the very young, may be developmental abnormalities, both mental and physical.
The detrimental effects of lead in the environment have long been recognized. Lead poisoning has been detected in waterfowl due to lead shot. The elimination of tetraethyl lead as an octane booster in gasoline was part of an effort to prevent this metal from further contamination of soil and water supplies. However, the use of lead in glazes, paints and coatings, to mention a few examples, has occurred over long periods of time; in fact, lead in pottery may have contributed to the demise of earlier civilizations
As a result of long-term use of lead in the past in so wide a range of products, it is difficult to avoid exposure to this element Lead solder joints in water pipes, for example, contribute to the lead content of drinking water. Modern interior paints are lead-free, but in older homes there may be significant exposure to lead in the surroundings from older lead-based paints, even when such paint layers are coated with the newer lead-free paints. Unfortunately, this has created a real risk of lead toxicity for those groups most susceptible; children.
The long term effect of lead on the health of children exposed to unacceptable levels is calculated to be very significant. This will ultimately reflect in higher health costs, due to increased disability and treatment required. This is of concern to health care professionals and to the federal government, to the extent that new rules related to a "threshold of concern" have been provided in guidelines set by Health & Human Services' Centers for Disease Control (C&E News, 1991). It is hoped that programs being developed to detect the presence of lead in groups at risk for the most damage from lead poisoning will lead to rapid, reliable methods of detecting low levels of lead in individuals. Unfortunately, it is difficult at best to detect lead in body fluids such as blood and it would be impractical to take tissue samples, for example brain tissue samples, to determine lead concentrations.
A simple, reliable method of detecting levels of lead in blood is not available. Current technology relies on time-consuming methods such as computerized stripping potentiometry (Almestrand, et aI., 1988). Although the instrumentation required for this determination is not unduly complex, highly skilled technicians are needed. This is also true for a commercially available hand held monitor, ElectraScan EC-1Pb monitor for lead detection, from Eutech cybernetics, Singapore (Gunasingham, et al., 1989).
Routine analysis of metals has generally relied on atomic absorption spectrometry. However, the equipment required for analysis is expensive. Moreover, the method exhibits lower accuracy and sensitivity toward lead as compared to anodic stripping voltammetry
Instruments currently available for monitoring trace metals generally require highly trained personnel to perform relatively sophisticated techniques. Consequently, analyses are performed in centralized laboratories set up for routine multiple sample analysis. However, there is no instrumentation available for use in the field or in the physician's office allowing rapid metal determinations with simple portable instruments that do not require highly technically trained personnel.
Trace metal determination based on metal-enzyme interaction has taken advantage of either activation or inhibition of an enzyme by a metal, usually specific for the enzyme. Fluoride has been measured by its inhibition of liver esterase catalysis of a butyrate substrate (Linde, 1959) and magnesium has been measured in plasma by isocitric dehydrogenase activation (Baum and Czok, 1959). Titration determinations or rates of TPNH formation measured spectrophotometrically have been reported to be useful for measuring levels of activating metals such as manganese, magnesium and cobalt. Inhibiting metals such as lead can also be measured (Kratochvil, et al., 1967).
Analysis of trace metals based on inhibition of an enzyme's ability to produce hydrogen peroxide and oxidize homovanillic acid to a fluorescent product has also been explored Horse radish peroxidase inhibition was linear over a range of 10-185 .mu.g/ml of lead (Guilbault, et al., 1968). Metal ion inhibition of the enzyme glucose oxidase with mercury (II), Ag(I) and Pb(II) has suggested that these metals are detectable at low levels although strong buffer-interactions were obtained when lead was present, casting doubt on the viability of the method generally to measure lead in trace amounts (Toren and Burger, 1968).
Of a few reported enzyme-inhibitor electrodes, most use CO.sub.2 and pH electrodes (Tran-Minh et al., 1990; Botre et al., 1983) which have a small, nonlinear response. Most potentiometric sensors detect the enzymatic reaction product, not the enzyme activity directly. The response time to inhibitors is usually long because the inhibition effect can show up only after the product has diffused away from the electrode surface. When a pH electrode is used, the signal largely depends on the pH and the buffer capacity of the sample solution.
Amperometric enzyme-inhibitor electrodes have been reported, either with rather classical configurations in which the enzyme and promoter solution are fixed on or near the electrode surface with a dialysis membrane (Albery et al., 1990) or with covalently bound enzymes and complicated electrochemical techniques with soluble electron transfer mediator in the sample solution (Smit and Cass, 1990). All the reported inhibitor sensors use wet enzymes, either on a membrane or electrode surface, or between a membrane and an electrode surface (or wet mediator in amperometric sensors). This makes it difficult for long-term storage. Moreover, none is reported to work with microcells using simple electrochemical techniques.