Detection of lead in blood

A device (10) is disclosed for measuring the concentration of metal ions in solution, particularly lead in blood. The device comprises a mercury free electrode (16), which is separated from the test solution (21) by a layer of material (20) which permits passage therethrough of the ions to be measured. In preferred embodiments an insulating layer (18) having an array of photoablated holes (19) is disposed between the electrode and the ion-permeable layer (20). Also disclosed are methods for operating the device and measuring ion concentration using anodic stripping voltametry, and assay kits incorporating devices as described together with appropriate meters and circuitry.

This application is a 371 of PCT/GB95/00269 filed on Feb. 10, 1995. 
FIELD OF THE INVENTION 
This invention relates to devices and methods for measuring the quantity of 
metal ions in solution, particularly (but not exclusively) the measurement 
of lead in blood. 
BACKGROUND TO THE INVENTION 
It has been well known for many years that lead is a toxic element and can 
have a number of serious health effects. Young children are especially at 
risk from lead poisoning. They can be exposed to lead from sources such as 
water (lead pipes), food, and air (leaded petrol). Old paint can contain a 
high concentration of lead and the most common cause of lead poisoning in 
young children is from eating paint chippings or dust from the walls or 
windows of old houses. 
In the United States of America the Center for Disease Control (CDC) 
considers lead poisoning such a serious problem that it recommends all 
children in the country under 6 years of age be screened for lead. The 
amount of lead which a child has been exposed to is determined by 
measuring the concentration of lead in the child's blood. Over the years, 
as more has been learned about the adverse effect of lead on children, the 
blood lead concentration considered safe has steadily declined. In 1985 
the CDC considered a lead concentration of less than 25 .mu.g/dl to be 
acceptable. In 1991 the CDC lowered the safe lead level to 10 .mu.g/dl. 
Until recently much of the screening for lead poisoning was done using a 
fluorometric method called the zinc protoporphyrin test. This method is 
quick and cheap but is not accurate or sensitive enough for measuring lead 
at the new lower limit of 10 .mu.g/dl. 
Atomic absorption spectroscopy (AA) can be used to measure lead in blood 
very accurately at low concentrations but the method is not practicable 
for screening because the instrument is large, very expensive, and 
requires a highly trained operator. 
The electrochemical method of anodic stripping voltametry (ASV) is another 
way of measuring lead in blood. In ASV, an electrode in contact with a 
solution to be tested is held at a negative potential for a sufficient 
period of time to reduce metal ions in the solution and concentrate them 
at the electrode. The potential is then ramped or scanned in the positive 
direction and any metals present will be stripped from the electrode when 
the unique oxidation potential of the respective metal is reached. The 
current produced during the stripping of each metal is proportional to the 
concentration of that metal in the test solution. Commercial ASV 
instruments are available but they are large, expensive, and not 
particularly accurate when used for measuring low concentrations of lead 
in blood. 
The CDC is now actively encouraging development of a system which is small, 
portable, cheap, and easy to use, but can measure low concentrations of 
lead in blood with good accuracy and precision. At the present time there 
are no commercially available systems which meet these criteria, but this 
invention relates to a system which shows promise of meeting the required 
criteria. 
SUMMARY OF THE INVENTION 
In one aspect, the invention provides a device for measuring the quantity 
of ions of a predetermined metallic element which are present in a 
solution, the device comprising a mercury-free electrode separated from 
the solution by a layer of material which permits passage therethrough of 
the ions to be measured. 
A preferred embodiment of the new system, designed for measuring lead in 
blood, uses a small, portable meter into which safely disposable cheap 
electrode strips are placed. A small volume of blood is added to a tube 
containing acid. This is mixed briefly to acidify the blood and release 
any bound lead. A drop of acidified blood is placed on the electrode strip 
and a button on the meter is pushed to start the analysis. In 2 or 3 
minutes, the lead concentration in that sample of blood is displayed on 
the meter. A fresh electrode strip is used for each blood lead test, the 
used strips being disposable without environmental problems. 
The improvements made possible by this invention are due to the use of 
mercury-free disposable electrode strips which have an ion exchange 
membrane covering the electrode from which the lead (or other metal) will 
be stripped. Conventional ASV equipment uses an electrode made from 
mercury or covered with mercury. The use of such an electrode has 
disadvantages because of the toxicity of mercury and the problems of its 
proper disposal. The electrodes used in a system according to this 
invention do not need to use mercury. The use of mercury-free disposable 
electrode strips opens the possibility of a rapid lead-in-blood test which 
is quick and simple to do. The invention eliminates the need for any 
cleaning, pre-plating, or preconditioning which is often necessary when 
conventional electrodes are used for ASV (although, as will be seen below, 
pre-plating electrodes according to the invention can have certain 
advantages). 
When conventional electrodes are used for measuring lead in blood it is 
difficult to measure low lead concentration due to fouling of the 
electrode surface by proteins and other blood components. The membrane 
covering an electrode in a system according to this invention prevents 
this fouling and enables the required low lead levels in blood to be 
easily measured. 
Devices according to the invention permit determination of lead-in-blood at 
levels below 25 .mu.g/dl. A preferred device comprises a disposable 
mercury-free electrode strip having a substrate supporting a working 
microelectrode array adjacent to a reference electrode, a respective 
conductive path for each electrode leading to a connection end thereof for 
each said path, at least the microelectrode array being coated with an ion 
exchange membrane. Desirably the microelectrode array comprises a 
conducting layer (e.g. carbon) overlain by an electrically insulating 
layer, the insulating layer being laser photoablated to exhibit an array 
of small holes which expose the conducting layer underneath. Between 100 
and 400 holes of some 40 micron diameter over a conducting area of some 5 
mm by 3 mm have been found to be satisfactory. Preferably, the electrode 
is a disposable electrode. 
The electrode strips used in our system preferably consist of a carbon 
working electrode and a silver/silver chloride reference electrode which 
are made by printing tracks of conductive ink onto a glass or plastics 
base. The preferred configuration of working electrode is a microelectrode 
array which gives a large signal to background ratio. Such an array can be 
made by printing an electrically insulating layer over the carbon working 
electrode. Laser photoablation is then used to make small holes in this 
insulating layer to expose the underlying carbon layer. The construction 
of a microelectrode array in this manner is described in WO91/08474. To 
complete the strip an ion exchange membrane is formed over the working 
electrode. 
Electrodes as described above work well at pH&gt;2, but they are less 
sensitive under more acid conditions. A convenient way to overcome this 
limitation is to preplate the carbon electrode with silver before the ion 
exchange layer is applied. -The amount of silver plated onto the electrode 
is critical, since if insufficient silver is deposited lead sensitivity is 
diminished, whilst if too much silver is deposited a large oxygen 
reduction background is observed. We have found very good lead stripping 
peaks can be obtained by preplating the electrode at approximately -1.0 V 
versus silver chloride for between 5 and 600 seconds using a solution 
containing between 1 and 100 ppm of silver ions. Ideally, silver is plated 
for 60 seconds using a 10 ppm silver solution. These conditions appear to 
lead to an ideal distribution of silver nuclei on which lead nucleation 
can initiate. It is also envisaged that other noble metals such as 
rhodium, palladium and platinum may be used in place of silver. 
Furthermore, it is believed that pre-plating with silver or another noble 
metal in this way will enhance the low pH sensitivity of electrodes in 
general, and this feature of the invention is therefore not restricted to 
the use of mercury-free electrodes, or those containing ion-permeable 
layers. 
A further convenient way to introduce silver is to incorporate a silver 
salt into a layer over the electrode. In this manner the silver is 
coplated with the target analyte during the test. A similar approach can 
be employed for electrodes which use mercury, in this case a mercury salt 
being incorporated into the layer. Alternatively, silver may be 
incorporated during the printing of the carbon electrode, by using an 
appropriate mixture of silver and carbon inks, or by printing carbon ink 
made from carbon particles metallized with silver. 
An additional benefit of using silver is that its presence appears to 
discriminate against the deposition of copper. Copper is present in quite 
large quantities in human blood samples and can interfere with the lead 
determination, because the oxidation potential of copper is similar to 
that of lead. The problem of copper interference may also be addressed by 
causing the copper to be unavailable to the electrode by, for example, 
precipitating the copper with ferricyanide (Fe.sup.3+ (CN.sup.-).sub.6 
!.sup.3-). The ferricyanide can be added to the sample during the 
pretreatment phase (for example by subjecting the sample to 1:10 dilution 
with a 5% potassium ferricyanide solution). Again, this aspect of the 
invention is not restricted to mercury-free electrodes, or those coated 
with an ion-permeable layer, and it is envisaged that the use of 
ferricyanide to remove copper ions will be of use with electrodes in 
general. 
A further way in which the problems associated with copper deposition may 
be diminished is by the addition of a reagent which affects the relative 
oxidation potentials of copper and lead, so that the anodic stripping 
voltametry peaks therefor are separated from each other, and therefore 
readily distinguishable. We have found that a good effect may be achieved 
with potassium iodide, which can be added, for example, by diluting the 
sample 9:1 with 0.2M potassium iodide solution. As with the use of 
ferricyanide, the use of potassium iodide is thought to have general 
applicability in the avoidance of detection difficulties due to copper, 
and this aspect of the invention is therefore not limited to mercury-free 
electrodes or those having ion-permeable layers. 
In another aspect, the invention extends to a method for determining the 
quantity of ions of a predetermined metallic element which are present in 
a solution, comprising the steps of contacting said solution with the 
layer of material overlying the mercury-free electrode, and determining 
the number of said ions coming into contact with said electrode by a 
voltametric method. 
The invention provides in a further aspect an assay kit comprising a device 
as described and a meter for connection to said mercury-free electrode and 
to a reference electrode. 
Suitably the meter is one into which the electrode strip can be inserted to 
put each connection end in electrical contact with a sensing circuit. 
Preferably, the sensing circuit is adapted to hold the microelectrode 
array at a negative potential relative to the reference electrode for a 
first period and then to apply a gradually increasing potential in the 
positive direction to the working electrode to strip lead from the 
microelectrode array when the oxidation potential for lead (or other metal 
under test) is reached. 
Conveniently means is provided in the meter to assess the integral of 
current against time at the stripping voltage for lead to determine a peak 
area for the stripped lead, since this peak area is related to the 
concentration of lead in the sample of blood fed to the strip. 
The assay kit can conveniently also include means to contain a blood sample 
while it is diluted with acid (e.g. HCl), a supply of acid for such 
dilutions and means to transfer a drop of acid-diluted blood to an 
electrode strip in the meter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The meter 10 shown in FIG. 1 (some 10 cm.times.20 cm.times.4 cm) has a 
socket 11 into which a sensing strip 12 shown in FIG. 2 can be inserted to 
put its contact areas 13, 14 in electrical connection with electronic 
circuitry within the meter. 
The meter 10 also includes an alpha-numeric display screen 25 and a bank 26 
of buttons 27 for controlling the electronic circuitry in the casing of 
the meter 10. The meter 10 also includes a port 28 for connection, if 
required, to a computer and VDU (not shown). 
The strip 12 includes two printed carbon tracks, one leading from area 13 
to a printed Ag/AgCl reference electrode 15 and the other leading from 
area 14 to a microelectrode array 16. The substrate can be a glass or 
plastics plate 17. 
The array 16 comprises an electrically insulating layer 18 over the carbon 
track which has been provided with an array (e.g. 280-14.times.20) of 
small (e.g. 40 microns diameter) holes 19 spaced apart (e.g. 160 microns 
apart) over a small area as shown. The pattern of holes can be punched by 
photoablation as described in WO91/08474. 
Overlying the open tops of the holes 19 is a layer 20 of a semipermeable 
ion exchange membrane. This may be manufactured for example from a 
perfluorosulphonated ion exchange resin (ionomer), such as NAFION (Trade 
Mark of Du Pont), or a poly(ester-sulphonic acid) film. In a practical 
embodiment the membrane was created from a dried drop (2 .mu.l) of a 3% 
NAFION solution in 75% ethanol/25% water. 
FIG. 3 also shows a drop 21 of acid-diluted blood on which a lead 
concentration test is to be conducted. 
The electronic circuitry within meter 10 first applies a negative potential 
to area 14 relative to area 13 for a period sufficient to reduce metal 
ions in the blood drop 21 applied to the strip 12 and concentrate them at 
the surface of the electrode 16 and then applies a ramped potential in the 
positive direction to strip metals from the electrode 16 when the specific 
oxidation potential of the metal in question is reached. 
FIG. 4 shows a typical plot of current flowing between electrodes 15 and 16 
as the ramp voltage increases, the peak 30, occurring between -500 and 
-600 mV, representing the presence of lead. 
The circuitry in the unit 10 can determine the area under the peak 30 and 
this can be displayed digitally on the screen 25 to give a measure of the 
concentration of lead in the blood sample 21. 
To check the accuracy of the meter 10, tests were conducted with samples of 
lead-free venous blood deliberately contaminated with 5, 10, 15, 20, 30 
and 40 micrograms of lead per deciliter. The blood samples were each added 
to dilute HCl in a 1:3 ratio and drops of acidified blood were tested on 
separate strips 12. The peak area values secured were plotted as crosses 
on the graph of FIG. 5. It will be noted that a good approximation to a 
linear relationship between peak area and lead concentration is obtained. 
Although we expect microelectrode areas faced by ion permeable layers will 
perform best in a system according to this invention it is expected that a 
lead-in-blood sensor capable of operating below the CDC safe lead level 
can be produced using a strip 12 much as shown in FIG. 2 but without the 
layer 18 or holes 19, the membrane 20 simply directly overlying the carbon 
area 16.