WO2001006235A1

WO2001006235A1 - Apparatus and method for sensing metal ions

Info

Publication number: WO2001006235A1
Application number: PCT/EP2000/005458
Authority: WO
Inventors: Peggy Lucienne Odette Amiot; Steven Howell; Peter David Lee; Hugh Rieley
Original assignee: Unilever Plc; Unilever Nv; Hindustan Lever Limited
Priority date: 1999-07-14
Filing date: 2000-06-13
Publication date: 2001-01-25
Also published as: GB9916569D0; AU5078000A

Abstract

A method and apparatus for sensing metal ions in a sample using an optical evanescent wave biosensor and its use in biological and chemical testing is disclosed.

Description

APPARATUS AND METHOD FOR SENSING METAL IONS

FIELD OF THE INVENTION

The present invention relates to a method and apparatus for sensing metal ions in a sample and their use in biological and chemical testing. In particular, the invention relates to the use of optical evanescent wave biosensors in detecting, monitoring, quantifying or studying the binding interactions of metal ions in a sample.

BACKGROUND TO THE INVENTION

Optical assay techniques based on monitoring the optical properties of an evanescent wave produced when a light beam is totally internally reflected at the boundary of a medium are well known and have been described in the literature. (See, for example, Mal qvist, M. Nature, 361, 186-187, 1993.)

Commercially available evanescent wave biosensors include the BIAcore sensor range (Biacore AB, Sweden) which makes use of the phenomenon of surface plasmon resonance (SPR) to detect changes in refractive index as molecules in a mobile phase interact with a binding partner or ligand immobilised on the surface of the sensor.

An alternative evanescent wave biosensor currently in commercial use is the IAsys biosensor (Affinity Sensors, Cambridge, UK) which is based on resonant mirror principles.

With both these sensors, binding of the molecule in a fluid (sample) phase to a derivatised sensor surface causes the optical properties of the sensor surface to become modified, monitoring of which change provides the basis of the detection method. To date, evanescent wave biosensors have been used to study macromolecular interactions in a number of fields, including antibody-antigen interactions, receptor-ligand interactions, virus research and ligand purification (see, for example, Myszka, Analytical biotechnology, 8, 50-57, 1997).

US 5,620,850 describes the derivatisation of surfaces such as surface plasmon resonance chips for the capture of biological materials using a biological binding partner of the biological molecule of interest. Metal ions are used as bridging elements in the construction of the sensor surface, linking a chelating agent immobilised at the surface of a solid phase with the biological binding partner of the biological molecule.

An advantage of using evanescent wave biosensors in studying molecular interactions is that as the monitored property is a refractive index change, there is no need to label the macromolecules . A further and particular advantage is that using this technique, it is possible to follow macromolecular interactions in real-time, allowing for quantitative characterisation of chemical binding kinetics and equilibria.

As the detector response is related to the molecular weight of the reactant that binds to the surface, it is not generally possible to observe directly interaction of a low molecular weight reactant with an immobilised binding partner using evanescent wave biosensors . This represents an important limitation to the use of this methodology. Reports in the literature suggest that the lower molecular weight limit for direct detection of reactants in such systems is in the order of 200 Da (see Myszka, above) .

WO 92/01939 describes a method for detecting metal ion concentration using an antibody which binds both antigen and metal ion, antibody affinity being reduced in the presence of metal ions. Surface plasmon resonance is used to detect the binding, giving an indirect measure (by perturbation of another binding equilibrium) of the presence of metal ions.

There are no reports in the literature of the direct detection of metal ions in solution using surface plasmon resonance biosensors or other such similar detection systems. Conventional methods for direct detection of metal ions such as mass spectrometry or atomic emission spectroscopy suffer from the disadvantage of requiring bulky and expensive equipment which is not suitable for "in field" use.

There remains a continuing need for the development of improved methods for sensing metal ions in solution. In particular, there is a need for the development of a method for direct detection of metal ions suitable for real-time application.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a method for sensing metal ions in a sample using an optical evanescent wave biosensor, wherein the interaction of metal ions and sensor surface is detected directly.

The invention also provides the use of a method according to the first aspect in biological or chemical testing.

In a further aspect, the invention provides an optical evanescent wave biosensor having a sensor surface wherein said sensor surface comprises an immobilised protein capable of binding a metal ion suitable for use in the method of the invention.

Also provided is a sensor surface comprising an immobilised protein capable of binding a metal ion suitable for use in the method of the invention. As used herein, the term 'sensing' includes within its scope the steps of detecting, monitoring and/or quantifying the presence of metal ions in a sample. It also includes studying the binding interactions of a sample comprising metal ions with a metal- binding ligand immobilised on a sensor surface. Direct detection of the interaction of metal ions and sensor surface refers to detection independently of other binding equilibria.

The term 'optical evanescent wave biosensor' is intended to encompass any sensor which operates by monitoring the optical properties of an evanescent wave produced when a light beam is totally internally reflected at the sensor surface.

The present invention may be more fully understood by reference to the following description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding that metal ions in a sample may be sensed using an optical evanescent wave sensor. Although widely reported in the literature to be of use in studying a variety of macromolecular interactions, examples of which are discussed above, there has previously been no suggestion that such biosensors could be used, other than in an indirect way, to study the presence or behaviour of metal ions in a sample. Indeed, given the teaching in the art that such optical biosensors cannot be used to detect directly low molecular weight molecules, there would be no expectation that interactions involving low molecular weight metal ions could be studied in this way.

It will be appreciated that the invention extends to the use of any optical evanescent wave biosensor. Particularly convenient biosensors for use according to the invention are the commercially available BIAcore (using surface plasmon resonance) and IAsys (resonant mirrors) instruments mentioned above. The method of the invention may be used qualitatively to detect the presence or absence of metal ions in the sample or quantitatively, for example, using conventional techniques such as measuring changes in rate of refractivity or by analysis of the absolute measurements at different times. The sample will generally be in fluid form, conveniently as a liquid, for example an aqueous solution, or may comprise a sample of body fluid, such as blood, urine, saliva, sweat.

The invention is applicable to any metal ion. Particularly suitable metals include iron, copper, aluminium, titanium and heavy metals such as lead and mercury.

As discussed above, with evanescent wave biosensors, the size of the monitored response is dependent upon the molecular weight of the species binding to the sensor surface; hitherto, it has generally been considered that a mass of around 200 Da represents a lower limit of detectability. In the case of metal ion binding, the mass of the entity binding will depend on the mass of the metal ion together with any other ligands that remain associated with it. Typically, according to the present invention, masses of metal ions and attendant ligands in the range of 50-200 Da are detectable .

Commercially available biosensors have a variety of sensor surfaces. A commonly used standard sensor surface is a carboxymethyl dextran layer. The carboxyl groups at the sensor surface can be activated, or functionalised, with a variety of ligands which may be immobilised at the sensor surface using conventional coupling chemistries.

In order to achieve specific binding of metal ions at the evanescent wave sensor surface in accordance with the present invention, the sensor surface may suitably be derivatised with metal-ion co-ordinating immobilised ligand groups which either contain a free electron pair or are negatively charged, such as thiol, phosphate, sulphate, thiocyanate, amino, cyano or halide groups. Alternatively, calixeranes may be used at surfaces to bind metal ions. Varying the nature of the ligand selected leads to a varying specificity for binding with different metal ions. A thiol derivatised sensor surface is suitable for binding metals such as zinc or gold, for example, whereas a sensor surface comprising a nitrogen donor group such as amine or cyano would be more suitable for binding a metal such as mercury. The average skilled person would have no difficulty in selecting an appropriate ligand depending on the particular metal concerned based on general chemical knowledge .

Alternatively, in a further aspect of the invention, co-ordinating ligands may be provided by a molecule such as a protein immobilised at the sensor surface. Examples of suitable proteins include transferrin, hemocyanin and apo-ferritin which can bind iron, ceruloplasmin which can bind copper, and keratin which can bind aluminium. Antibodies or antibody fragments represent another suitable group of proteins. In addition to metal ion- binding proteins, shorter stretches of peptide sequence capable of binding metal ions may also be used, for example, zinc finger domains .

The method of the present invention may suitably be applied to sensing metal ions in a variety of biological and chemical applications. One such situation where the ability to detect and/or monitor the presence of metal ions in real-time would be advantageous is in monitoring or detecting the presence of pollutants, especially heavy metal ion pollutants in the water supply. Other applications of the method of the invention include testing of effluent from sewage/industrial plants, for example testing/monitoring of solutions released into the environment by the mining and nuclear fuel industries for the presence of such metals as lead and uranium. Alternatively, the method of the invention may conveniently be applied to monitoring the interaction of metal salts with protein coated surfaces. By way of example, the irreversible binding of materials to skin proteins is a common cause of skin irritancy and hence the study of the interaction of metal species with skin proteins such as keratin gives a useful indication of the potential irritancy of skin care products comprising such metal species on application to the skin. Such a method is particularly useful, although not limited in application, to testing or predicting the irritancy of topically applied personal care products .

It will, of course, be appreciated that optical evanescent wave sensors having an appropriate protein coated sensor surface can conveniently be used to predict the irritancy of skin care products, irrespective of whether or not they contain metal species, based on their ability to bind skin proteins.

The method of the invention may conveniently be used to analyse the metal content of cosmetic products. Levels of trace metals such as nickel, chromium and cobalt (which are sensitisers) or neurotoxins such as antimony or mercury in cosmetic products are regulated and it is important that convenient methods for their detection are available.

Other applications of the method of the present invention include clinical applications where metal ions are detected from body fluids or tissue biopsies . This method may be used to assess systemic toxicity of metal ions in the body, for example, by studying the binding of metal ions to the blood serum protein transferrin, or used to aid clinical diagnosis.

The invention will be further described by the following illustrative examples and with reference to the accompanying drawings, in which: Figure 1 shows a sensogram (arbitrary Response Units against time) tracking the preparation of a keratin coated biosensor chip.

Figure 2 shows a control sensogram in which ammonium titantium lactate solution was passed over an untreated biosensor chip surface.

Figure 3 shows a sensogram tracking the preparation of a transferrin coated biosensor chip.

Figure 4 shows a sensogram tracking the binding of iron ions to a transferrin coated biosensor chip.

Figure 5 shows a sensogram in which ammonium titanium lactate solution was passed over a standard carboxymethyl dextran sensor surface.

EXAMPLES

Example 1: Interaction of a metal ion salt with keratin

Keratin was extracted from human skin taken from the underside of the foot. The skin sample was placed in an aqueous solution of 4 M urea and 20 mM dithiothreitol for 3 hours at 50°C. This extracts the keratin from the skin. The resulting mixture was spun in a centrifuge at 14000 g for 10 minutes. The supernatant solution was then removed and the remaining solid keratin re-dissolved in distilled water.

A sensorgram trace of the preparation of a keratin chip surface is shown in Figure 1. A Cl chip (Pioneer Products, Biacore AB) was treated initially with 60μl of a freshly mixed solution of N- hydroxysuccinimide (NHS) and l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) . Two aliquots of 30 μl keratin solution were then passed over the chip. The increase in the level of the baseline observed indicates the mass of bound keratin. Finally 60 μl of ethanolamine was passed over the chip to block any excess NHS-EDC.

Although analysis is possible at a wide range of pHs, experiments were carried out at pH 5.5, in order to mimic conditions on the skin. The Cl (flat carboxymethylated gold surface) biosensor chip (Biacore AB) coated in keratin was then subjected to a coinject of 20 μl [2- (N-morpholino)ethane-sulfonic acid] (MES) solution at pH 5.5 immediately followed by 40 μl of 1% w/v aqueous A1C1₃.6H₂0 solution also at pH 5.5. Experiments were carried out in duplicate to ensure reproducibility and each was carried out on a fresh keratin surface. It is, however, possible to regenerate the surface by treatment with either dilute HC1 or ethanolamine. Figure 2 shows the binding of A1C1₃ to keratin at the sensor surface .

Example 2 : Monitoring the binding of metals to apo-human transferrin

The human apo-Transferrin (apo-hTf) used in this example was purchased from SIGMA (catalogue No. T1147) . The powder was diluted in distilled water to obtain a 10 g/1 solution.

Before being used, the apo-hTf solution was purified by filtration, using Microcon centrifugal filter devices. This was done to remove low molecular mass contaminants. The Microcon ultracentrifugation membranes contain trace amounts of glycerine, so in order to remove this, they were spin-rinsed with de-ionised water (500 μl per filter) .

Microcon reservoirs were inserted into vials and 500 μl of the apo-hTf solution was pipetted into them. The Microcons were then centrifuged for 12 minutes at 13,000 g. The filtrate was disposed of. To suspend any transferrin, 70 μl of water was added to each Microcon and agitated gently. The suspension was then transferred to new microcentrifuge tubes. Following this manipulation, the vials were separated from reservoirs, which were placed upside down in the new microcentrifuge tubes. In order to retrieve the remaining protein and to transfer concentrate to vials, the latter were spined for 3 minutes at 13,000 g. The substrate contained in ten vials was then collected together in the same tube.

The transferrin coated Cl chip was prepared as described in Example 1, except with transferrin in place of keratin. This example was run at pH 7.4 in order to mimic physiological conditions . Figure 3 shows a sensorgram trace of the preparation of a transferrin chip. The surface was treated with 20 μl of an aqueous 0.2 M NaHC0₃ solution immediately before 40 μl of FeCl₃.6H₂0 (supplied by Aldrich and used as received) . The increase in the baseline before and after treatment indicates the mass of iron bound to transferrin (shown in Figure 4) and the rising slope during the FeCl₃.6H₂0 treatment provides kinetic data on the binding interaction.

Using this method, it is possible to monitor the interaction of a range of metal ions, in addition to iron, with the blood serum protein transferrin. This has relevance for studying the systemic toxicity of metals.

Example 3: Monitoring levels of metal ions in a water supply/effluent stream

An FI (flat gold surface with short dextran chains) biosensor chip (Pioneer Products, Biacore AB) was used as supplied. An aqueous solution of ammonium titanium lactate (available from Du Pont under the trade name Tyzor LA) was prepared (0.5% w/v) . 40 μl of which was passed over the biosensor chip at a rate of 10 μl.min^"1. The resulting sensorgram (Figure 5) clearly shows the presence of titanium ions by an increase in baseline. Control runs in which plain water was passed over the surface gave no change in the baseline. This procedure has been followed successfully for the detection of a number of metal ions (e.g. iron, aluminium) and using a number of different chips (e.g. CM5, Cl, Jl) .

Claims

1. A method for sensing metal ions in a sample using an optical evanescent wave biosensor, wherein the interaction of metal ions and sensor surface is detected directly.

2. A method according to claim 1, wherein the evanescent wave is produced by surface plasmon resonance or by resonant mirror principles .

3. A method according to claim 1 or 2, wherein a protein capable of binding a metal ion is immobilised at the sensor surface of the biosensor.

4. A method according to claim 3, wherein the protein is keratin, transferrin, ceruloplasmin, apo-ferratin, hemocyanin or albumin.

5. A method according to claim 1, wherein a metal-ion co- ordinating ligand group which either comprises a free electron pair or is negatively charged is immobilised at the sensor surface of the biosensor.

6. Use of a method according to any one of claims 1 to 5 in biological or chemical testing.

7. Use according to claim 6 in detecting and/or predicting irritancy of skin care products.

8. Use according to claim 6 in clinical diagnosis.

9. Use according to claim 6 in monitoring and/or detecting metal ion pollution of water supplies or industrial effluent.

10. An optical evanescent wave biosensor suitable for use in a method according to any of claims 1 to 5 having a sensor surface comprising an immobilised protein capable of binding a metal ion.

11. A sensor surface suitable for use in a method according to any one of claims 1 to 5 comprising an immobilised protein capable of binding a metal ion.

12. A sensor surface comprising an immobilised protein capable of binding a metal ion, wherein the protein is keratin, transferrin, ceruloplasmin, apo-ferratin, hemocyanin or albumin.