WO2013030542A1

WO2013030542A1 - Detection of neurodegenerative disease

Info

Publication number: WO2013030542A1
Application number: PCT/GB2012/052064
Authority: WO
Inventors: David Newman
Original assignee: University Of Exeter
Priority date: 2011-08-26
Filing date: 2012-08-23
Publication date: 2013-03-07
Also published as: GB2507705A; GB201403183D0; GB201114733D0

Abstract

The application is directed to apparatus and methods for detecting iron containing proteins, eg. ferritin, transferrin etc., in bodily fluids, eg. whole blood, serum, cerebrospinal fluid (CSF), using a magneto-optic technique, especially based on the Cotton-Mouton effect. These proteins serve as sensitive biomarkers for disruption of iron homeostasis and neurodegenerative diseases, such as Alzheimer's, Parkinson's, ALS etc. In one embodiment, an apparatus for transmission measurements (10) comprises a light source (12), a polariser (14), and a sample cell (16) for containing a liquid sample under analysis. A photodetector (18) is positioned near the rear face of the cell to detect light transmitted through the cell. The output of the detector is analysed by a computer or microprocessor (20) as a function of a modulating magnet field generated by magnet (22).

Description

Detection of Neurodegenerative Disease

This invention relates to methods and devices for detecting neurodegenerative diseases, and also to methods and devices for detecting certain iron-containing analytes.

Age-related neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis have a profound social and public health impact. In countries such as the UK, in which the population is increasingly aged, neurodegenerative conditions such as these are of increasing importance. With the exception of riluzole, which is licensed for use in slowing the development of amyotrophic lateral sclerosis, all other current therapies for these conditions are purely symptomatic in nature. However, it is hoped that in the future neuroprotective or restorative therapies will become available, and any such interventions are likely to be most effective if applied at the earliest possible point in the course of the disease. In these circumstances, pre-symptomatic or pre-clinical diagnosis of disease in those at risk will become of premium value. Mild cognitive impairment (MCI) is a term which is used to refer to the transitional state between the cognitive changes of normal aging and very early dementia such as occurs in Alzheimer's disease. As such, this offers a unique opportunity for pre-clinical diagnosis of Alzheimer's disease.

From the forgoing discussion, it will be apparent that there is a substantial need for a technique which can be used to detect neurodegenerative disease, especially the onset of neurodegenerative condition. The present invention, in at least some of its embodiments, addresses these needs.

The present inventors have recognised that the disruption of iron homeostasis (including disruption through deposition or accumulation) is known to have significance to or at least an association with neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis (1 -5). Some researchers think that the disruption of iron homeostasis plays a central role in the pathogenesis of several common age-related neurodegenerative conditions including Alzheimer's disease (1 ), Parkinson's disease (2) and amyotrophic lateral sclerosis (3).

In a broad aspect, the present invention proposes that neurodegenerative diseases can be detected using a magneto-optic optic detection technique to detect certain iron-containing species which are associated with the disruption of iron homeostasis, such as through deposition or accumulation.

According to a first aspect of the invention there is provided a method of detecting a neurodegenerative disease including steps of:

i) providing a sample of a bodily fluid which contains an analyte, in which the analyte is an iron containing molecule related to iron homeostasis;

ii) detecting a disruption of normal iron homeostasis by detecting the analyte in the sample using a magneto-optic detection technique; and iii) correlating the disruption of normal iron homeostasis detected by magneto-optic detection technique with the occurrence of the neurodegenerative disease.

Advantageously, step ii) includes:

applying a magnetic field across at least a portion of the sample; and detecting a physical property of the sample, the detected physical property corresponding to a physical property of the analyte which varies in dependence with the applied magnetic field.

Typically, the applied magnetic field orients the analyte with respect to the applied magnetic field, and the detected physical property corresponds to a physical property of the analyte which varies in dependence on the orientation of the analyte with respect to the applied magnetic field.

Preferably, the magneto-optic detection technique utilises the Cotton-Mouton effect. The Cotton-Mouton effect arises from a magneto- optic interaction in the presence of a transverse magnetic field. The magneto-optic interaction can be sensitive to the spin state or physical arrangement of molecular or macro-molecular structural units contained in the medium under study. The Cotton-Mouton effect gives rise to magnetic birefringence or dichroism. The term "classical Cotton-Mouton effect" is used herein to describe optical phenomena induced by the Cotton-Mouton effect via the deformation of the linear optical polarisability of a medium's molecular constituents. This can be regarded as the magneto-optic analogue of the Kerr effect. Measurements of this type are with in the scope of the invention. However, preferably an effect which is herein after termed the "Extraordinary Cotton-Mouton effect" is exploited. In the Extraordinary Cotton-Mouton effect, the medium under analysis is supra- molecular in nature, and can physically rotate to align with the applied magnetic field. This can give rise to a much more substantial effect in comparison to the classical Cotton-Mouton effect.

Generally, step ii) includes introducing electromagnetic radiation into the sample. Advantageously, the physical property detected is a property associated with the interaction of electromagnetic radiation with the analyte.

Preferably, the electromagnetic radiation propagates through the sample along an axis which is transverse to the direction of the applied magnetic field.

The electromagnetic radiation may be of any suitable wavelength. Typically, electromagnetic radiation in the range 400 to 2,500nm is used.

Advantageously, the electromagnetic radiation is polarised. In these embodiments, the physical properties detected may be related to a polarisation state of the electromagnetic radiation. The physical properties detected may be the phase of the electromagnetic radiation. Linearly or circularly polarised light may be utilised. The ellipticity of the electromagnetic radiation after interaction with the sample could be measured. Both amplitude and phase may be detected.

Alternatively, the polarisation state of the electromagnetic radiation may be varied, and a dependence of the detected physical property on the polarisation state may be detected.

In some embodiments, the transmission of the electromagnetic radiation through the sample is detected.

The magnitude and/or the direction of the applied magnetic field may be varied, and a dependence of the physical property on the variation of the applied magnetic field may be detected.

Typically, the detection of the analyte in the sample is performed ex vivo.

Preferably, the sample is a sample of blood, blood serum or cerebrospinal fluid.

The neurodegenerative disease detected may be Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or multiple sclerosis.

Advantageously, the analyte is transferrin or ferritin. In biological systems, iron acts an essential cofactor to a wide range of enzymes, and exists either bound to proteins, or as part of a larger prosthetic group such as iron-sulphur clusters or haem groups. In humans, non-enzymatic iron is predominantly found either bound to transferrin (for transportation), or in storage forms (bound to ferritin or haemosiderin). The present invention proposes that transferrin and ferritin are particularly useful markers for the presence of neurodegenerative disease via magneto-optic detection. It is known that iron plays a role in Alzheimer's disease, a characteristic of which is the presence of iron (ferritin) bearing senile plaques and neurofibrillary tangles in the post-mortem brains of patients. A range of magneto-optic based detection schemes might be utilised. However, it is known that both ferritin (10) and transferrin (1 1 ) can agglomerate in solution. Ghosh et al (1 1 ) have demonstrated that human transferrin readily forms protein fibrils under conditions typical of those present in living organisms. These fibrils exhibit periodic iron nanomineralisation along their length which is of the same order as haemozoin crystals produced during malarial infection. The present inventors have previously demonstrated that haemozoin crystals produced during malerial infection can be easily detected by the Cotton-Mouton effect at concentrations (1 ng/ml) well below required for reliable diagnosis (International Patent Application PCT/GB07/004300; references12-14, the entire contents of all of which are hereby incorporated by reference). Other iron containing molecules which are present as fibrils or other agglomerations, in particular substantially linear agglomerations such as rods, might be detected by the Extraordinary Cotton-Mouton effect, or by other magneto- optic techniques.

Thus, the analyte may be present as an agglomeration, such as a fibril structure, supra-molecular structures of this type can give rise to enhanced magneto-optic effects, such as the Extraordinary Cotton- Mouton effect.

According to a second aspect of the invention there is provided a device for detecting a neurodegenerative disease in a subject by interrogating a sample of the subject's bodily fluid which contains an analyte, wherein the analyte is an iron containing molecule related to iron homeostasis, the device including:

a detection system which can detect a disruption of normal iron homeostasis by detecting the analyte in the sample using a magneto-optic detection technique; and

a correlator for correlating the detection of the analyte with the presence of the neurodegenerative disease.

The detection system may include a magnetic field production device for applying a magnetic field across at least a portion of the sample, and a detector for detecting a physical property of the sample, the detected physical property corresponding to a physical property of the analyte which varies in dependence with the applied magnetic field. The device may be configured so that magnitude and/or the direction of the applied magnetic field is controllably variable. In these embodiments, the magnetic field production device may include an electromagnet. Alternatively, the magnetic field production device may be a permanent magnet having a movement device for moving the permanent magnet with respect of the sample. For example, the permanent magnet may be rotated around the sample.

The detection system may include a source of electromagnetic radiation which is introduced into the sample. A convenient, although non-limiting, example is a laser, such as a diode laser. The laser or other source of electromagnetic radiation is selected so as to produce electromagnetic radiation of a desired wavelength, which is typically in the visible or near infra-red region of the electromagnetic spectrum. In some embodiments, the device is configured so that the electromagnetic radiation propagates into the sample along an axis which is transverse to the direction to the applied magnetic field.

The detector may detect a property associated with the absorption of magnetic radiation by the analyte. For example, the detector may detect electromagnetic radiation transmitted through the sample.

Advantageously, the source of electromagnetic radiation provides polarised electromagnetic radiation. The detector may detect the polarisation state of the electromagnetic radiation. The amplitude of the electromagnetic radiation may be detected also. The source of electromagnetic radiation may provide linearly or circularly polarised electromagnetic radiation, and the detector may detect a phase angle.

To the best of our knowledge, the only previous Cotton-Mouton measurements of molecules responsible for the transportation or storage of iron are those of Dobek et al (15) on ferritin in solution at concentrations down to about 1 .5mg/ml. Analysis of the results led these researchers to conclude that the magnetic birefringence observed via the Cotton-Mouton effect arose only from deformation of the linear optical polarisability of the ferritin molecule. There was no suggestion in this reference that neurodegenerative disease might be detected. The present inventors have realised that enhanced detection of ferritin and transferrin per se can be achieved when these analytes are present as agglomerations. Thus, according to a third aspect of the invention, there is provided a method of detecting an analyte in a sample, in which the analyte is an agglomeration of transferrin or ferritin, the method including the steps of:

i) applying a magnetic field across at least a portion of the sample to orient the analyte with respect to the applied magnetic field; ii) detecting a physical property of the sample which corresponds to a physical property of the analyte which varies in dependence of the orientation of the analyte with respect to the applied magnetic field; and

iii) correlating the detected physical property with the presence of the analyte.

According to a fourth aspect of the invention there is provided a device for detecting an analyte in a sample, in which the analyte is an agglomeration of transferrin or ferrritin, the device including:

a magnetic field production device for applying a magnetic field across at least a portion of the sample to orient the analyte;

a detector for detecting a physical property of the sample which corresponds to a physical property of the analyte which varies in dependence with the orientation of the analyte with respect to the applied magnetic field; and

a correlator for correlating the detected physical property with the presence of the analyte.

Whilst the invention has been described above, it extends to any inventive combination of the features set out above, or in the following description, drawings or claims. For example, any feature described with reference to one aspect of the invention may be applied or utilised in other aspects of the invention.

Methods and devices in accordance with the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:-

Figure 1 shows a first detection arrangement of the invention; and

Figure 2 shows a second detection arrangement of the invention.

Figure 1 shows a detection arrangement, depicted generally at 10, for transmission measurements of iron containing compounds which are associated with iron homeostasis. The arrangement 10 comprises a light source 12 producing light at a desired wavelength or range of wavelengths, the output of which passes through a polariser 14 before entering a suitable cell 16 containing a liquid sample under analysis. A suitable photo-detector 18 is positioned at or near to the rear face of the cell 16 so that light from the light source 12 which is transmitted through the cell 16 is detected by the photo-detector 18. The output of the photo- detector 18 is analysed by a suitable analysis device 20 such as a computer or other microprocessor containing device. The detection arrangement 10 further comprises a magnetic field production device 22. The analysis device 20 may also function as a controller, or a physically separate controller device may be utilised. The controller can be used to control the operation of the light source 12 and/or the magnetic field production device 22 so that detection is performed using magnetic field modulation. This can be achieved by employing an electromagnet as the magnetic field production device 22 and operating the electromagnet so as to modulate the strength of the magnetic field applied across the sample in the cell 16. Alternatively, an arrangement can be utilised in which a permanent magnet applies the magnetic field across the sample in the cell 16. The permanent magnet can be moved by a suitable mechanical arrangement, for example utilising one of the actuators which are controlled by the controller thereby modulating the direction of the applied magnetic field. Rotation of the permanent magnet around the sample is possible. Modulation sensitive detection of the magnetically meditated absorption of the light is performed.

The response of the iron-containing analyte can be determined in a straightforward manor by using a broadband light source 12 together with some form of variable wavelength selection, such as appropriate filters or a monochromator. Alternatively, one or more tuneable laser sources may be used. In the case of transferrin fibrils, detection in the red region of the visible spectrum is likely to be possible.

Figure 2 shows a second detection arrangement depicted generally at 30, which shares many features with the detection arrangement 10 shown in Figure 1 . Identical numerals are used to depict such shared features. The arrangement 30 again detects light which is transmitted through the sample 16. In the arrangement 30, measurements are made of the polarisation state of the light after interaction with an iron-containing marker analyte such as ferritin or transferrin. The arrangement 30 further comprises a phase modulator 32 positioned between the polariser 14 and the cell 16. The phase modulator 32 modulates the polarisation state of the light incident on the cell 32. For example, the phase modulator may modulate the light between left and right-handed circular polarisation, or between two orthogonal states of linear polarisation. A second polariser 34 may be provided between the cell 16 and photo-detector 18, and this second polariser 34 may be adjustable so as to determine the polarisation state of the light transmitted through the cell 16. The operation of the light source 12, phase modulator 32 and, if required, second polariser 34 can be controlled by analysis device 20 or by a separate controller. In the arrangement 30, detection of the iron-containing molecule is achieved by investigating the polarisation of the light after interaction with the iron- containing molecule. Linear or circular dichroism can be detected. For example, ellipticity can be measured. Other techniques which involve the measurement of a phase angle can be employed, as will be known to the skilled reader. Both amplitude and phase can be measured and analysed by the analysis device 20. The magnetic field applied by the magnetic field production device 22 may be modulated under the control of the analysis device 20 or a separate controller in order to enhance sensitivity with respect to the magneto-optically sensitive iron-containing analyte molecule.

A detection technique such as interferometric coherent detection may be used if the sample introduces a significant scattering dependent depolarisation of the light. Red blood cells are a source of such depolarisation, and therefore the use of interferometric coherent detection may be contemplated if analysis of blood samples is envisaged. However, it is not envisaged that techniques such as this are required in embodiments in which the sample is blood serum obtained after separation of red bloods cells, or cerebrospinal fluid.

References

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Claims

1 . A method of detecting a neurodegenerative disease including the steps of:

ii) detecting a disruption of normal iron homeostasis by detecting the analyte in the sample using a magneto-optic detection technique; and

iii) correlating the disruption of normal iron homeostasis detected by the magneto-optic detection technique with the occurrence of the neurodegenerative disease.

2. A method according to claim 1 in which step ii) includes:

applying a magnetic field across at least a portion of the sample; and

detecting a physical property of the sample, the detected physical property to corresponding to a physical property of the analyte which varies in dependence with the applied magnetic field.

3. A method according to claim 2 in which the applied magnetic field orients the analyte with respect to the applied magnetic field, and the detected physical property corresponds to a physical property of the analyte which varies in dependence on the orientation of the analyte with respect to the applied magnetic field.

4. A method of detecting a neurodegenerative disease according to any one of claims 1 to 3 in which the magneto-optic detection technique utilises the Cotton-Mouton effect.

5. A method of detecting a neurodegenerative disease according to any previous claim in which step ii) includes introducing electromagnetic radiation into the sample.

6. A method of detecting a neurodegenerative disease according to claim 5 in which the physical property detected is a property associated with the interaction of electromagnetic radiation with the analyte.

7. A method of detecting a neurodegenerative disease according to claim 5 when dependent on claim 2 in which the electromagnetic radiation propagates through the sample along an axis which is transverse to the direction of the applied magnetic field.

8. A method of detecting a neurodegenerative disease according to any one of claims 5 to 7 in which the electromagnetic radiation is in the range 400 to 2500nm.

9. A method of detecting a neurodegenerative disease according to any one of claims 5 to 8 in which the electromagnetic radiation is polarised.

10. A method of detecting a neurodegenerative disease according to any one of claims 9 in which physical property detected is related to a polarisation state of the electromagnetic radiation.

1 1 . A method of detecting a neurodegenerative disease according to claim 10 in which the physical property detected is the phase of the electromagnetic radiation.

12. A method of detecting a neurodegenerative disease according to any one of claims 5 to 1 1 in which the transmission of the electromagnetic radiation through the sample is detected.

13. A method according to any of claims 3 to 12 when dependent on claim 2 in which the magnitude and/or the direction of the applied magnetic field is varied, and a dependence of the physical property on the variation of the applied magnetic field is detected.

14. A method according to any previous claim in which the detection of the analyte in the sample is performed ex vivo.

15. A method according to any previous claim in which the sample is a sample of blood, blood serum or cerebrospinal fluid.

16. A method according to any previous claim in which the neurodegenerative disease detected is Alzheimer's disease.

17. A method according to any one of claims 1 to 15 in which the neurodegenerative disease detected is Parkinsons's disease, amyotrophic lateral sclerosis, or multiple sclerosis.

18. A method according to any previous claim in which the analyte is transferrin or ferritin.

19. A method to any previous claim in which the analyte is present as an agglomeration such as a fibril structure.

20. A method of detecting an analyte in a sample, in which the analyte is an agglomeration of transferrin or ferritin, the method including the steps of: i) applying a magnetic field across at least a portion of the sample to orient the analyte with respect to the applied magnetic field; ii) detecting a physical property of the sample which corresponds to a physical property of the analyte which varies in dependence on the orientation of the analyte with respect to the applied magnetic field; and

21 . A device for detecting a neurodegenerative disease in a subject by interrogating a sample of the subject's bodily fluid which contains an analyte, wherein the analyte is an iron containing molecule related to iron homeostasis, the device including:

22. A device according to claim 21 in which the detection system includes a magnetic field production device for applying a magnetic field across at least a portion of the sample, and a detector for detecting a physical property of the sample, the detected physical property corresponding to a physical property of the analyte which varies in dependence with the applied magnetic field.

23. A device for detecting a neurodegenerative disease according to claim 22 which is configured so that the magnitude and/or the direction of the applied magnetic field is controllably variable.

24. A device according to claim 23 in which the magnetic field production device includes an electromagnet.

25. A device for detecting a neurodegenerative disease according to claim 23 in which the magnetic field production device is a permanent magnet having a movement device for moving the permanent magnet with respect to the sample.

26. A device for detecting a neurodegenerative disease according any one of claims 21 to 25 in which the detection system includes a source of electromagnetic radiation which is introduced into the sample.

27. A device for detecting a neurodegenerative disease according to claim 26 when dependent on claim 22 which is configured so that the electromagnetic radiation propagates into the sample along an axis which is transverse to the direction of the applied magnetic field.

28. A device for detecting a neurodegenerative disease according to claim 26 or claim 27 in which the detector detects a property associated with the absorption of electromagnetic radiation by the analyte.

29. A device for detecting a neurodegenerative disease according to claim 28 in which the detector detects electromagnetic radiation transmitted through the sample.

30. A device for detecting a neurodegenerative disease according to any one of claims 26 to 29 in which the source of electromagnetic radiation provides polarised electromagnetic radiation.

31 . A device for detecting a neurodegenerative disease according to claim 30 when dependent on claim 22 in which the detector detects the polarisation state of the electromagnetic radiation

32. A device for detecting a neurodegenerative disease according to claim 31 in which the source of the electromagnetic radiation provides circularly polarised electromagnetic radiation, and the detector detects a phase angle.

33. A device for detecting an analyte in a sample, in which the analyte is an agglomeration of transferrin or ferritin, the device including: a magnetic field production device for applying a magnetic field across at least a portion of the sample to orient the analyte;