WO2006002483A1

WO2006002483A1 - Dialysis-assisted optical fibre spectroscopy probe

Info

Publication number: WO2006002483A1
Application number: PCT/AU2005/000981
Authority: WO
Inventors: Andrei Zvyagin; Yuri German Anissimov; Michael Stephan Roberts; Amelie Verhaege
Original assignee: The University Of Queensland
Priority date: 2004-07-05
Filing date: 2005-07-05
Publication date: 2006-01-12

Abstract

Dialysis assisted optical fibre probes (2), comprising a dialysis membrane (7) defining a sample volume (8); and at least one optical fibre (3, 4), for transmitting illumination to the sample volume (8) and receiving a spectroscopic signature of one or more analytes (16) within the sample volume. The dialysis assisted optical fibre probe (2) can be use to obtain the spectroscopic signature, e.g. fluorescence, absorption or Raman spectra, from a biological or chemical sample.

Description

TITLE "DIALYSIS-ASSISTED OPTICAL FIBRE SPECTROSCOPY PROBE"

FIELD OF THE INVENTION This invention relates to an optical fibre probe for analysis of biological samples. In particular the invention relates to a micro-dialysis assisted optical fibre probe for conducting biological and chemical analysis.

BACKGROUND OF THE INVENTION The testing of biological samples often requires the taking of tissue or blood samples for analysis. The taking of samples may be painful, inconvenient and/or may compromise the health of the patient, in extreme cases. There is a constant search for improved techniques and new apparatus which reduce time, cost, inconvenience and pain of analysing biological samples.

Microdialysis is a well-established technique, initially employed to research the pharmacokinetics of neurotransmitters and opioids, in particular the time required for pharmaceuticals to cross the blood-brain barrier. M. Muller, Advanced Drug Delivery Reviews, 45, 2000, 255-269, discusses a range of clinical applications of microdialysis. Microdialysis involves the insertion of a catheter or cannula having a dialysis membrane extending from its distal end into the tissue of interest; the tissue is perfused with a buffer solution (commonly known as Ringers solution). Some endogenous and exogenous compounds migrate to the inner volume of the microdialysis membrane. Samples are pumped from the inner volume and analysed using a range of techniques. Depending on the exogenous compounds under investigation the samples may be analysed using standard HPLC, GS, UV- vis spectral techniques, biological assays. One of the disadvantages of current microdialysis techniques is that they require the use of pumps to extract samples from the relevant tissue site, making the apparatus bulky and expensive. Also, the external testing of samples adds to the time required to analyse samples. In addition, the microdialysis outflow concentration is not usually at equilibrium with the site of measurement, and therefore does not perfectly describe compound concentrations of the tissue site due to continuous flow of buffer through the microdialysis probe. This necessitates additional study of recovery vs. flow rate to be performed and makes interpretation of the results difficult. Overall, the relationship of drug concentration in the dialysate and the real concentration in the tissue is not simple and is subject to many factors adding to the complexity of analysis.

Microdialysis techniques use relatively slow perfusion rates, in order to achieve near equilibration between tissue and perfusate, which creates an inherent lag time and dispersion of drug within the microdialysis catheters. As a result the time resolution of sampling tissue fluid concentrations is often more than 10 minutes, therefore risking loss of important pharmacokinetic and pharmacodynamic information if investigating fast acting drugs. Optical spectroscopy provides an effective analysis technique for a wide spectrum of compounds. U. Utzinger and R.R. Richards-Kortum, J. Biomedical Optics, 8(1), 121-147, January 2003, reviews a range of optical spectroscopy techniques that can be used in testing biological samples, both in vivo and in vitro. Utzinger describes the use of optical fibre probes for use in reflectance, polarized reflectance, fluorescence and Raman spectroscopy; where with the appropriate selection of optical fibres and spectral analysis biological samples may be obtained. In essence all optical spectroscopy of biological samples requires illumination of the biological sample or tissue fluid, and collecting and analysing the absorption, fluorescence or Raman scatter. Utzinger alludes to that the use of in vivo optical spectroscopy but indicates that it may only be used to indicate the presence of abnormal or cancerous cells. Current optical spectroscopy techniques are limited in their ability to identify and quantify the amount of exogenous compounds present in particular tissue, due to the interference of absorbing chromophores, endogenous fluorophores and/or scattering particles, such as tissue proteins.

Zhao, Y. et al, Anal. Chem. 1999, 71 , 3887-3893 describes the measurement of oxygen consumption in perfused mouse hearts using optic fibre probes wherein the tips have a layer of fluorescent tris(1 ,10- phenanthroline)ruthenium(ll)chloride hydrate (Rh(phen)₃ ²⁺) entrapped near or on the tip by a gas permeable photopolymerizable siloxane membrane. A single wavelength of illumination excites the Rh(phen)₃ ²⁺ to fluoresce. As the binding rate of Rh(phen)₃ ²⁺ with the oxygen in heart alters so does the intensity of fluorescence of the Rh(phen)₃ ²⁺. In essence, the rate of decay of fluorescence is measured as Rh(phen)₃ ²⁺ binds with oxygen.

Similarly, Werner, T. et al, Mikrochim Acta, 131 , 25-28 (1999) describes a fibre optic microsensor where the fibre tip is coated with an ion- selective lipophilic ion carrier, plasticised PVC, and ruthenium (II) tris-4,4¹- diphenyl-2,2'-bipyridyl ion-pair with bromothymol blue [Ru(dibipy)₃(BTB)₂] as a proton donor to measure chloride and potassium ion concentration by recording the decrease or increase of fluorescence of the excited Ru(dibipy)₃(BTB)₂ as a function of pH. Wolfbeis, O.S., Anal. Chem. 2002, 74, 2663-2678 reviews numerous articles and texts relating to fibre-optic chemical sensors and biosensors, all of which, like those described in Zhao, Y. and Werner, T. (supra), involve the entrapment of a selectively reactive species, such as dyes, metal complexes and enzymes, at the tip of an optical fibre by a membrane. The known optical fibre sensors are limited to testing for one characteristic, such as pH, or a select group of ions or compounds which react with the entrapped selectively reactive species. Currently available optical fibre sensors are limited in their usefulness as analytical techniques due to the selective nature of the reactive species located on the tip of the optical fibre. Furthermore, they suffer from the same disadvantages as the optical spectroscopy techniques when used on biological samples, as discussed above with reference to Utzinger.

OBJECT OF THE INVENTION It is an object of the invention to overcome or alleviate one or more of the above disadvantages or to provide the consumer with a useful or commercial choice. SUMMARY OF THE INVENTION

In a first aspect of the invention there is provided a dialysis assisted optical fibre probe for conducting assays of biological and chemical samples which comprises: a dialysis membrane defining a sample volume; and at least one optical fibre, for transmitting illumination to the sample volume and receiving a spectroscopic signature of analytes within the sample volume.

The dialysis assisted optical probe may further comprise a guide means to facilitate placing said at least one photonic device means in contact with the sample volume.

Said at least one optical fibre may be a single fibre which both transmits illumination and collects spectroscopic signature or may comprise two or more optical fibres in which one or more fibres transmit an illumination to the sample volume whilst one or more fibres collects spectroscopic signature. More suitably, said at least one optical fibre is preferably a multimode fibre having an outer diameter between 125-250μm.

The guide means is preferably a rigid cylindrical body adapted to retain the photonic device, having an open end which allows the photonic device to contact the sample volume. Preferably the guide means is a catheter, cannula, needle or the like

The dialysis membrane may extend from the open end of the guide means or from said at least one optical fibre. Preferably the dialysis membrane is a microdialysis membrane having a molecular weight cut off between 3 - 200 kDa.

In a second aspect of the invention there is provided a dialysis assisted optical fibre probe system, for conducting assays of biological and chemical samples comprising;

• the dialysis assisted optical fibre probe including; o a dialysis membrane defining a sample volume; and o at least one optical fibre, for transmitting illumination to the sample volume and receiving a spectroscopic signature of analytes within the sample volume

• an illumination source connected to said at least one optical fibre, for illuminating the sample volume;

• a collection means connected to said at least one optical fibre, for receiving the spectroscopic signature from the sample volume; and

• an analysis means connected to the collection means, for analysing the spectroscopic signature and producing a spectrum of one or more analytes within the sample volume. The illumination source may be any illumination source known to be used in optical spectroscopy including lasers, filtered white light, infra-red lamps, UV lamps and the like. Preferably the illumination source is a filtered UV lamp.

The at least one optical fibre probe of the first aspect may be selected from a single fibre which both transmits illumination and collects spectroscopic signature, or may comprise two or more optical fibres in which one or more fibres transmit an illumination to the sample volume whilst one or more fibres collects spectroscopic signature.

The analysis means may comprise matching lens, spectrometers, analogue to digital converters and personal computers having standard spectral analysis software. The analysis means preferably allows the recording and analysis of absorption, fluorescence or Raman spectra.

In a third aspect of the invention there is provided a method of analysing a biological and chemical sample including the steps of; • inserting a dialysis assisted optical fibre probe comprising; o a dialysis membrane defining a sample volume; and o at least one optical fibre, for transmitting illumination to the sample volume and receiving a spectroscopic signature of analytes within the sample volume; • passing an illumination, from an illumination source, through said at least one optical fibre, to the sample; and • collecting a spectroscopic signature of one or more analytes within sample; and

• analysing said spectroscopic signature to produce a spectrum of said one or more analytes.

The method of analysing biological samples may include a further step of perfusing the dialysis assisted optical probe with a buffer and allowing equilibration prior to illumination of the sample.

The buffer may be selected from physiological saline solution (0.9%

NaCI); phosphate buffered saline solution (PBS); HEPES buffer, MOPS buffer or other buffers or their modified formulation to be compatible with a site of sampling and capable of enhancing sampling and its further analysis.

The method of testing biological samples may be carried out in vitro or in vivo. Preferably the analysis of biological samples determines the presence of fluorophores, such as endogenous compounds and/or exogenous compounds. Endogenous compounds may include porphyrins and the like. Exogenous compounds of interest may include any pharmaceutically active compounds, which are administered to a subject to treat or control one or more diseases, conditions, ailments and the like.

Exogenous compounds may include, but not be limited to, aspirin, acetaminophen, acyclovir, albuterol, 3-n-butylphthalide, ciprofloxacin, diclofenac, dihydroergotamine, domperidone, dopamine, Elsamitrucin, epirubicin, fenofibrate, fluorescin, gliquidone, grepafloxacin, homovanillic acid, ibuprofen, indomethacine, ICG (indocyanine green), ketanserin, ketanserinol, lisinopril, lomefloxacin, meptazinol, metaproterenol, mirtazapine, Mycophenolic acid, naproxen, penicillin, pimozide, pirlindole, piroxicam, quinacrine, salidroside, sulpiride, sulphanilamide, streptomycin, tyrosine hydroxylase, Zaleplon, Zolpidem and the like.

BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention will be described, by way of example only, with reference to the accompanying drawings in which:

FIG 1 is a schematic representation of an embodiment of a dialysis assisted optical probe system of the invention, in use; FIG 2 is a graph showing the relationship between sample volume depth, defined by the distance between the free ends of the optical fibres and the bottom of the dialysis membrane, and optical fibre separation on collected fluorescence intensity. FIG 3 is a differential curve of the data from FIG 3 showing the relationship between sample volume depth and fluorescence intensity;

FIG 4 is a schematic representation of the sample depth and angles used in the mathematical modelling to determine sensitivity of the dialysis assisted optical probe of the invention. FIG 5(a) is a plot of fluorescence signal power versus sampling volume depth showing correlation of experimental results against theoretical fit (solid line).

FIG 5(b) is a plot of the fluorescence signal power versus the fluorophore (Rh:6G) concentration: linear fitting (solid line). FIG 6. is a plot of the normalized dialysis assisted optical fibre probe signal versus time showing the fit of experiment results against theoretical curve (solid line).

FIG 7A is a fluorescence spectra comparing Rhodamine:6G

(Rh:6G) and Rh:6G in combination with PC Red using standard fluorescence spectroscopy techniques;

FIG 7B is a fluorescence spectrum comparing Rh:6G and Rh:6G in combination with PC Red using the dialysis assisted optical fibre spectroscopy of FIG 1 ;

FIG 8A is a fluorescence spectrum comparing Rhodamine B (Rh:B) and Rh:B in combination with polystyrene spheres using standard fluorescence spectroscopy techniques; and

FIG 8B is a fluorescence spectrum comparing Rh:B and Rh:B in combination with polystyrene spheres using the dialysis assisted optical fibre spectroscopy of FIG 1. FIG 9 is a plot of (a) scattering origin, and (b) endogenous fluorophores, in which the spectra of aqueous Rh:6G is a reference (solid line) and the effects of (a) scattering tissue phantom, (b) endogenous fluorophore phantom without a membrane (clashed line), using a membrane (dotted line) are shown.

FIG 10 is a schematic representation of an alternative configuration of the optical fibre used in a dialysis assisted optical fibre probe of the invention.

DETAILED DESCRIPTION

FIG 1 shows an embodiment of the dialysis assisted optical fibre probe system 1 , in use in vivo. The microdialysis assisted optical fibre probe system 1 comprises photonic device 2, in this example an optical fibre probe. The optical fibre probe 2 has an illumination fibre 3 and a collection fibre 4 located within a guide means 5, in this example representing a cannula or needle. Needle 5 has a first or open end 6. A dialysis membrane 7 extends from or over the open end 6 of the needle 5 to define a sample volume 8. It will be appreciated that the dialysis membrane 7 may alternatively extend from or over the photonic device or optical fibre probe 2.

The dialysis membrane 7 may be selected from cuprophane, polycarbonate and polyethersylphone membranes with a molecular weight cut off between 3 and 20OkDa, depending on the application. An illumination source 9 provides illumination via a filter or fibre coupler 10 thorough the illumination fibre 3. The illumination source 9 for these examples is a 2nd harmonic of Nd:YAG laser of wavelength 532 nm. The illumination source 9, may also be a He-Ne laser of wavelength 543.5nm. The collection fibre 4 is connected to a spectrometer 11 via matching optics 12. In these examples the spectrometer 26, is an Action Research Spectra-Pro 275. The spectrometer 11 is in turn connected to an analysis means 13 which may comprise an analogue to digital converter 13 and a personal computer 14. The personal computer would be preloaded with known signal processing software used for spectral analysis.

The illumination fibre 3 and the collection fibre 4 for these examples are both multimode fibres having a core diameter of 200μm and numerical aperture (NA) of 0.39.

The optical fibre probe 2 is shown in FIG 1 as being inserted into the skin 14, by either placing the needle 5 into the skin or tissue and then locating the optical fibre probe 2 within the bore of the needle 5, or pre- locating the optical fibre probe 2 within the bore of the needle 5 and inserting both at the same time. The needle 2 protects the illumination fibre 3 and collection fibre 4 from damage during insertion into the skin 14. The needle 5 is inserted into the skin 14 in the normal manner used for inserting catheters, cannulas and the like. Once inserted the optical fibre probe 2 is allowed to come to equilibrium with the fluids of the surrounding skin or tissue sample 14. The skin 14, is shown schematically as being made up of tissue components 15, such as muscle fibres and proteins, and containing fluorophores 16.

Passing an appropriate illumination down the illumination fibre 3 illuminates the sample volume 8. The sample volume 8 will generate a spectroscopic signature, due to fluorescence from the fluorophores 16 within the sample volume 8. The spectroscopic signature, or fluorescence, is collected by the collection fibre 4 and analysed using standard fluorescence spectroscopic methods. Fluorophores 16 may include both endogenous compounds inherent within the tissue or skin and exogenous compounds, or pharmaceuticals, which are capable of fluorescing under the appropriate excitation.

The optical fibre probe 2 may be located into a sample container, for in vitro testing, so that the dialysis membrane is immersed in the sample, the probe 2 is allowed to come to equilibrium with the sample prior to illumination of the sample and collection of the optical spectra occurs in a similar manner to that outlined above.

It will be appreciated that in the instances that the dialysis assisted optical probe is to be used for in vitro analysis the guide means 5 may be any rigid surround and/or support that protects the photonic device 2 from damage during use. It is also possible for the guide means 5 to be employed for in vitro testing if the photonic device 2 has sufficient robustness and resilience.

In another embodiment the dialysis assisted optical fibre probe system of the invention may be used in an absorption mode. In an absorption mode the optical fibre probe may be formed from a single fibre in combination with a photonic device with a reflective coating on its distal end. The photonic device has a light guide track that extends out a preferably appreciable amount of radiation outside to form an evanescent field. The combined device acts in both illumination and collection modes. Exogenous compounds within a tissue or skin sample are illuminated at a particular wavelength and the amount of energy absorbed by the exogenous compounds (spectroscopic signature) is measured. Absorption data and/or fluorescence spectral analysis can be conducted using known techniques and in a similar manner to that described with reference to FIG 1.

It will be appreciated by a person skilled in the art that the schematic arrangement of FIG 1 may be also readily used for analysing exogenous compounds utilising Raman scatter spectroscopy methods, without departing from the essence of the invention.

EXAMPLE 1 - Effect of the sample volume depth As a preliminary investigation the effect of sample volume depth, spatial separation of the optical fibres within the optical fibre probe 21 was tested using the bench top arrangement similar to the schematic arrangement discussed above with reference to FIG 1 . A sample containing 1 μM Rhodamine 6G (Rh:6G) in water was placed in the sample container 25. Rh:6G was used as a model for exogenous fluorophores such as aspirin, quinacrine and the like. The optical fibre probe was placed into the sample prior to the sample being illuminated with light having a wavelength of 532 nm from the green second-harmonic Nd:YAG a dialysis membrane defining a sample volume; and at least one optical fibre, for transmitting illumination to the sample volume and receiving a spectroscopic signature of one or more analytes within the sample volume, laser. The fluorescence from the Rh:6G was collected and analysed using a spectrometer.

The optical fibres utilised within the optical fibre probe were Thorlabs Multimode fibres having a core diameter of 200μm (radius r_c); a clad diameter of 225μm and a numerical aperture (NA) of 0.39.

The Rhodamine 6G solution used in this example has a quantum efficiency of η~ 1 and a molar absorption coefficient (ε) of 0.50e6 L/mol.m.

Variation in the sample volume depth and fibre separation were analysed for their effect on fluorescent spectra readings.

It was found that a majority of the fluorescence signal was collected with the sample volume proximal to the free end of the collector fibre. The sensitivity of the optical fibre probe was calculated utilising the below formulae.

W W_o xεxC_M xη _{

" total det ^'-{z-z₀ xcos0_β + Jz₀ ² + r² -Jz² + r²^

wherein : n = concentration of fluorophores

CM = moral concentration of the solution [mol/L] NA = Avogadro's Number (6,022045x10²³mol^'1) I = intensity at illumination fibre end [W/cm²] W₀ = power of illumination fibre end [W] R₀ = radius core diameter of the illumination fibre η = quantum efficiency ε = molar absorption coefficient FIG 2 shows the relationship between the strength of the fluorescence signal with variation in the sample depth and the spatial separation of the illumination and collection fibres. It can be seen from FIG 2 that it is preferable to have the collection and illumination fibres as close as possible. The excessive sample volume, i.e. over 1 mm, can be seen to have little effect on the signal accumulation, thus proving the useful local property of the technique.

FIG 3 is a differential fluorescence curve for the results of FIG 2. FIG 3 shows that the main contribution to the fluorescent signal comes from 0.5mm sample depth, where the preferential sampling occurs.

Example 2 - Mathematical modelling; Sensitivity and Response Time

The dialysis assisted optical fibre probe of the invention can briefly be described as comprising an illumination fiber which delivers excitation, e.g. laser light, of power P_1n to a sampling volume that contains analytes, preferably drug fluorophores, of concentration n [cm^"3] which permeated through the membrane from the tissue. The analyte (or fluorophores) can be characterized by quantum efficiency η , and absorption cross-section σ_a

[cm²]. The analyte excitation or induced fluorescence is collected by the collection fiber and analyzed to determine the analytes spectroscopic signature.

Fiber optic spectrometer collection efficiency

The spectra signal power of the dialysis assisted optical probe system depends on the power of the excited fluorescence, the fibre collection efficiency, and the spectrometer throughput. The power of the excited fluorescence P_βuo , in the dialysis assisted optical probe system sampling volume V, is expressed as:

P_fluo

, (1)

where I_m(r) denotes the excitation irradiation [W.cm^"2] emitted from the distal end of the fiber, which, in general, is a three-dimensional function. An optical field emerging from the distal end of a multimode fiber is conventionally modelled as a Gaussian beam. For the estimation purpose, however, it suffices to model this optical field as:

(1 ) a uniform optical beam of a constant diameter equal to that of the multimode fiber core, r_c, in the near-field approximation, and;

(2) a cone of light with uniform irradiance across each cross- section, in the far-field approximation.

The half angle of this cone is given by the acceptance angle θ_a, shown in FIG 4, here θ_a = diSm{NA_f / n_m) where NA_f is the numerical aperture of the fiber and n_m is the refractive index of the solute. Under these simplifying assumptions, I_m(r) is reduced to:

_{j (r)}J^p.A^πr?)>^nearf^ιeld ₍₂₎

[P,_n/{π4_Z ²NA_f ²), far field

where z is the distance from the distal end of the fiber. The integration in

Eq. (1) is simplified to one-dimensional integration over the z-axis under the conditions of Eq. (2). The combination of the fiber core diameter and NA_f determines the fiber collection efficiency, since only light reaching the fiber core within its acceptance cone will be guided. Taking into account Eq. (2) and cylindrical symmetry of the configuration, the collected fluorescence power is given by:

where ΔΩ(p,z) is the solid angle of fluorescence light that reaches the fiber core within its acceptance cone; p, z denote two cylindrical coordinates with the origin at the fiber tip center, z₀ denotes the maximum axial distance at which fluorescence is collected. The functional representation of ΔΩ(p,z) depends on the position of the analyte or fluorophore with respect to the optical fiber tip.

FIG 4 represents three geometric constructions for the collection of spectral signatures from biological samples. In Case 1 , the near-field approximation [see Eq. (2)] is represented, where the fluorophores located within one Rayleigh range from the fiber-tip, and within a cylinder of radius r_c centered about the fiber axis. In Case 2, the far field case for fluorophores satisfying p ≤ r_c is depicted and in Case 3, the fluorophores are located in the far-field, p > r_c . In Cases 2 and 3, the projected fluorescence emission angle subtended by the core is smaller than the acceptance angle of the fiber. The dependence of P_cf(z₀) versus the sampling volume depth z₀ , is plotted in

FIG 5(a) as a solid curve. It can be seen from FIG 5(a) that fluorophores in the close vicinity of the fibre (1 - 2 mm) primarily contribute to the signal under typical experimental conditions. Clearly showing that the dialysis assisted optical probe system only requires enclosure of several millimeters to be sufficient to collect most of the detectable fluorescence signal in the sampling volume. Finally, the spectrometer throughput needs to be taken into consideration for the dialysis assisted optical probe system detection sensitivity. If the numerical aperture of the coupling optics is matched to that of the spectrometer, the power detected by the photoreceiver is given by

where η_g is the grating diffraction efficiency, η_opt is the transmission efficiency characterizing losses on the optical surfaces of the spectrometer. dP_cf(z₀)/dλ [W.nnrf¹] is the collected fluorescence power spectral density and λ_s is the sampled wavelength. From Eq (4), the throughput of the spectrometer is also determined by the ratio of the input slit area A_slit to that of the focused spot A_spot , and the width of its spectral wavelength bandpass.

Response time constant of dialysis assisted optical probe system

In order to estimate the expected response time, a model of the dialysis assisted optical probe system as a long tube of radius r_Q , characterized by the permeation constant P [cm/s]. Consider the fluorophore concentration inside the probe c(t), versus time t, and assuming the initial concentration of fluorescent drug molecules outside the tube c_m, the equilibrium between inner and outer compartments is established via permeation and diffusion. In the standard diffusion theory framework, the equilibrium process can be approximated by the following relationship:

C(O = J- le^~{t-^τ)/'^«c_m(τ)dτ , (5)

^•eq

where t_eq is the equilibrium time. t_eq is the sum of the diffusion and permeation times: t_eg = t_ώff +t_pem , where t_dlff = r_o ²/(5.76/J>) represents the diffusion time, D is the diffusion constant [cm²/s], and; t_pem = 2r_o/P is the permeation time. If t_dlff » t_pem , and using typical parameters of the dialysis assisted optical probe system, of r₀ = 0.05 cm, D = 2.8 χ lθ^~6 cm²/s for

Rh:6G in water [Brock, R. et al, Biophysical Journal, 75(5), 2547-2557, (1998).] and D = 3.6 χlθ^~6 cm²/s for Rh:B in water [Rani, S. A. et al,

Antimocrobial Agents and Chemotherapy, 49(2), 728-732, (2005).], the t_dlff equilibration time is calculated to be 155 s and 121 s respectively. Such a short response time constant represents a very attractive target for biomedical drug sensing application, and is believed to be achievable upon optimization of the dialysis membrane parameters.

A theoretical curve numerically calculated from Eq. (3) [FIG 5(a), solid line] was fit to the experimental data points by using the spectrometer efficiency as an adjustable parameter. FIG 5(a) confirms that the major contribution to fluorescent signal received by the dialysis assisted optical probe system when used in fluorescence mode signal comes from the volume within 1.4 mm from the tip of the optical fibre, measured at the 90% signal fall off.

To determine the detection sensitivity of the fluorescence dialysis assisted optical probe system, the detected fluorescence power was plotted versus Rh:6G molar concentration over the range of 10 - 100 μM, as shown in FIG 5(b).

To confirm the above modelling the following experiment was carried out. The miniature dialysis assisted fibre-optic spectrometer was configured using a single illumination/collection multimode fiber, similar to that shown in

FIG 1. The outer cladding diameter, core diameter, and numerical aperture

{NA_f ) of this fiber were 125 μm, 105 μm, and 0.21 , respectively. A 1.2-mW laser of wavelength of 532 nm, was used as the illumination source. This illumination source was coupled into the multimode fiber (coupling efficiency of 69%) and delivered to the sampling head. A dichroic filter (wavelength cut-off, 540-nm) was used to separate the illumination radiation from the longer-wavelength fluorescence radiation collected by the distal fiber-tip. The collected fluorescence radiation was coupled into a computer-controlled scanning single-grating Czerny-Turner spectrometer (Acton Research Corporation, SpectraPro® 275, f/3.8, wavelength resolution 1.6 nm) terminated by a PIN photoreceiver.

The sampling head comprised a sealed membrane tube filled with buffer solution, in which the fiber distal end was immersed. The filtering performance of the dialysis membrane is determined by its molecular cut-off defined as the molecular weight, measured in Daltons, at which 80% of the molecules are prevented from passing through the membrane. The selected dialysis membrane had a molecular cut-off of 20,000 Daltons, which was sufficient to pass fluorophores of interest (fluorescent dyes, molecular weight < 500 Daltons), but prevent passing micron-sized particles that represented agents obscuring the spectroscopic signature in our study.

A biological phantom of small-molecular weight exogenous fluorophores, such as drug molecules, was represented by Rhodamine:6G dye (Rh:6G). Endogenous tissue fluorophores were mimicked by micron- sized dye-doped beads (PC-Red™, Fluoresbrite® Polychromatic Red

= 584 nm) that were unable to penetrate through the membrane. The scattering tissue was mimicked by aqueous suspension of 2- and 5-μm polystyrene spheres (15% by weight). In our experiments, 1 μM of Rh:6G was dissolved in water/methanol solution of equal volume ratio. This concentration is easily achievable in live tissue via the common drug delivery pathways. The liquid biological phantom was placed in a vial into which the sampling head was immersed. The spectroscopic signal was acquired by operating the spectrometer and transferring data to a PC for display, analysis, and archiving. The acquired spectra were processed to correct for the dichroic filter wavelength response, and also, to remove the instrumental high- frequency noise by low-pass filtering of the signal. The acquired spectroscopic signals using different biological phantoms were normalized for comparison.

As anticipated by the above modelling and confirmed by the above experiment, this dependence was linear with a slope of 0.431 pW/μM . Intersection of this signal line with the fluorescence signal detection threshold (noise floor) yielded a minimum detectable concentration of 340 nM, as applied to the described dialysis assisted optical probe system.

Response Time

Since the operation rate represents an important performance indicator of the proposed technique, we carried out measurement of the equilibrium time whose theoretical estimate is presented in Eq. (5). The peak detected fluorescence signal in a 1-mM aqueous solution of

Rhodamine B (Rh:B) was measured versus time using the dialysis assisted optical probe system, and the result is plotted in FIG 6. The data points were normalized to the asymptotic value of the signal saturation.

A theoretical curve (solid line) was fitted to the data using Eq. (5). The estimated time constant of this exponential theoretical curve yields an equilibrium time t_eq = 157 s. This measured time constant of the dialysis assisted optical probe system prototype was found to be 36 s longer than the theoretical value of 121 s, assuming a negligible permeation time constant.

It is possible to increase the dialysis assisted optical probe system operation rate and reduce the permeation time by reducing the radius of the dialysis membrane tube or using a membrane with a larger permeability. Whilst the permeation time constant was found to be approximately 2 minutes, the demonstrated equilibrium time is already superior benchmarked against the existing tissue assaying techniques, including microdialysis [P. Lonnroth, P. A. Jansson, and U. Smith, Am. J. Physiol. 253, E228-231 (1987).].

EXAMPLE 3 - Effect of tissue constituents and endogenous fluorophores Utilising the microdialysis assisted optical fibre probe system of FIG 1 the effectiveness of the optical fibre probe 2 and the effect of tissue constituents and endogenous fluorophores were investigated. A- Endogenous fluorophores

Tissue naturally contains a number of compounds, which fluoresce, including flavin, tryptophan, collagen and the like. These compounds are likely to interfere in any analysis which is attempting to measure the fluorescence of an exogenous compound.

To determine the effect of endogenous fluorophores using the optical fibre probe of the invention, the fluorescence spectra of a 10μM solution of Rh:6G (modelling an exogenous compound) and a solution of 10μM Rh:6G with 10μM PC Red where measured using standard fluorescence spectrometric techniques. The PC Red, which has a diameter of approximately 2 μm, is used to model the effect of endogenous compounds. The initial spectra are shown in FIG 7A. It can be seen in FIG 7A that the presence of PC Red significantly alters the spectrum, resulting in a shift in spectral peak intensity from approximately 562nm, for Rh:6G alone, to approximately 580nm for the Rh:6G and PC Red. In addition, there is a significant suppression of intensity at about 562nm in the spectra for Rh:6G and PC Red indicating that endogenous constituents may results in spectrum from exogenous compounds being swamped or distorted to the point where no meaningful analytical data can be obtained.

In contrast, if the fluorescent spectra of the above samples where taken utilising the dialysis assisted optical fibre system of FIG 1 , shown in FIG 7B, the spectra of Rh:6G is barely altered by the presence of PC Red in the solution. It is believed that the microdialysis membrane prevented the PC Red from entering the sample volume 8 and therefore minimised interference arising from endogenous fluorophores.

B - Tissue constituents

It is known that tissue constituents, such as proteins, scatter illumination thus interfering with the spectrum that may be able to be obtained from exogenous compounds. To analyse the effectiveness of the optical fibre probe system of FIG 1 in minimising the scattering effect of tissue constituents, a fluorescence spectra were obtained for a solution of Rh:B and a solution of Rh:B and polystyrene spheres, using standard fluorescence spectral techniques. The Rh:B is used to model an exogenous compound whilst the polystyrene spheres are used to model the scattering effect of tissue constituents. The comparative spectra are shown at FIG 8A.

It can be seen that there is significant distortion of the spectra when polystyrene spheres are present in the solution with a shift in peak intensity from 580nm for Rh:B alone to approximately 590nm for Rh:B in the presence of polystyrene spheres. The fluorescent spectra were then recorded using the microdialysis assisted optical fibre probe system of FIG 1. The comparative spectra are shown in FIG 8B. It can readily seen that the scattering effect of the polystyrene spheres is almost eliminated when the optical fibre probe of the invention is used. The spectral results indicate that the microdialysis membrane 7 prevent scattering particles from entering the sample volume.

Example 4

Using the experimental conditions of example 2 above the dialysis assisted optical probe was investigated to determine the ability of the dialysis assisted optical probe system technique to suppress spectroscopic artefacts of scattering origin. The spectral shape of Rh:6G fluorescence signal acquired in clear aqueous solution (FIG 9, solid line) was markedly distorted in the presence of non-absorbing scatterers, as shown in FIG 9(a), dashed line, when dialysis assisted optical probe system was operated without a membrane. The enclosure of the sampling head with a dialysis membrane resulted in efficient suppression of the spectroscopic artefacts due to the scatterers (FIG. 9(a), dotted line), which would represent a considerable hindrance in the spectroscopic measurements in vivo.

The scatterer-affected peak was observed to occur either red-shifted in wavelength, as in FIG 9(a), or blue-shifted, depending on the scatterer size and concentration. It is hypothesised that the spectral shift may result from the intricate interplay of several scattering properties of non-absorbing scatterers versus wavelength: scattering cross-section, scattering anisotropy factor g, and probability of multiple scattering.

The immunity of dialysis assisted optical probe system to the presence of endogenous fluorophores or scatter particles was tested by using the liquid biological phantom that contained fluorescent polystyrene spheres PC-red. The fluorescence peaks of the exogenous and endogenous fluorophore phantoms were chosen to be well separated in wavelength (~30 nm), so that the obscuring effect was profound. Indeed, the spectroscopic signature of the exogenous fluorophores phantom [FIG 9(b), solid line] was completely distorted in the presence of the high-concentration endogenous fluorophores phantom (PC-red), as shown in FIG 9(b), dashed curve. The enclosure of the sampling head with a dialysis membrane caused considerable suppression of these spectroscopic artefacts [FIG 9(b), dotted line], and was a result of the filtering property of the dialysis membrane that prevented micron-sized PC-red particles from entering the sampling head. The residual red-shift of the filtered spectroscopic signal in dialysis assisted optical probe system is believed to be due to the contribution of the endogenous fluorophores phantom situated outside the sampling head. Example 5 - Absorption DAFOS

The dialysis assisted optical probe may be used to collect absorption signatures from a biological sample by altering the dialysis assisted optical fibre probe 2 to comprise a fibre optic guide and exposure devices whose distal end is enclosed with a dialysis membrane. FIG 10 is a schematic representation of an alternative optical fibre probe 2 having an illumination- collection optical fibre coupling 101 located in a wave guide 102. A reflective surface 103 is located at the free end of illumination-collection optical fibre coupling 101. A broadband source illumination is delivered via an illumination-collection optical fibre coupling 101 and guided into the sample volume by the wave guide or exposure device 102. The reflective surface 103 causes the illumination to be reflected or bounced back up the illumination-collection fibre coupling 101. The sample volume is exposed to the evanescent field 104 created by illumination passing through and reflected back into the optical fibre 101. The sample volume absorbs some of the evanescent field 104. The change in evanescent field 104 can be measured and analysed to record the absorption spectral signature of the analyte within the sample volume. In an alternative embodiment the dialysis assisted optical fibre probe may have the optical fibre tip tapered for a section to result in the tapered section used for collection of the spectral signature. Illumination is delivered along the length of the optical fibre to the tapered portion, which is in contact with the sample volume. This light is absorbed by the sample volume, the absorption is then recorded by the tapered optical fibre.

The Applicants believe that the absorption spectroscopic signatures can be simulated by biomedically-relevant absorbers, e.g. lndocyanine Green dye. In shot-noise limit, the minimum detectable molar concentration of the absorbing drug is given by

c_M^ = [e_MLη(θD,λ)l^ι[P₀pτl^1/2 , (2) where ε_u - the molar extinction coefficient, L - the taper length, η{θD,λ) is the fraction of optical power that extends out into the solute, η is a function of optical diameter of the taper and λ is the light wavelength. P₀ - the total guided power, p is the detector photoresponsivity, and τ is its integration time. The system sensitivity depends critically on the taper parameters, L and η . The preliminary numerical simulations carried out have yielded

?7 « 0.01 at OD=3.75, λ = 800nm considering existing tapering technology. The ultimate sub-nM sensitivity is theoretically attainable using reasonable parameters of the absorption dialysis assisted optical probe system. It is hypothesised by the Applicants that the optical fibre probe of the invention can be used in combination with a UV light source. To date UV light sources are not used for in vivo testing due to the mutagenesis properties of UV. It is anticipated that the optical fibre probe may enable the UV light source to be fully contained to such an extent that only the sample volume is exposed to the UV light. Such containment of the UV light will allow UV-visible spectra to be obtained for a range of exogenous compounds without exposing the patient to unnecessary mutagenesis side effects of the UV.

The experimental results available to date indicate that the microdialysis assisted optical fibre probe system of the invention is highly sensitive and can measure exogenous compound concentrations of <1 μM. As such the Applicants believe that the dialysis assisted optical fibre probe of the invention can be effectively used in monitoring transdermal delivery of pharmaceuticals, in particular exogenous fluorophores. It will be appreciated by the person skilled in the art that analysis of the spectroscopic signature of a biological and/or chemical sample will allow for the detection of analytes and/or determination of the concentration of analytes present in within the sample.

The Applicants have found that the optical fibre probe of the invention surprisingly provides significant advantages over the prior art methods of microdialysis or optical spectroscopy. The combination of fibre-optic spectroscopy and dialysis-assisted assaying methodologies enables efficient suppression of the spectral background that has plagued in vivo biomedical sensing applications of fibre-optic spectroscopy. The optical fibre probe and microdialysis assisted optical fibre probe system of the invention provide a simple, cost effective analysis technique which provides rapid real time analysis results, sensitive to low concentrations of pharmaceuticals or their metabolites, can be used locally and in a variety of tissue types and locations.

It should be appreciated that various other changes and modifications may be made to the invention described without departing from the spirit or scope of the invention.

Claims

CLAIMS:

1. A dialysis assisted optical fibre probe for conducting assays of biological and chemical samples which comprises: a dialysis membrane defining a sample volume; and at least one optical fibre, for transmitting illumination to the sample volume and receiving a spectroscopic signature of one or more analytes within the sample volume.

2. A dialysis assisted optical fibre probe of claim 1 , further comprising a guide means, having an open end, to facilitate the location of said at least one optical fibre in contact with the sample volume.

3. The dialysis assisted optical fibre probe of claim 1 , wherein said at least one optical fibre is a single fibre which both transmits illumination and collects spectroscopic signature.

4. The dialysis assisted optical fibre probe of claim 1 , wherein said at least one optical fibre comprises two or more optical fibres in which one or more fibres transmit an illumination to the sample volume whilst one or more fibres collects spectroscopic signature.

5. The dialysis assisted optical fibre probe of claim 1 , wherein said at least one optical fibre is a multimode fibre having an outer diameter between 125-250μm.

6. The dialysis assisted optical probe of claim 2, wherein the guide means is a rigid cylindrical body adapted to retain said at least one optical fibre and having an open end which allows said at least one optical fibre to contact the sample volume.

7. The dialysis assisted optical fibre probe of claim 2, wherein the guide means is a catheter, cannula or needle.

8. The dialysis assisted optical fibre probe of claim 2, wherein the dialysis membrane extends from the open end of the guide means.

9. The dialysis assisted optical fibre probe of claim 1 , wherein the microdialysis membrane has a molecular weight cut off between 3 - 200 kDa.

10. The dialysis assisted optical fibre probe of claim 1 , further comprising an evanescent waveguide at a sample end.

11. A dialysis assisted optical fibre probe system comprising;

• a dialysis assisted optical fibre probe including; o a dialysis membrane defining a sample volume; and o at least one optical fibre, for transmitting illumination to the sample volume and receiving a spectroscopic signature of analytes within the sample volume

• an analysis means connected to the collection means, for analysing the spectroscopic signature and producing a spectrum of one or more analytes within the sample volume.

12. The dialysis assisted optical fibre probe system of claim 10, wherein the illumination source is selected from lasers, filtered white light, infra-red lamps and UV lamps.

13. The dialysis assisted optical fibre probe system of claim 10, wherein the illumination source is a filtered UV lamp.

14. The dialysis assisted optical fibre probe system of claim 10, wherein said at least one optical fibre is a single fibre which both transmits illumination and collects spectroscopic signature.

15. The dialysis assisted optical fibre probe system of claim 10, wherein said at least one optical fibre is two or more optical fibres in which one or more fibres transmit an illumination to the sample volume whilst one or more fibres collects spectroscopic signature.

16. The dialysis assisted optical probe system of claim 10, wherein the analysis means comprises matching lens, spectrometers, analogue to digital converters and personal computers having standard spectral analysis software.

17. A method of analysing biological and chemical samples including the steps of;

• inserting a dialysis assisted optical fibre probe comprising; o a dialysis membrane defining a sample volume; and o at least one optical fibre, for transmitting illumination to the sample volume and receiving a spectroscopic signature of analytes within the sample volume;

• passing an illumination, from an illumination source, through said at least one optical fibre, to the sample; and • collecting a spectroscopic signature of one or more analytes within sample; and

18. The method of analysis of biological samples of claim 17, further comprises the step of perfusing the dialysis assisted optical probe with a buffer until equilibrium with the sample is achieved.