DEVICE FOR DETECTING AND/OR DETERMINING THE CONCENTRATION OF AN ANALYTE PRESENT IN A TISSUE AND A METHOD AND USE OF THIS DEVICE

Abstract

The device for detecting and/or determining the concentration of an analyte present in a tissue includes a sensor which is an optical fibre interferometer, and one interferometer arm being coated with an immobilised binding agent enabling selective binding of the analyte. The interferometer arm is mounted inside a guide enabling puncturing the tissue and performing an in situ measurement without the necessity to collect or prepare a sample. The guide is provided with a closed guide face, longitudinal perforations on sidewalls enabling the analyte to reach the binding agent, and an opening in the input end of the guide for introducing the interferometer with the arm into the guide. At the input end, the opening is sealed, enabling the isolation of the interior of the guide from the surroundings. The interferometer is mounted in a position in which the interferometer does not touch the inside walls of the guide.

Claims

1. A device for detecting and/or determining the concentration of an analyte present in a tissue, comprising: a sensor being comprised of an optical fibre interferometer, having one interferometer arm being coated with an immobilised binding agent so as to enable a selective binding of the analyte present in the tissue; a guide, wherein said interferometer arm is mounted inside a guide so as to enable puncturing the tissue and performing the measurement in situ, without the necessity to collect or prepare the sample, wherein said guide is comprised of a closed guide face, longitudinal perforations on sidewalls enabling the analyte to reach the binding agent and an opening in the input end of the guide for introducing the interferometer into the guide at the input end of the guide, the opening being sealed, enabling the isolation of the interior of the guide from the surroundings, and the interferometer being mounted in a position in which the interferometer does not touch the inside walls of the guide.

2. The device according to claim 1, wherein the interferometer is mounted centrally inside the guide, so that the axis of the interferometer core is not shifted relative to the guide axis by a value higher than 100 m.

3. The device according to claim 1, wherein the interferometer is mounted in the guide by epoxy glue.

4. The device according to claim 3, wherein setting of the epoxy glue is achieved by heating, upon which cooling of the interferometer combined with the guide was conducted in a controlled manner, so that the temperature drop would not exceed 10 C./s.

5. (canceled)

6. The device according to claim 1, wherein said guide is comprised of metal.

7. The device according to claim 1, wherein said guide has a length in a range of 0.5-30 cm, an internal diameter of 0.1 mm-5 mm, an external diameter of 0.2-7 mm and a shape resembling a biopsy needle.

8. The device according to claim 1, wherein the binding agent are comprised of antibodies or antigen binding fragments or antigen binding affibodies.

9. The device according to claim 8, wherein the analyte is a tumour marker or another disease marker, which can be used in diagnostics of diseases or treatment monitoring.

10. The device according to claim 9, wherein the analyte is an HER2, PSA, AFP, CA19-9, CA125 or CEA tumour marker.

11. The device according to claim 1, wherein the binding agent is a nucleic acid.

12. A method of detection and/or quantitative determination of an analyte present in a tissue, the method comprising the steps of: analysing the presence and/or concentration of said substance by means of an interferometer in the device as defined in claim 1.

13. The method according to claim 12, wherein the step of analysing the presence and/or amount of said analyte is preceded by a step of isolating a tissue sample and a step of puncturing the collected sample using the device as defined in claim 1.

14-17. (canceled)

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0076] FIG. 1 presents an interferogram for the case of an interferometer known in state of the art with equal arms and a change in this interferogram upon binding with an antigen layer 10 nm in thickness.

[0077] FIG. 2 presents an interferogram for the case of an interferometer with an imbalance of arms L=1 mm and a change in this interferogram upon binding with an antigen layer 10 nm in thickness.

[0078] FIG. 3 presents an interferogram for an imbalance of L=20 m and a comparison of the spectrum before and after binding with an antigen layer 10 nm in thickness.

[0079] FIG. 4 presents an embodiment of an optical fibre sensor for detecting the analyte.

[0080] FIG. 5 presents a spectral distribution observed on the detector for the sensor of FIG. 4.

[0081] FIG. 6 presents an alternative, preferred embodiment of the optical fibre sensor for detecting the analyte.

[0082] FIG. 7 presents a spectral distribution observed on the detector for the sensor of FIG. 6.

[0083] FIG. 8 presents an alternative, preferred embodiment of the optical fibre sensor for detecting the analyte.

[0084] FIG. 9 presents a spectral distribution observed on the detector for the sensor of FIG. 8.

[0085] FIG. 10 presents a construction layout of the device according to an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Examples

Example 1. A Sensor for Detecting an Analyte

Example 1A

[0086] In a preferred embodiment presented in FIG. 4, a sensor for detecting an analyte comprises a light source 1, a detector 2 and a coupler 4 connected to a circulator 3. The light source 1, in this particular case, is a superluminescent diode SLED 1550 nm Exalos number EXS210048-02, and the detector 2, in this particular case, is an optical spectrum analyser OSA ANDO AQ-6315B. A Corning SMF-28e+ optical fibre and an FBT 50:50 coupler produced by Fibrain were used.

[0087] The signal from the light source 1 is delivered to the circulator 3, from which it is subsequently delivered to the coupler 4 by single-mode pieces of optical fibre. The signal behind the coupler 4 is split into two optical fibresinterferometer arms. The signal is reflected from optical fibre faces, one of the arms 5 being coated with antibodies. The signal returns through the coupler 4 to the circulator 3, from which it is further led into the detector 2.

[0088] The interferometer arm whose end is not coated is longer by L=38.9 m.

[0089] One of the interferometer arms is coated with antibodies, which in this particular case is immunoglobulin G (IgG) from rabbit serum. These antibodies enable the detection of an antigen, which in this particular case is anti-rabbit immunoglobulin (obtained as a result of placing rabbit IgG in goat's blood plasma and separating the antigen) tagged with a luminescent marker FITC (Anti-IgG-FITC). Interference fringes in the wavelength domain, whose shift indicates the binding of antigens with antibodies, are visible on the detector.

[0090] The process of coating a piece of optical fibre with antibodies was conducted according to the method presented below. The coated end of the optical fibre was submerged in a solution of NaOH with a concentration above 1 M for 10 min., and subsequently rinsed in purified water and dried in the air for 15 min. It was then kept for hours in a temperature of 90 C. in a 10% solution of (3-glycidyloxypropyl)trimethoxysilane (GOPS) with water (volume fraction) with pH=2, established by means of a 1 M solution of HCl. Subsequently, the tip of the optical fibre was rinsed in ethanol, upon which it was dried for 13 h in a temperature of 105 C. in vacuum or in an argon atmosphere. Subsequently, it was submerged in a 100 mg/ml solution of 1,1-carbonyldiimidazole (CDI) in acetonitrile (ACN) for 20 min., upon which it was rinsed in acetonitrile and a solution of phosphate buffer (PBS) and incubated in a 1.2 mg/ml solution of PBS comprising the antibodies of immunoglobulin G from rabbit serum for 4 days in a temperature of 4 C., upon which it was stored in a solution of PBS for 12 h in a temperature of 4 C.

[0091] In order to perform the measurement, the device is brought into contact with the sample, so that the analyte present in the sample could be bound with the binding agent.

[0092] FIG. 5 presents the spectral distribution observed on the detector. The binding of antigens caused a spectral shift of 0.6 nm. In order to determine the amount of the analyte, standard solutions with known concentrations of the analyte are prepared and a calibration curve is established for the dependence of the change in the spectral shift on the amount of the analyte. The concentration of the analyte in the sample is determined by means of a calibration curve established in this manner.

[0093] Calculation of the optical thickness of layers bound in an interferometer system:

LIST OF REFERENCES

[0094] 1The wavelength at which a spectral minimum occurs before an increase in the thickness of the layers [0095] 2The wavelength at which a spectral minimum occurs after an increase in the thickness of the layers [0096] Light phase for the given wavelength upon passing through the optical system [0097] LpInitial interferometer imbalance length [0098] npThe refractive index of the material ensuring the initial interferometer imbalance [0099] Lchange in the length of the material at the face of the interferometer [0100] nThe refractive index of the material which is the increasing material

[0101] The minimum before the growth (increase) of the layers is considered to be the initial light phase 1. After the growth of the optical thickness of the layer, the same phase condition 2 will be fulfilled for another wavelength. Such a condition can be written down as:

[00001]${}_1=\frac{2.Math.\left(L_p.Math.n_p\right)}{{}_1}$${}_2=\frac{2.Math.\left(L_p.Math.n_p+.Math..Math.L\left(n_{}-1\right)\right)}{{}_2}$

Assuming

[0102]
.sub.1=.sub.2

We obtain

[00002]$\frac{2.Math.\left(L_p.Math.n_p\right)}{{}_1}=\frac{2.Math.\left(L_p.Math.n_p+.Math..Math.L\left(n_{}-1\right)\right)}{{}_2}$

[0103] Due to this, the following dependence can be reached after transformations:

[00003]$.Math..Math..Math.L.Math.=\frac{L_p.Math.n_p\left(\frac{{}_2}{{}_1}-1\right)}{n_{}-1}$

[0104] This dependence describes the change in the physical length of the layer which was bound in the system. More detailed explanations involving the calculation method can also be found in Hariharan, P. (2007). Basics of Interferometry. Elsevier Inc.

Example 1B

[0105] In an alternative, preferred embodiment presented in FIG. 6, the sensor for detecting the analyte comprises a light source 1, a detector 2 and a coupler 4. The light source 1 in this particular case is a supercontinuum-type source SLED 1540 nm Yenista OSICS SLD 1550, and the detector 2 in this particular case is an OSA Yokogawa AQ6370D spectrometer. A Standard Singlemode Fibre (SSMF) optical fibre fulfilling the guidelines of ITU-T, G.652 from the Draka company and a 10202A-50 coupler from the Thorlabs company were used.

[0106] The signal from the light source 1 is delivered to the coupler 4 via optical fibres. The signal behind the coupler 4 is split into two optical fibresinterferometer arms. The signal is reflected from the optical fibre faces, one of the arms 5 being coated with antibodies. The signal returns through the coupler 4 to the detector 2.

[0107] The interferometer arm whose end is coated is longer by L=10 m.

[0108] One of the interferometer arms is coated with antibodies, which in this particular case is immunoglobulin G (IgG) from rabbit serum. These antibodies enable the detection of an antigen, which in this particular case is anti-rabbit immunoglobulin (obtained as a result of placing rabbit IgG in goat's blood plasma and separating the antigen) tagged with a luminescent marker FITC (Anti-IgG-FITC). Interference fringes in the wavelength domain, whose shift indicates the binding of antigens with antibodies, are visible on the detector.

[0109] The process of covering one of the interferometer arms with antibodies consisted of several steps. The optical fibre was first degreased by means of isopropyl alcohol in order to remove contaminants. Subsequently, the optical fibre was submerged in distilled water and boiled for 15 minutes. Upon removal from water, the optical fibre was placed in a 2% solution of (3-mercaptopropyl)trimethoxysilane (MPTS) in toluene. After 30 minutes the optical fibre was removed and rinsed in toluene, and subsequently submerged in a solution of N-succinimidyl 4-20 maleimido-butyrate (GMBS, no. CAS 8-307-12-6) with a concentration of 6 mg/10 ml of ethanol with a concentration of 96%. The optical fibre was kept in this solution for 60 minutes. It was subsequently rinsed once in ethanol and three times in distilled water. The optical fibre was later placed in a solution of antibodies with a concentration of 1 mg/ml in a solution of phosphate buffer. The coated optical fibre was removed from the solution after 12 hours and rinsed in distilled water.

[0110] FIG. 7 presents the spectral distribution observed on the detector. The binding of antigens caused a spectral shift of 0.5 nm. In order to determine the amount of the analyte, standard solutions with known concentrations of the analyte are prepared and a calibration curve is established for the dependence of the change in the spectral shift on the amount of the analyte. The concentration of the analyte in the sample is determined by means of the calibration curve established in this manner. The method for calculating the thickness of the layer of the bound analyte is such as in Example 1A.

Example 1C

[0111] In a preferred embodiment presented in FIG. 8, the device for detecting the analyte comprises a superluminescent diode 1 (S5FC1018SSM Benchtop SLD Source) emitting a polarised light with a central spectral wavelength of 1310 nm and a spectral half-width of 50 nm, connected to a PANDA-type single-mode at a wavelength of 1260 nm or more optical fibre maintaining the polarity (PM1300-XP from the Thorlabs company), a PLC-type optical power splitter 6 with an optical power division of 50%:50% for a wavelength of 1310 nm, two PANDA-type single-mode at a wavelength of 1260 nm and more optical fibres maintaining the polarity with lengths of L1=50 mm, L2=50 mm, one of which comprises in the middle of its length a break 9, an optical power splitter 7 maintaining the polarity and an optical spectrum analyser 8. A PMC1310-50B-FC coupler from the Thorlabs company was used.

[0112] The signal from the light source 1 is delivered to the splitter 6. The signal behind the splitter 6 is split into two optical fibresinterferometer arms. The signal propagates through the interferometer arms, whose different lengths ensure the initial imbalance of the interferometer. One of the optical fibres comprises a break 9 in the middle of its length, and the ends on both sides of the break 9 are placed at a distance of 0.1 mm from each other. Through the coupler 7 the signal enters the optical spectrum analyser 8.

[0113] One of the tips of the cut optical fibre is coated with antibodies like in Example 1A, which in this particular case is immunoglobulin G (IgG) from rabbit serum. These antibodies enable the detection of an antigen, which in this particular case is anti-rabbit immunoglobulin (obtained as a result of placing rabbit IgG in goat's blood plasma and separating the antigen) tagged with a luminescent marker FITC (Anti-IgG-FITC). The coated end of the optical fibre has been moved back to the second tip at the same distance as before.

[0114] The measurement is performed by contacting the coated end of the optical fibre with the studied sample, so that the analyte could by bound by the binding agent.

[0115] Interference fringes in the wavelength domain, whose shift by 0.5 nm indicates the binding of an antigen layer with a thickness of 115 nm with antibodies, are visible on the detector (FIG. 9). In order to determine the amount of the analyte, standard solutions with known concentrations of the analyte are prepared and a calibration curve is established for the dependence of the change in the spectral shift on the amount of the analyte. The concentration of the analyte in the sample is determined by means of the calibration curve established in this manner. The method for calculating the thickness of the layer of the bound analyte is such as in Example 1A.

Example 1D. Coating with Affibodies

[0116] In a preferred embodiment presented in FIG. 4, the device for detecting the analyte comprises a light source 1, a detector 2 and a coupler 4 connected to a circulator 3. The light source 1 in this particular case is a superluminescent diode SLED 1550 nm Exalos number EXS210048-02, and the detector 2 in this particular case is an optical spectrum analyser OSA ANDO AQ-6315B. A Corning SMF-28e+ optical fibre and an FBT 50:50 coupler produced by Fibrain were used.

[0117] The signal from the light source 1 is delivered to the circulator 3, from which it is subsequently delivered to the coupler 4 by single-mode pieces of optical fibre. The signal behind the coupler 4 is split into two optical fibresinterferometer arms. The signal is reflected from the optical fibre faces, one of the arms 5 being coated with affibodies. The signal returns through the coupler 4 5 to the circulator 3, from which it is further led into the detector 2.

[0118] The interferometer arm whose end is not coated is longer by L=38.9 m.

[0119] In the case of the affibodies, immobilisation by means of thiol groups is applied using the available reagents (Thermofisher Scientific, Waltham, Mass.). To this end, the end of the interferometer is submerged in 1 M sulphuric acid for 1 h and subsequently rinsed with distilled water and submerged for 15 min in ethanol and for another 15 min in acetone. The cleaned end of the optical fibre is subsequently submerged in anhydrous acetone comprising 2% of APTES (3-aminopropyltriethoxysilane) for 0.5-5 min. Upon the end of the reaction, the optical fibre is rinsed in anhydrous acetone and dried in the air for 15-30 min. Subsequently, the optical fibre is incubated in a cross-linking buffer (50 mM of sodium phosphate, 0.15 M of NaCl, 10 mM of EDTA, pH 7.2) comprising 2 mg/ml of Sulfo-SMCC (sulphosuccinim idyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) for 1 h in room temperature. After rinsing in the cross-linking buffer and drying in the air for 30 min., the optical fibre is stored in 4 C. in a dessicator until the moment of incubation with the affibody.

[0120] In order to reduce the terminal thiol groups, to a solution of affibody with a concentration of 1 mg/ml in a reducing buffer (50 mM of sodium phosphate, 150 mM of NaCl, 2 mM of EDTA, pH 7.5), DTT (dithiothreitol) is added to obtain the final concentration of 20 mM with a subsequent incubation for 1-2 hours in room temperature or for 45 min in 37 C. The surplus of DTT should be removed by column filtration, e.g. with Sephadex G-25, equilibrated with the cross-linking buffer. The collected eluent comprising the affibody with a reduced thiol group should be immediately used to modify the previously prepared optical fibre. To this end, the end of the optical fibre is incubated in a solution of the Affibody for 2-4 h in room temperature and subsequently rinsed by means of the cross-linking buffer and stored in a solution of physiological saline (PBS) in 4 C.

Example 2. The Device for Performing Measurements Directly in Tissue

[0121] The interferometer according to Example 1A or 1B or 1C or 1D is placed in a metal guide 30 with a length ranging between 0.5 and 30 cm, an internal diameter of 0.1 mm-5 mm, an external diameter of 0.2-7 mm and a shape resembling a biopsy needle. The interferometer 10 is introduced through the opening 32 in the input end 34 of the guide and is attached to the input end 34 of the guide 30 by specialised epoxy glue, e.g. MULTIBOND-1101 from the MULTIBOND company, so that the surface of the interferometer does not touch the walls of the guide (like in FIG. 10). The joining of the interferometer with the guide was accomplished in such a manner that the interferometer was placed centrally inside the guide, so that the axis of the interferometer core would not be shifted relative to the guide axis by a value higher than 100 m (micrometres). Subsequently, the space between the interferometer and the guide was filled with epoxy glue and subjected to heating in a temperature range between +50 C. and +200 C. until achieving complete setting of the glue. Cooling the interferometer combined with the guide was conducted in a controlled manner, i.e. the temperature drop was no faster than 10 C./s. Controlling the rate of cooling the interferometer combined with the guide allowed obtaining a loss in the optical power/attenuation of the interferometer at a level below 0.02 dB.

[0122] Tests presenting the impact of sealing on attenuation/loss have been performed. The used measurement equipment:

[0123] A set for the measurement and monitoring of the changes in insertion loss (insertion attenuation):

[0124] The measurements used a device from the EXFO-IQ203 company integrated in a panel form, provided with:

[0125] a laser source (IQ2123 ORL) with two wavelengths: 1310 nm+20/25 nm and 1550 nm+10/40 nm,

[0126] with a spectral width of 45/65 nm (1310/1550 nm),

[0127] with a temperature stability of 0.03 dB (t=8 h T=0 . . . 50 C., and

[0128] an InGaAs (IQ1103) detector

[0129] measurement range 800-1700 nm,

[0130] relative precision of power measurement 0.015 dB,

[0131] The results of the conducted tests are presented in Table 1 below:

TABLE-US-00001 TABLE 1 The results of the studies of changes in insertion loss. A IL at airtight connections performed after the test Test results (The change in attenuation after the test should P.sub.in P.sub.out not exceed 0.2 dB for the criteria [|W] [W] of Telcordia GR1221) Sample [before [after A IL P-positive no. sealing] sealing] [dB] N-negative 1 5.373 5.342 0.00 P 2 6.338 6.338 0.00 P 3 6.695 6.679 0.01 P 4 6.203 6.177 0.02 P 5 6.811 6.790 0.01 P 6 6.510 6.513 0.00 P 7 6.708 6.709 0.00 P 8 6.614 6.613 0.00 P 9 6.844 6.834 0.01 P 10 6.371 6.359 0.01 P 11 5.831 5.827 0.00 P 12 5.750 5.755 0.00 P 13 6.227 6.231 0.00 P 14 6.285 6.275 0.01 P 15 6.502 6.488 0.01 P 16 5.968 5.945 0.02 P 17 6.281 6.285 0.00 P 18 6.330 6.324 0.00 P 19 6.480 6.451 0.02 P 20 6.524 6.520 0.00 P 21 6.174 6.173 0.00 P mean 0.01

[0132] The interferometer attached to the input end of the guide ensures the ability to sterilise the sensor and ensures free inflow of the tested substance, and at the same time ensures keeping the optical fibre sensor at a distance from the guide walls, so that the movement of the guide does not affect the performed measurement. Such a method of attachment provides a barrier against the penetration of biological and chemical contaminants and substances which could disrupt the measurement.

[0133] The guide 30 has a closed guide face 31 and longitudinal perforations 33 in sidewalls, enabling the analyte to reach the interferometer 10. The opening 32 in the input end of the guide, constituting an entrance for the piece of optical fibre, of the interferometer 10 into the guide 30 is sealed, enabling a tight isolation of the interior of the guide 30 from the surroundings.

[0134] The device in this embodiment also comprises, besides the light source 1 and the detector 2, a monitor 21 and a printer 22.

Example 3. Measurement of the Concentration of the HER2 Tumour Marker

Example 3A. Measurement with Antibodies

[0135] For the measurement of the HER2 tumour marker, a piece of optical fibre is prepared by immobilising the antibodies which selectively bind the HER2 tumour marker (anti-HER2 monoclonal antibodies, R&D Systems, Minneapolis, Minn.), as described in Example 1, the difference being that the binding agent is immunoglobulin selectively binding the HER2 tumour marker (anti-HER2 monoclonal antibodies, R&D Systems, Minneapolis, Minn.).

[0136] The measurement of the concentration of the tumour marker is performed by introducing for 5 sec.-15 min. the optical probe described in Example 1A or 1B or 1C, placed inside a metal guide, as described in Example 2, into the tissue by direct puncturing therewith the tumour tissue in the patient's body (similar as during a diagnostic biopsy performed with a biopsy needle) or directly after cutting off the tumour or by submerging it in a tissue homogenate obtained by mechanical dispersion thereof. The concentration of the marker present in the tissue corresponds to the amount of markers bound to the surface of the interferometer, and the value of the concentration is determined based on the spectral shift.

Example 3B. Measurement with Affibodies

[0137] The method for making an optical probe is different from the one from Example 3A in that instead of the antibodies, affibodies are immobilised using thiol groups, as described in Example 1D. The sensor is placed in the guide as described in Example 2. The measurement of a tumour marker, e.g. HER2, is performed similarly to Example 3A.

LIST OF REFERENCES

[0138] 1light source [0139] 2detector [0140] 3circulator [0141] 4coupler [0142] 5interferometer arm coated with a binding agent [0143] 6optical power splitter [0144] 7optical power splitter maintaining the polarity [0145] 8optical spectrum analyser [0146] 9break in the course of the optical fibre [0147] 10interferometer [0148] 21monitor [0149] 22printer [0150] 30guide [0151] 31guide face [0152] 32opening in the input end of the guide [0153] 33perforations [0154] 34input end of the guide