Device for measuring the concentration of an analyte in the blood or tissue of an animal or a human, particularly a premature infant, in a self-calibrating manner

Abstract

The invention relates to a device for measuring the concentration of an analyte in the blood or tissue of a an animal or a human, particularly a premature infant, wherein for measuring said concentration the device comprises a means (30) comprising at least a first and a second permeability with respect to said analyte, wherein the first permeability for said analyte differs from the second permeability for the analyte. Further, the invention relates to a corresponding method.

Claims

1. Device for measuring the concentration of an analyte in the blood or tissue of an animal or a human, which analyte passively diffused through the skin of said animal or human, wherein for measuring said concentration the device comprises a membrane (30) comprising at least a first and a second permeability with respect to said analyte, wherein the first permeability for said analyte differs from the second permeability for said analyte, wherein the membrane (30) is designed to be switched between a first state, in which said membrane (30) comprises said first permeability with respect to said analyte, and a second state, in which said membrane (30) comprises said second permeability with respect to said analyte, such that the mass flow rate of the analyte that diffuses through said membrane (30) when the membrane (30) resides in the first state differs from the mass flow rate of the analyte that diffuses through said membrane (30) when the membrane (30) resides in the second state, and wherein the device (1) comprises an analyzing means (20) for measuring the concentration of said analyte in the blood or tissue of said animal or said human, wherein said analyzing means (20) is designed to measure a first concentration of the analyte diffused through the membrane (30) residing in the first state, and wherein said analyzing means (20) is designed to measure a second concentration of the analyte diffused through the membrane (30) residing in the second state.

2. Device according to claim 1, characterized in that said means (20) comprises a probe head (10) for contacting the skin of said animal or said human, wherein the probe head (10) comprises said membrane (30).

3. Device according to claim 1, characterized in that the device comprises a switching means (40) for switching said membrane (30) between said two states, wherein for switching said membrane (30) between said two states, the switching means is designed to one of: irradiate the membrane (30) with electromagnetic radiation, irradiate the membrane (30) with light, apply an electric and/or a magnetic field to the membrane (30), change the pH-value of a medium contacting said membrane (30), change the temperature of the membrane (30), or exert a pressure and/or a shear stress on said membrane (30).

4. Device according to claim 3, characterized in that said switching means comprises a light source (40) designed to irradiate said membrane (30) with light.

5. Device according to claim 4, characterized in that the switching means comprises a light guide (401) for guiding said light of the light source (40) towards the membrane (30).

6. Device according to claim 2, characterized in that the probe head (10) comprises a diffusion chamber (100) adjacent to said membrane (30) for receiving said analyte.

7. Device according to claim 6, characterized in that the diffusion chamber (100) comprises an inlet (101) for feeding a perfusion medium (3) into the diffusion chamber (100), as well as an outlet (102) for discharging said perfusion medium (3) out of the diffusion chamber (100).

8. Device according to claim 7, characterized in that the analyzing means (20) is connected to said outlet (102), so that said perfusion medium (3) together with the respective analyte can be transported to the analyzing means (20).

9. Device according to claim 1, characterized in that said membrane (30) comprises a photochromic compound.

10. Device according to claim 1, characterized in that the membrane (30) comprises a spirobenzopyran moiety or a spirooxazine moiety, wherein particularly the membrane (30) is formed out of a polycarbonate coated or grafted with a compound or a polymer comprising said spirobenzopyran moiety or spirooxazine moiety.

11. Device according to claim 1, characterized in that said membrane (30) comprises an amphiphilic network comprising a spirooxazine moiety or a spirobenzopyran moiety.

12. Device according to claim 2, characterized in that the analyzing means (20) comprises a microfluidic chip (201) configured for measuring said concentrations using a fluorescence measurement, which microfluidic chip (201) is integrated into the probe head (10).

13. Device according to claim 12, characterized in that the microfluidic chip (201) is arranged on top of a diffusion chamber (100) of the probe head (10) on a side facing away from the membrane (30).

14. Method for measuring the concentration of an analyte in the blood or tissue of an animal or a human, the method comprising the steps of: letting the analyte diffuse through a membrane (30) comprising a first permeability with respect to said analyte, and letting said analyte diffuse through said membrane (30) comprising a second permeability with respect to said analyte, wherein the first permeability differs from the second permeability, and measuring a first concentration of the analyte diffused through the membrane (30) comprising the first permeability, and measuring a second concentration of the analyte diffused through the membrane comprising the second permeability, and determining the concentration of said analyte in the blood or tissue using said first and second concentration.

15. Method for measuring the concentration of an analyte in the blood or tissue of an animal or a human, comprising the steps of: letting the analyte diffuse through a membrane (30) comprising a first permeability with respect to said analyte, and letting said analyte diffuse through a further membrane comprising a second permeability with respect to said analyte, wherein the first permeability differs from the second permeability, and measuring a first concentration of the analyte diffused through the membrane comprising the first permeability, and measuring a second concentration of the analyte diffused through the further membrane comprising the second permeability, and determining the concentration of said analyte in the blood or tissue using said first and second concentration.

16. Device for measuring the concentration of an analyte in the blood or tissue of an animal or a human, which analyte passively diffused through the skin of said animal or human, wherein for measuring said concentration the device comprises a membrane comprising a first permeability with respect to the analyte and a further membrane comprising a second permeability with respect to said analyte, wherein the first permeability for said analyte differs from the second permeability for said analyte such that the mass flow rate of the analyte that diffuses through the membrane differs from the mass flow rate of the analyte that diffuses through the further membrane, and wherein the device (1) comprises an analyzing means (20) for measuring the concentration of said analyte in the blood or tissue of said animal or said human, wherein the analyzing means is designed to determine the concentration C.sub.g,body of the analyte in the blood of the animal or human with help of measuring a concentration of the analyte C.sub.ml for the first permeability and a concentration C.sub.mh of the analyte for a lower second permeability and calculating C.sub.mlC.sub.mh/(C.sub.ml−C.sub.mh).

17. Device for measuring the concentration of an analyte in the blood or tissue of an animal or a human, which analyte passively diffused through the skin of said animal or human, wherein for measuring said concentration the device comprises a membrane (30) comprising at least a first and a second permeability with respect to said analyte, wherein the first permeability for said analyte differs from the second permeability for said analyte, wherein the membrane (30) is designed to be switched between a first state, in which said membrane (30) comprises said first permeability with respect to said analyte, and a second state, in which said membrane (30) comprises said second permeability with respect to said analyte, such that the mass flow rate of the analyte that diffuses through said membrane (30) when the membrane (30) resides in the first state differs from the mass flow rate of the analyte that diffuses through said membrane (30) when the membrane (30) resides in the second state, and wherein the device (1) comprises an analyzing means (20) for measuring the concentration of said analyte in the blood or tissue of said animal or said human, wherein the analyzing means is designed to determine the concentration C.sub.g,body of the analyte in the blood of the animal or human with help of measuring a concentration of the analyte C.sub.ml for the first permeability and a concentration C.sub.mh of the analyte for a lower second permeability and calculating C.sub.mlC.sub.mh/(C.sub.ml−C.sub.mh).

Description

(1) Further features and advantages of the invention shall be described by means of a detailed description of embodiments with reference to the Figures, wherein

(2) FIG. 1 shows a schematical view (not to scale) of an embodiment of the device according to the invention;

(3) FIG. 2 shows a schematical cross section (not to scale) of a probe head of the device according to the invention;

(4) FIG. 3 shows a schematical view (not to scale) of the probe head positioned on an infant;

(5) FIG. 4 shows a perspective view of a probe head of a device according to the invention;

(6) FIG. 5 shows a top view of the probe head shown in FIG. 4;

(7) FIG. 6 shows a cross sectional view of the probe head along the line A-A of FIG. 5;

(8) FIG. 7 shows a cross sectional view of the probe head detail encompassed by circle B of FIG. 6;

(9) FIG. 8 shows a cross sectional view of the further probe head detail encompassed by circle C of FIG. 6;

(10) FIG. 9 shows a perspective view of a part of the probe head containing the diffusion chamber;

(11) FIG. 10 shows a lateral view of the part shown in FIG. 9;

(12) FIG. 11 shows a plan view onto the diffusion chamber according to FIG. 9;

(13) FIG. 12 shows a cross section A-A of FIG. 10;

(14) FIG. 13 shows a cross section B-B of FIG. 11;

(15) FIG. 14 shows detail C of FIG. 13;

(16) FIG. 15 shows detail D of FIG. 13;

(17) FIG. 16 shows a schematic overview of the device according to the invention,

(18) FIG. 17 shows a microfluidic chip of the device according to the invention for measuring glucose concentration;

(19) FIG. 18 shows a mold for a microfluidic chip of the kind shown in FIG. 17;

(20) FIG. 19 shows a mold for a microfluidic chip of the kind shown in FIG. 17;

(21) FIG. 20 shows a synthesized spiro-compound SP5 (2-(3′,3′-dimethyl-6-nitro-spiro[chromene-2,2′-indoline]-1′-yl) ethanol) that can be used in the present invention;

(22) FIG. 21 shows a synthesized spiro-compound SP7 with R=Me (2-(3′,3′-dimethyl-6-nitro-spiro[chromene-2,2′-indoline]-1′-yl)ethyl 2-methylprop-2-enoate) and SP9 with R═H (2-(3′,3′-dimethyl-6-nitro-spiro[chromene-2,2′-indoline]-1′-yl)ethyl prop-2-enoate) that can be used in the present invention;

(23) FIG. 22 shows a synthesized spiro-compound SP12 (3-(3′,3′-dimethyl-6-nitro-spiro[chromene-2,2′-indoline]-1′-yl)propanoic acid) that can be used in the present invention;

(24) FIG. 23 shows a synthesized spiro-compound SP14 with R=Me (2-[3-(3′,3′-dimethyl-6-nitro-spiro[chromene-2,2′-indoline]-1′-yl)propanoyloxy]ethyl 2-methylprop-2-enoate) and SP16 with R═H (2-prop-2-enoyloxyethyl 3-(3′,3′-dimethyl-6-nitro-spiro[chromene-2,2′-indoline]-1′-yl)propanoate) that can be used in the present invention;

(25) FIG. 24 shows a synthesized spiro-compound SO37 (1,3′,3′-trimethylspiro[benzo[f][1,4]benzoxazine-3,2′-indoline]-9-ol) that can be used in the present used in the present invention;

(26) FIG. 25 shows a synthesized spiro-compound SO39 with R=Me ((1′,3′,3′-trimethylspiro[benzo[f][1,4]benzoxazine-3,2′-indoline]-9-yl) 2-methylprop-2-enoate) and SO50 ((1′,3′,3′-trimethylspiro[benzo[f][1,4]benzoxazine-3,2′-indoline]-9-yl) prop-2-enoate) with R═H that can be used in the present invention;

(27) FIG. 26 shows a synthesized spiro-compound SO49 ((5′-acetoxy-1,3′,3′-trimethyl-spiro[benzo[f][1,4]benzoxazine-3,2′-indoline]-9-yl) prop-2-enoate) that can be used in the present invention;

(28) FIG. 27 shows the 3D flow streamlines inside the diffusion chamber as seen from the top, the flow being laminar;

(29) FIG. 28 shows a cross-section of the diffusion chamber showing the local dialysate extraction fraction having values between 0 (no substance extracted) and 1 (same concentration as in the blood). Shown are the results from a 3D Finite Element Method with a low permeability of the membrane;

(30) FIG. 29 same as in FIG. 28 simulated with a high permeability of the membrane;

(31) FIG. 30 shows the total dialysate extraction fraction at the outlet of the diffusion chamber as determined from the theory or from a FEM simulation. The two results are linearly proportional R.sup.2=9.99999;

(32) FIG. 31 shows that in the application range the dialysate extraction fraction is linearly proportional to the membrane permeability R.sup.2=9.99997;

(33) FIG. 32 shows the glucose concentration measurement with a standard fluorimeter and the kinetic enzymatic UV-method. The upper bound and the lower bound, between which 95% of the measurements should be located, are also shown (±2*RMSE);

(34) FIG. 33 shows the glucose concentration measurement with a microfluidics microfluorimeter. The upper bound and the lower bound between which 95% of the measurements should be located are also shown (±2*RMSE);

(35) FIG. 34 shows the permeability change of a plasma-induced spirobenzopyran and PHEMA coated track-edged polycarbonate membrane (pore diameter: 200 nm);

(36) FIG. 35 shows a glucose diffusion experiment through taped pig skin from the ear. The absorption of light is proportional to the glucose concentration;

(37) FIG. 36 shows a schematical view (not to scale) of an embodiment of the device according to the invention;

(38) FIG. 37 shows a schematical view (not to scale) of the probe head of FIG. 36 positioned on an infant;

(39) FIG. 38 shows a perspective view of the probe head of FIGS. 36 and 37;

(40) FIG. 39 shows an exploded view of the probe head of FIGS. 36 to 38;

(41) FIG. 40 shows a view of a top side of the probe head of FIG. 39 which faces away from the membrane;

(42) FIG. 41 shows a cross sectional view along the line A-A of FIG. 40;

(43) FIG. 42 shows the detail C of FIG. 41;

(44) FIG. 43 shows a view of the diffusion chamber of the probe head of FIG. 38 (without membrane);

(45) FIG. 44 shows a plan view onto the diffusion chamber of the probe head;

(46) FIG. 45 shows a cross sectional view along the line A-A of FIG. 44;

(47) FIG. 46 shows the detail B of FIG. 45;

(48) FIG. 47 shows an exploded view of an embodiment of the probe head according to the invention wherein an analyzing means in the form of a microfluidic chip is integrated into the probe head;

(49) FIG. 48 shows a plan view onto the upper side of the probe head of FIG. 47;

(50) FIG. 49 shows a plan view of the probe head of FIGS. 47 and 48;

(51) FIG. 50 shows the diffusion chamber of the probe head of FIGS. 47 to 49;

(52) FIG. 51 shows a modification of the device according to the invention of FIG. 16 with an extra vacuum pump with a vacuum regulator in the end,

(53) FIG. 52 shows an SEM micrograph of a mold for a microfluidic chip of the kind shown in FIGS. 17 and 47;

(54) FIG. 53 shows an SEM micrograph of a mold for a microfluidic chip of the kind shown in FIGS. 17 and 47;

(55) FIG. 54 shows an SEM micrograph of a mold for a diffusion (microdialysis) chamber of the kind shown in FIG. 47;

(56) FIG. 55 shows the 3D flow streamlines inside the diffusion chamber of the probe head shown in FIGS. 36 to 46, the flow being laminar;

(57) FIG. 56 shows a cross-section of a model of the diffusion chamber of the kind in FIG. 38, 44 showing the local dialysate extraction fraction having values between 0 (no substance extracted) and 1 (same concentration as in the blood). Shown are the results from a 3D Finite Element Method with a high permeability of the membrane;

(58) FIG. 57 shows a cross-section of a model of the diffusion chamber of the kind in FIG. 38, 44 showing the local dialysate extraction fraction having values between 0 (no substance extracted) and 1 (same concentration as in the blood). Shown are the results from a 3D Finite Element Method with a lower permeability of the membrane;

(59) FIG. 58 shows the total dialysate extraction fraction at the outlet of the diffusion chamber of the kind shown in FIGS. 38 and 44 as determined from the theory or from a FEM simulation. The two results are linearly proportional R.sup.2=1;

(60) FIG. 59 shows that in the application range the dialysate extraction fraction at the outlet of the diffusion chamber of the kind shown in FIGS. 38 and 44 is linearly proportional to the membrane permeability for both the theory R.sup.2=0.9996 and the simulation R.sup.2=0.9998;

(61) FIG. 60 shows that as predicted from the theory, the dialysate extraction fraction at the outlet of the diffusion chamber of the kind shown in FIGS. 38 and 44 does not depend on the height if the diffusion chamber in the 3D FEM simulation. There is no significant correlation between the chamber height and the dialysate extraction fraction. The differences in the dialysate extraction fraction between the different points come from computing errors; and

(62) FIG. 61 shows the dialysate extraction fraction of a probe head of the kind of FIG. 36-46 measured with two different states, once exposed with white light, once exposed with UV light.

(63) FIG. 1 shows a schematical illustration of a device 1 according to the invention. The device 1 comprises a probe head 10 (cf. also FIG. 4) comprising a diffusion chamber (also denoted as microdialysis chamber) 100 delimited by a membrane 30 for contacting the skin 2 of a patient P, wherein the diffusion chamber 100 comprises an inlet 101 as well as an outlet 102 for feeding a perfusion medium 3 into the diffusion chamber 100 and for discharging it out of the diffusion chamber 100, wherein the outlet 102 is connected to an analyzing means 20 for measuring the concentration of an analyte (e.g. glucose) in a perfusion medium 3, which analyte diffused through the membrane 30 in a first state of the latter, as well of the analyte in the perfusion medium 3, when the analyte diffused through the membrane 30 in a second state of the membrane, wherein the permeability of the membrane 30 with respect to said analyte differs in said two states such that the two concentrations of the analyte differ.

(64) Further, reagents 4 are provided for the ex-vivo measurement of the concentration of the analyte in said mixture with the perfusion medium 3 that flows to the analyzing means 20. For measuring said concentrations, the analyzing means 20 preferably comprises a microfluorimeter (optional if a different concentration measurement is chosen) 200, which comprises a microfluidic chip 201 that determines the glucose concentration, as well as a computer 202 or a dedicated embedded computer for computing the glucose concentration from the microfluorimeter 200, for displaying it, and for controlling an excitation for switching the membrane 30 from the first state to the second state and vice versa. Preferably, the membrane 30 is switched to the first state by irradiating it by means of a light source 40 with light, particularly UV light, having a first wavelength, preferably in the range from 300 nm to 400 nm, and to the second state by irradiating it by means of the light source 2 with light, particularly visible light, having a second wavelength, particularly in the range from 500 nm to 650 nm.

(65) FIG. 2 shows a schematical cross section of the probe head 10 of the device 1 according to the invention. According thereto, the light source 40 can be either embedded directly into the probe head 10 or can be located remotely and connected to the probe head 10 with a light guide 401. When the probe head 10 contacts the skin 2 of the patient P, said analytes (e.g. glucose) A′ of the ex-vivo body fluid of the patient P can diffuse through the membrane 30, which delimits the diffusion chamber 100, into the chamber 100.

(66) As can be seen from FIG. 3, preferably merely the probe head 10 comes into contact with the patient P.

(67) FIGS. 4 to 15 show a preferred embodiment of the probe head 10 of the device 1 according to invention.

(68) The probe head 10 comprises a body 11 (cf. FIG. 6) that is preferably formed out of a UV transparent PMMA. The body 11 comprises a first recess 12 on a rear side of the body 11 for insertion of a free end 401a of the light guide 401 designed to guide UV/visible light from the light source 40 to the membrane 30, which light guide 401 can be positioned and fastened with respect to the body 11 by means of three screws 402 protruding through associated apertures 403 (cf. FIG. 12) having matching threads into said first recess 12 so that the free end 401a can be clamped by means of said three screws 402. The body 11 further comprises a U-shaped second recess 100 forming said diffusion chamber 100 on a front side of the body facing away from said rear side (cf. FIGS. 8, 9, 13 and 14), which second recess 100 is covered by said light switchable membrane 30 which is releasably clamped to the body 11 by means of an elastic O-ring 13 that is arranged in a circumferential groove 14 formed an a lateral side of said body 11 when fastening the membrane 30 to the body 11 so as to cover the second recess/inflation chamber 100 with the membrane 30 (cf. FIGS. 6, 7, 9, and 10). The switchable membrane 30 could also be releasably self-glueing itself to the probe head 10, therefore not requiring the elastic O-ring 13 and the circumferential groove 14.

(69) As shown in FIGS. 4, 5, and 6 the probe head 10 further comprises a first conduit 110 being in flow connection to said inlet 101 as well as a second conduit 111 being in flow connection with to said outlet 102, wherein said inlet 101 and outlet 102 are formed in a bottom of said first recess, wherein said inlet 101 and said outlet 102 are formed as through-openings each opening out into an end of said U-shaped diffusion chamber 100 as shown in FIGS. 12, 13, 14 and 15.

(70) For determining the actual concentration of the analyte in the blood of the patient P, the ex-vivo analytes passively diffusing through the skin and the membrane 30 are analyzed. For this, these analytes are carried away from the probe head 10 to the analyzing means 20 by means of the perfusion medium 3. Appropriate analysis allows the measurement of analyte concentration in the perfusion medium 3. Thereafter, light as external stimulus is applied to change the diffusion resistance of the membrane, i.e., the membrane 30 is brought from the first state into its second state. After a second run of analyte analysis the blood analyte level can be calculated from the two consecutive concentration measurements with the different membrane states.

(71) For glucose analysis, the glucose containing perfusion medium/fluid can be mixed with an enzymatic solution for the stoichiometric conversion of glucose by the analyzing means 20. This final solution will then be spectroscopically analysed by a fluorimeter of the analyzing means 20. This is transferred into the microfluidic chip 201 connected to the outlet 102 of the probe head 10 as shown in FIGS. 1 and 16.

(72) FIG. 16 shows a block diagram of a device 1 according to the invention. The perfusion medium 3 is pumped via a particle filter 114 and a liquid flow meter 115 through inlet 101 into the diffusion chamber 100 of the probe head 10 and transports the analyte to the analyzing means 20, namely to the microfluidic chip 201 of the microfluorimeter 200 that is designed to measure the clucose concentration in the repective perfusion medium 3. Since a known flow of perfusion medium 3 is used, the concentration of the analyte in the respective fluid 3 can be determined by the analyzing means 20.

(73) Further, the reagents or reagent fluid 4 (preferably a Hexokinase/Glucose-6-phosphate deydrogenase/ATP/buffer/Mg2+ solution in water) for the ex-vivo measurement of the concentration that flows to the microfluorimeter 200 (both optional if a different concentration measurement is chosen; the analyzing means should just allow to monitor preferably continuously (or almost) the concentration within the required range with sufficient precision) are pumped via a flow meter 116 and a particle filter 117 to the microfluorimeter 200 of the analyzing means 20.

(74) The microfluidic chip 201 that determines the respective glucose concentration is connected to a computer or a dedicated embedded computer for computing the glucose concentration from the two concentrations for the two different membrane states as measured by the microfluorimeter 200. The computer 202 is further designed to display the computed concentration of the analyte in the blood of the patient P, as well as for controlling the switching means (light source 40) that switches the membrane 30 between the two states. Analyzed dialysate is discharged into a waste container 203.

(75) Further, the air compressor 120 or compressed air line, the precision air pressure regulators 118 (e.g. with feedback and vent), as well as the air pressure measurement devices 119 allow a pressure driven flow of the perfusion medium 3 through the probe head 10/diffusion chamber 100 into the microfluorimeter 200 and of the reagent fluid 4 into said microfluorimeter 200 with a very low pressure variation, leading to a very low flow variation and thus a very small error in equation (9) below. The pressure from the air compressor/compressed air line 120 is brought to two the two precision pressure regulators 118 so that the two reservoirs 3, 4 for the reagent fluid and the perfusion medium (or perfusate) are pressurized. Controlling now the pressure difference of each reservoir 3, 4 in comparison with the pressure of the analyzing means outlet (at the atmospheric pressure) the pressure difference will drive laminar flows into the probe head 10, tubing, and the microfluidic chip.

(76) As already introduced above, Fick's law models the passive diffusion across the skin of the patient and the membrane 30:
C.sub.g,body−C.sub.g,sensor=F.sub.g(R.sub.g+R.sub.m)  (1),
where C.sub.g,body is he blood glucose concentration, C.sub.g,sensors the glucose concentration in the diffusion chamber 100, F.sub.g the glucose flow through the skin and the membrane 30, R.sub.g the skin resistance to glucose diffusion, R.sub.m the membrane resistance to glucose diffusion which has two values, one for each state. The unknowns are the blood glucose concentration C.sub.g and the skin resistance to glucose diffusion R.sub.g. With measuring the concentration and the glucose diffusion flow for two membrane resistances, both the skin resistance and the blood glucose concentration can be determined.

(77) So to keep the concentration gradient high, and thus the glucose diffusion flow high, the sensor is flushed with said perfusion medium in the above described microdialyis setup. Thus, once integrated over the flow streamline through the diffusion chamber 100 equation (1) becomes:

(78) $\begin{array}{cc}{C}_{g,\mathrm{sensor}}=\left(1-\exp \left[-\frac{A_m}{Q_d\left(R_g+R_m\right)}\right]\right)C_{g,\mathrm{body}}& \left(2\right)\end{array}$
the term

(79) $\begin{array}{cc}\left(1-\exp \left[-\frac{A_m}{Q_d\left(R_g+R_m\right)}\right]\right)& \left(3\right)\end{array}$
is also called the dialysate extraction fraction, where Qd ist he dialysate flow and Am the microdialysis chamber area in contact with the membrane.

(80) If

(81) $Q_d\frac{R_g+R_m}{A_m}>>\frac{1}{2}$
the dialysate extraction fraction in (3) can be linearized. Then Eqn. (2) becomes:

(82) $\begin{array}{cc}{C}_{g,\mathrm{sensor}}=\frac{A_m}{Q_d}\frac{1}{R_g+R_m}{C}_{g,\mathrm{body}}& \left(4\right)\end{array}$

(83) Then, with measuring the two analyte concentrations C.sub.ml and C.sub.mh for two corresponding membrane states, or two membrane resistance values R.sub.ml and R.sub.mh, one gets the blood glucose concentration, from (4):

(84) 0$\begin{array}{cc}{C}_{g,\mathrm{body}}=\frac{Q_d}{A_m}\left(R_{\mathrm{mh}}+R_{\mathrm{ml}}\right)\frac{C_{\mathrm{ml}}{C}_{\mathrm{mh}}}{C_{\mathrm{ml}}-C_{\mathrm{mh}}}& \left(5\right)\end{array}$

(85) The theoretical measurement errors are calculated as follows:

(86) $\begin{array}{cc}\Delta C_{G,\mathrm{Body}}=\frac{Q_d}{A_m}\sqrt{{\left(\frac{{\left(R_g+R_{ml}\right)}^2}{R_{\mathrm{mh}}-R_{\mathrm{ml}}}\right)}^2+{\left(\frac{{\left(R_g+R_{\mathrm{mh}}\right)}^2}{R_{\mathrm{mh}}-R_{\mathrm{ml}}}\right)}^2}\Delta C_{g,\mathrm{sensor}}& \left(6\right)\\ \frac{\Delta C_{g,\mathrm{body}}}{C_{G,\mathrm{body}}}=+\frac{1}{R_{\mathrm{mh}}-R_{\mathrm{ml}}}\Delta R_{\mathrm{ml}}& \left(7\right)\\ \frac{\Delta C_{g,\mathrm{body}}}{C_{G,\mathrm{body}}}=-\frac{1}{R_{\mathrm{mh}}-R_{\mathrm{ml}}}\Delta R_{\mathrm{mh}}& \left(8\right)\\ \frac{\Delta C_{G,\mathrm{Body}}}{C_{G,\mathrm{Body}}}=\frac{\Delta Q_d}{Q_d}\sqrt{{\left(\frac{R_g+R_{ml}}{R_{\mathrm{mh}}-R_{\mathrm{ml}}}\right)}^2+{\left(\frac{R_g+R_{\mathrm{mh}}}{R_{\mathrm{mh}}-R_{\mathrm{ml}}}\right)}^2}& \left(9\right)\\ \frac{\Delta C_{G,\mathrm{Body}}}{C_{G,\mathrm{Body}}}=\frac{\Delta C_{G,\mathrm{sensors}}}{C_{G,\mathrm{sensors}}}\sqrt{{\left(\frac{R_g+R_{ml}}{R_{\mathrm{mh}}-R_{\mathrm{ml}}}\right)}^2+{\left(\frac{R_g+R_{\mathrm{mh}}}{R_{\mathrm{mh}}-R_{\mathrm{ml}}}\right)}^2}& \left(10\right)\end{array}$

(87) Thus, with the following values (R.sub.g is approximate), a negligible R.sub.ml<<R.sub.mh, R.sub.mh=R.sub.g:

(88) R.sub.G=1,200,000 scm.sup.−1

(89) Q.sub.d=5 μL min.sup.−1

(90) A.sub.m=4 cm.sup.2

(91) The equations 6-10 give:

(92) $\Delta C_{\mathrm{GB}}=200\Delta C_{\mathrm{meas}}$$\frac{{\sigma }_{\mathrm{meas}}}{C_{\mathrm{meas}}}<0.32\%$$\frac{{\sigma }_{Q_d}}{Q_d}<0.32\%$$\frac{{\sigma }_{R_{\mathrm{mh}}}}{R_{\mathrm{mh}}}<0.32\%$$\frac{\Delta C_{g,\mathrm{body}}}{C_{G,\mathrm{body}}}=\frac{\Delta R_{\mathrm{mh}}}{R_{\mathrm{mh}}}$

(93) The principle of the microfluidic chip 201 for concentration measurement by means of with fluorescence measurement is shown in FIG. 17. The chip 201 is preferably made out of polydimethylsiloxane (PDMS) that is molded onto a SU-8 on silicon mold fabricated by a non-standard photolithographic process described in detail in [1]. The chip 201 may also be made out of polycarbonate (PC), e.g., by means of injection molding. The chip 201 uses continuous flow rates. It first passively mixes the diluted analytes in the dialysate (e.g. in the respective mix with the perfusion medium 3) with reagent fluid 4 for the standard hexokinase glucose assay. The mixing duration with passive diffusion is negligible compared with the reaction lag time. Thus, the enzymatic reaction is not diffusion limited and follows a pseudo-first-order reaction kinetic. There is a delay channel (60 s) 24 for the lag time of the coupled enzyme reaction. The fabricated channel height is controlled and uniform so that the delays in the channels are not altered. With a channel uniformity of ±2.5% on thick structures this is a non-standard fabrication process with multilayers UV patterned SU-8 photoresist using only the central part of the silicon wafer for the mold. The flow inside the channels is kept laminar so that little transverse mixing occurs, i.e., so that the flow is uniform over the channel length.

(94) In detail, as shown in FIG. 17, the microfluidic chip 201 for monitoring the glucose concentration in the dialysate comprises a reagent inlet 21 for the reagent fluid 4, a dialysate inlet 22 used for said dialysates, a micromixer 23 for mixing dialysate and reagent fluid 4, said delay channel 24, a the first fluorescence chamber 25, a second delay channel 26, a second fluorescence chamber 27, as well as a waste outlet 28 for discharging the measured dialysate.

(95) FIG. 18 shows the fluorescence chamber 25 of the manufactured mold with the probed volume 251, the visible light (VIS) fluorescence emission collection fibers 252 and the ultra-violet UV fluorescence excitation light 253.

(96) FIG. 19 shows a variation of the fluorescence chamber 25 of the manufactured mold with probed volume 251, the visible light (VIS) fluorescence emission collection fibers 252 and the ultra-violet UV fluorescence excitation light 253.

(97) After the first delay channel 24, the pseudo-first order reaction is monitored by the fixed-point method using the two fluorescence chambers 25, 27 separated by the 7 s second delay channel 26. The difference between the two fluorescence signals of the enzymatic reaction product is proportional to the glucose concentration. The fluorescence chamber 25 has three embedded optical fibers, one for the fluorescence excitation of at 340 nm, and two for the emission at 450 nm. The excitation comes from a filtered UV lamp, a laser, or a LED and the emission is measured on a cooled silicon photomultiplier (SiPM).

(98) In this specific example, two different wavelengths were used to change from the first state of permeability of the membrane 30 to the second one.

(99) As photochromic compounds spirobenzopyran- and spirooxazine-compounds were used as shown in FIGS. 20 to 26.

(100) The spirocompounds were integrated in the grafted polymer either by copolymerization of an acrylate derivative of the spirocompound with MMA (methyl methacrylate), AEMA (aminoethyl methacrylate), HEMA (hydroxyethyl methacrylate) or HEA (hydroxyethyl acrylate), HEA-TMS ([trimethylsilyloxy]hydroxyethyl) or PDMS ((α,ω)-methacryloxypropyl poly(dimethylsiloxane)) or by postmodification of a grafted PAEMA (poly(N-amino)ethyl methacrylate), PHEMA (polyhydroxyethyl methacrylate) or PHEA (polyhydroxyethyl acrylate) layer or polymer network with a carboxylic acid derivative of the spirocompound.

(101) FIG. 20 shows a synthesized spiro-compound SP5 according to a first example.

(102) FIG. 21 shows a synthesized spiro-compound SP7 with R=Me and SP9 with R═H according to a second and third example.

(103) FIG. 22 shows a synthesized spiro-compound SP12 according to a fourth example.

(104) FIG. 23 shows a synthesized spiro-compound SP14 with R=Me and SP16 with R═H according to a fifth and sixth example.

(105) FIG. 23 shows a synthesized spiro-compound SO37 according to a seventh example.

(106) FIG. 24 shows a synthesized spiro-compound SO39 with R=Me and SO50 with R=H according to an eighth and ninth example.

(107) Finally, FIG. 25 shows a synthesized spiro-compound SO49 according to a tenth example.

(108) The light-responsive membranes 30 were prepared in different ways:

(109) Commercially available track-etched polycarbonate membranes with a pore size of 15 nm, 30 nm, 50 nm, 100 nm, 200 nm, 400 nm, 1000 nm were dip-coated with a polymer solution containing a co-polymer of a spirobenzopyran acrylate and PMMA. The spirobenzopyran acrylate monomer itself can also be polymerized. The co-monomer primarily serves for enhancing the effect of the spirobenzopyran switch and the photostability of the spiropyrans. The copolymer contained between 1-10% of spirobenzopyran according to FIG. 21 (the CAS-No. is 89908-23-6 for R═H and 25952-50-5 for R═CH.sub.3) or FIG. 23. Best switching was observed with 4% of spirobenzopyran. Other membranes, which were coated, are track-etched polyester membranes and PVDF membranes which showed a lower permeability change. The permeability change was demonstrated with aqueous solutions of caffeine and glucose.

(110) ##STR00005##

(111) Further, plasma-induced graft surface polymerization of track-etched polycarbonate membranes with a pore size of 15 nm, 30 nm, 50 nm, 100 nm, 200 nm, 400 nm, 1000 nm were produced. A copolymer consisting of a spirobenzopyran acrylate and a comonomer (2-hydroxy acrylate, 2-hydroxy methacrylate, 2-amino methacrylate, methyl methacrylate) was grafted from a plasma treated membrane. The amount of spirobenzopyran in the grafted polymer was between 1-100%. Spirobenzopyran acrylate was replaced by spirooxazine acrylate in some experiments. The permeability change was demonstrated with aqueous solutions of caffeine and glucose.

(112) Further, surface-induced atom transfer radical polymerization (SI-ATRP) was performed from track-edged polyester membranes with a pore size of 200 nm. A copolymer consisting of a spirobenzopyran acrylate and a comonomer (2-hydroxy acrylate (HEA), 2-hydroxy methacrylate (HEMA), 2-amino methacrylate (AEMA), methyl methacrylate (MMA)) was grafted from a plasma treated membrane. The amount of spirobenzopyran in the grafted polymer was between 0.4-13%. The permeability change was demonstrated with aqueous solutions of caffeine.

(113) Further, amphiphilic co-networks were used as membranes consisting of poly (2-hydroxy ethylacrylate) as the hydrophilic domain and (α,ω)-methacryloxypropyl poly(dimethylsiloxane) (PDMS) as hydrophobic domain [2, recipe of amphiphilic conetwork without photochromic unit]. Membranes with different weight ratios (wt.-%) of HEA:PDMS (70:30, 60:40, 50:50, 40:60) and spirobenzopyran acrylate were produced (0.05-2 wt.-%). Spirobenzopyran was replaced by spirooxazine acrylate in some experiments. The permeability change was demonstrated with aqueous solutions of caffeine. In case the membrane 30 sticks only to the probe head 10, but not to the skin of the patient (e.g. in case of the amphiphilic co-network) the O-ring 13 may be omitted. Generally, the inlet and outlet 101, 102 (cf. FIG. 2 for instance) may also switch positions, with the outlet still being connected to the analyzing means 20.

(114) The dimensions of the probe head 10 can of course be varied. The material can be changed, especially in case the switching stimulus of the membrane permeability is changed to e.g. electrical stimulation. Thus, transparency to UV-light would not be needed anymore.

(115) A standard fluorimeter could be used instead of the microfluorimeter on a chip 201. It would also be connected with capillaries to the probe head 10. The material of the microfluidic chip 201 could also be changed. Instead of fluorescence, absorption spectroscopy could be used. Instead of SiPM, photomultiplier tubes or other sensitive photodetectors could be used. In general, any online glucose concentration accurate enough could be used instead of the enzymatic reaction. It also has to be changed if other metabolites have to be measured.

(116) By including a drug into the perfusion medium 3, the device 1 could be used for controlled drug delivery.

(117) The working principle of the sensor could also work by using side by side two membranes with different permeabilities instead of using an intelligent membrane which changes permeability by applying a stimulus. Such a change in permeability can be triggered either by a change of light, temperature, pH or electricity.

(118) Other photochromic molecules, which could be integrated in a light-responsive membrane are: Azobenzene Coumarin, and Dithienylethene.

(119) Further, extensive Finite Element Method Simulations of the probe head 10 were conducted. The real 3D geometry was used for the simulation. The boundary conditions for the flow were chosen as follows: no-slip flow onto the chamber walls with pressure driven flow and a laminar flow pattern into the inlet 101 and atmospheric pressure at the outlet 102. The 3D pattern of the flow inside the microdialysis chamber 100 is laminar as shown in FIG. 27.

(120) Further, FIG. 28 shows a cross-section of the diffusion chamber 100 showing the local dialysate extraction fraction with a dimensionless value between 0 (no substance extracted to 1 (same concentration as in the blood) as resulting from a 3D Finite Element Method with a low permeability of the membrane 30.

(121) Further, FIG. 29 shows the same simulation as FIG. 28, but with a higher permeability of the membrane 30.

(122) Diffusion of a glucose concentration of 5 mmol/L through the skin and the smart membrane 30 according to the invention was simulated with different permeability states. FIG. 30 shows a cross-section of the dialysate extraction fraction (concentration of the substance in the dialysate divided by the concentration of the same substance in the blood) pattern inside the diffusion chamber 100 with glucose diffused through a membrane 30 with a low permeability state. FIG. 31 shows the same with a high permeability state. FIG. 30 plots the dialysate extraction fraction at the outlet as simulated compared with the theory. The two are highly correlated R.sup.2=0.99999, but the relation is not exactly one to one. The curved laminar flow pattern with decreased flow velocity closer to the walls explains the difference. This difference is good, because it means higher concentration to be measured continuously after the microdialysis head 10 and better accuracy.

(123) FIG. 31 shows that the dialysate extraction fraction is highly linearly proportional R.sup.2=0.99997 to the membrane permeability in the application expected range for glucose. This means we can use only two membrane states for measuring the blood glucose.

(124) It has been shown that glucose concentration could be determined with the appropriate accuracy with a fluorimeter. FIG. 32 shows the fluorescence measurement with enzymatic reaction of very low glucose concentration.

(125) Glucose concentration could be measured with the same principle using the microfluorimeter as shown in FIG. 33.

(126) It was also demonstrated that the membrane permeability to glucose can be switched by exposure to UV light at 366 nm, as opposed to daylight. The results are shown in FIG. 34 using a plasma-induced spirobenzopyran and PHEMA coated track-edged polycarbonate membrane (pore diameter: 200 nm).

(127) Finally, in-vitro testing with taped pigskin from the ear exhibited passive diffusion processes of glucose with a Franz cell experiment as shown in FIG. 35.

(128) In the above, the glucose measurement was described as one possible application of the device 1 according to the invention, because it has a tremendous clinical value. However, the same principle can be applied to any analyte that diffuses through the skin and thus the device 1 enables a broad new approach of non-invasive diagnostics.

(129) Examples of possible analytes: Lactate, Glucose, Creatinine, Bilirubin, Urea, Ammonia, Opioid, Cocaine, Cortisone, Hydrocortisone, Caffeine, drugs (to control the taking of medication)

(130) FIGS. 36 to 46 show a further embodiment of the device 1/probe head 10 according to the invention.

(131) Again, the probe head 10 comprises a diffusion chamber (also denoted as microdialysis chamber) 100 that is delimited by a membrane 30 for contacting the skin 2 of a patient P, wherein the diffusion chamber 100 comprises an inlet 101 as well as an outlet 102 for feeding a perfusion medium 3 into the diffusion chamber 100 and for discharging it out of the diffusion chamber 100. The probe head 10 further comprises a first conduit 110 (e.g. a capillary) being in flow connection with the inlet 101 as well as second conduit 111 (e.g. a capillary) being in flow connection with the outlet 102. Via said second conduit 111, the outlet 102 is connected to an analyzing means 20 for measuring the concentration of an analyte (e.g. glucose) in a perfusion medium 3, which analyte diffused through the membrane 30 in a first state of the latter, as well of the analyte in the perfusion medium 3, when the analyte diffused through the membrane 30 in a second state of the membrane, wherein the permeability of the membrane 30 with respect to said analyte differs in said two states such that the two concentrations of the analyte differ.

(132) The probe head 10 may be used with the device 1 described in conjunction with FIG. 16. Further, all probe heads 10 described herein may be used in conjunction with the device shown in FIG. 51. The device 1 shown in FIG. 51 corresponds to the device of FIG. 16, with the difference that the device 1 according to FIG. 51 comprises an additional vacuum pump 204 connected via a pressure regulator 118 to the waste container 203 for receiving the analyzed dialysate. The pressure generated by the pump 204 can be measured by a pressure measurement device 205. The reagent fluid countainer could also be connected to this additional vacuum pump 204 via a pressure regulator 118 instead of the air compressor/compressed air line 120.

(133) Further, the probe head 10 of FIGS. 36 to 46 comprises a body 11 that is preferably formed out of molded PDMS. The body 11 comprises a preferably circular recess 100 forming said diffusion chamber 100 on a front side of the body 11 facing away from a rear side of the body 11 on which said conduits 110, 111 are arranged. The recess 100 is covered by a light switchable membrane 30 as described herein, particularly an amphiphilic co-network mentioned above.

(134) As the hydrophilic domain of the membrane 30 is e.g. made of PDMS, one can also bond the membrane 30 onto the body 11 of the probe head 10 with a surface treatment with oxygen plasma. The membrane 30 could be also bonded with UV curable resist.

(135) Furthermore, the diffusion chamber 100 comprises a plurality of posts 113 for supporting the membrane 30. Said posts 113 may be formed by a grid structure 113 (e.g. of the type shown in FIG. 54 as a micrograph).

(136) FIGS. 47 to 50 shows a further embodiment of the probe head 10 according to the invention wherein a microfluidic chip 201 (analyzing means 20), that may be designed as described above, is integrated into the probe head 10.

(137) Here, the probe head 10 comprises (from top to bottom) again a membrane 30 (which may a suitable membrane described herein), e.g. in the form of an rectangular layer, for contacting the skin 2 of a person P (the contact surface of the membrane 30 is the shown upper side of the membrane 30 in FIG. 47), a layer (body) 11 that is preferably formed out of molded PDMS forming a diffusion chamber 100 comprising a plurality of posts (e.g. a grid structure) 113 for supporting the membrane 30, a scattering layer 300 that is preferably formed out of molded PDMS for uniformizing the membrane excitation light coming from fibers 260 for guiding light needed to switch the membrane 30 as described herein, a light guide plate 301 that is preferably formed out of molded PDMS and preferably consists of or comprises spherical mirrors for reflecting light with 90° with a density of the square of the distance to the fibers 260 for correcting the decrease of the intensity of the light emitted from the fibers 260, a mirror layer 302 that is preferably formed out of molded PDMS for reflecting all the light emitted from the fibers 260 into the light guide plate 301 onto the membrane 30 made of a hollow cavity supported by a plurality of posts 303 (e.g. in the form of a grid structure 303), an optical shield 304 that is preferably formed out of molded PDMS mixed with a black light absorbing dye for absorbing light and preventing light to reach the microfluidic chip (or analyzing means) 201 as described herein, fibers 252a for guiding visible light (VIS) from the fluorescence chambers 25 and 27 as well as fibers 253a for guiding UV light to the fluorescence chambers 25 and 27 (see also above), and another optical shield 305 that is preferably formed out of molded PDMS mixed with a black light absorbing dye for again preventing light to reach the microfluidic chip (or analyzing means) 201 and enclosing the embedded probe head 10.

(138) The probe head 10 further comprises a conduit 240 (e.g. capillary) for guiding reagent fluid 4 to reagent inlet 21 of chip 201 (the dialysate inlet 22 is connected to the diffusion chamber 100), as well as a conduit (e.g. capillary) 110 for guiding the perfusion medium 3 into the diffusion chamber 100, and a conduit (e.g. capillary) 280 for drawing off the measured analyte from the chip 100.

(139) The above-described components of the probe head 10 are particularly designed in the form of layers that are stacked on top of each other, as can be seen in FIG. 47. Further, FIGS. 52 and 53 show molds for a microfluidic chip of the kind shown in FIGS. 17 and 47, with regions for the probed volume 251, the visible light (VIS) fluorescence emission collection fibers 252 and the fibers for ultra-violet UV fluorescence excitation light 253

(140) Furthermore, FIG. 54 shows an SEM micrograph of a diffusion (microdialysis) chamber 100 of the kind shown in FIG. 47 with the grid structure 113 for supporting the adjacent membrane 30.

(141) Further. FIG. 55 shows the 3D flow streamlines inside the diffusion chamber of the probe head shown in FIGS. 36 to 46, the flow being laminar;

(142) FIG. 56 shows a cross-section of a model of the diffusion chamber 100 of the kind in FIG. 38, 44 showing the local dialysate extraction fraction having values between 0 (no substance extracted) and 1 (same concentration as in the blood). Shown are the results from a 3D Finite Element Method with a high permeability of the membrane 30.

(143) FIG. 57 shows the same simulation as FIG. 56, but with a lower permeability of the membrane 30

(144) FIG. 58 shows the total dialysate extraction fraction at the outlet of the diffusion chamber of the kind shown in FIGS. 38 and 44 as determined from the theory or from a FEM simulation. The two results are linearly proportional R.sup.2=1. This means that the simulated probe head 10 even with more complicated laminar flow pattern can be well explained by the theory.

(145) FIG. 59 shows that in the application range the dialysate extraction fraction at the outlet of the diffusion chamber of the kind shown in FIGS. 38 and 44 is linearly proportional to the membrane permeability for both the theory R.sup.2=0.9996 and the simulation R.sup.2=0.9998.

(146) FIG. 60 shows that as predicted from the theory, the dialysate extraction fraction at the outlet of the diffusion chamber of the kind shown in FIGS. 38 and 44 does not depend on the height if the diffusion chamber in the 3D FEM simulation. There is no significant correlation between the chamber height and the dialysate extraction fraction. The differences in the dialysate extraction fraction between the different points come from computing errors. This means that the if the height of the diffusion chamber 100 changes, it does not influence the dialysate extraction fraction nor the measurement, and

(147) FIG. 61 shows the dialysate extraction fraction of a probe head of the kind of FIG. 36-46 measured with two different states, once exposed with white light, once exposed with UV light. This shows that exposing the membrane 30 to UV light or white light controls the dialysate extraction fraction.

REFERENCES

(148) [1] de Courten, D., Baumann, L., Scherer, L. J., & Wolf, M. (2012). Opto-Fluidic Chip for Continuous Inline Monitoring of Glucose with Kinetic Enzymatic Fluorescence Detection. Procedia Engineering, 47, 1203-1206. [2] N. Bruns, J. Scherble, L. Hartmann, R. Thomann, B. Iván, R. Mülhaupt, J. C. Tiller, Macromolecules, 2005, 38, 2431-2438.