DEVICE AND METHOD FOR DETECTING A SPECIFIC ANALYTE IN A LIQUID SAMPLE AND USES OF SAID DEVICE

Abstract

The invention relates to a device and method for detecting a specific analyte in a liquid sample. The device that can be used in the method contains at least one fluid line, at least one receiving region for receiving a liquid sample, at least one enzymes region containing at least one determined enzyme and/or at least one acidification region containing at least one acid. The device also contains at least one reaction region used to form gas bubbles. The fluid line transports the liquid sample from the receiving region via the enzyme region and/or the acidification region to the reaction region by means of capillary forces and/or at least one micropump allowing fast, simple and cost-effective detection of a specific analyte in a liquid sample, the detection being carried out with a high level of sensitivity, specificity and precision. The invention further relates to uses of the device.

Claims

1. A device for detecting a particular analyte in a liquid sample by means of at least one conversion of the analyte selected from the group consisting of an enzyme-catalyzed conversion of the analyte and an acid-mediated conversion of the analyte to yield, at least one gas, the device comprising at least one fluid line; at least one receiving zone for the receiving of a liquid sample; at least one zone selected from the group consisting of at least one enzyme zone comprising at least one particular enzyme suitable for catalyzing the conversion of the analyte to be determined to yield at least one gas, and at least one acidification zone comprising at least one acid; and at least one reaction zone for the formation of gas bubbles, wherein the reaction zone comprises or consists of a chamber which is fluidically connected to the at least one fluid line and which has liquid-tight walls; wherein the at least one fluid line is suitable for transporting the liquid sample from the receiving zone to the reaction zone via at least one zone selected from the group consisting of the at least one enzyme zone and/or the at least one acidification zone, by means of at least one selected from the group consisting of capillary forces and at least one micropump in the fluid line.

2. The device as claimed in claim 1, wherein the device comprises at least one selected from the group consisting of multiple fluid lines; and multiple reaction zones.

3. The device as claimed in claim 1, wherein the chambers comprises at least one wall, which exhibits at least one selected from the group consisting of a transparency for light of a wavelength within a region selected from the group consisting of IR region, visible region, UV region and combinations thereof; and a tightness for liquids, gases and combinations thereof.

4. The device as claimed in claim 1, wherein the at least one receiving zone is suitable for receiving a liquid sample selected from the group consisting of aqueous solutions comprising or consisting of blood, urine, sputum, foodstuffs, river water, saltwater, seawater, groundwater, drinking water, wastewater and mixtures thereof.

5. The device as claimed in claim 1, wherein the at least one enzyme zone comprises at least one selected from the group consisting of at least one enzyme selected from the group consisting of urease, lactate oxidase, lactate dehydrogenase, catalase, pyruvate decarboxylase, thyreoperoxidase and combinations thereof; at least one further enzyme; and at least one cofactor of an enzyme.

6. The device as claimed in claim 1, wherein in that the at least one enzyme zone comprises the at least one enzyme and/or at least one cofactor of the at least one enzyme, or both, in dry form; or in aqueous form.

7. The device as claimed in claim 1, wherein the at least one enzyme zone comprises at least one selected from the group consisting of biological cells and cell lysate.

8. The device as claimed in claim 1, wherein in that the at least one fluid line comprises a membrane which has at least one selected from the group consisting of a pore diameter of ≤20 μm; a suitability for the removal of biological cells, and an arrangement between the receiving zone for the receiving of a liquid sample and the reaction zone for the formation of gas bubbles.

9. The device as claimed in claim 1, wherein the acidification zone has at least one selected from the group consisting of an acid selected from the group consisting of acids solid at room temperature and standard pressure, HCl, H.sub.2SO.sub.4, H.sub.3PO.sub.4 and mixtures thereof; and an arrangement between the receiving zone for the receiving of a liquid sample and the reaction zone for the formation of gas bubbles or is arranged within the reaction zone for the formation of gas bubbles.

10. The device as claimed in claim 1, wherein the at least one fluid comprises at least one oxidation zone, wherein the oxidation zone comprises at least one oxidant.

11. The device as claimed in claim 1, wherein the at least one fluid line has at least one selected from the group consisting of a length of from 0.1 to 20 cm; a width of from 0.05 to 20 mm; a height of from 0.05 to 2 mm; and a maximum diameter within the range from 0.05 to 20 mm.

12. The device as claimed in claim 1, wherein the particular analyte is at least one is selected from the group consisting of a small organic molecule having a mass of <500 Da, peptide, protein and mixtures thereof; and an analyte which is a marker for a state selected from the group consisting of disease, water pollution, food contamination and combinations thereof.

13. The device as claimed in claim 1, wherein the device is arranged in or on an optical detection instrument.

14. A method for detecting a particular analyte in a liquid sample by means of at least one conversion of the analyte selected from the group consisting of an enzyme-catalyzed conversion of the analyte and an acid-mediated conversion of the analyte, to yield at least one gas, the method comprising the steps of applying a liquid sample possibly comprising the analyte to be determined to a receiving zone of a device, wherein the device is a device for detecting a particular analyte in a liquid sample by means of at least one conversion of the analyte selected from the group consisting of an enzyme-catalyzed conversion of the analyte and an acid-mediated conversion of the analyte, to yield at least one gas, wherein the device comprises at least one fluid line; at least one receiving zone for the receiving of a liquid sample; at least one zone selected from the group consisting of at least one enzyme zone comprising at least one particular enzyme suitable for catalyzing the conversion of the analyte to be determined to yield at least one gas, and at least one acidification zone comprising at least one acid; and at least one reaction zone for the formation of gas bubbles, wherein the reaction zone comprises or consists of a chamber which is fluidically connected to the at least one fluid line and which has liquid-tight walls; wherein the at least one fluid line is suitable for transporting the liquid sample from the receiving zone to the reaction zone via at least one zone selected from the group consisting of the at least one enzyme zone and the at lease one acidification zone, by means of at least one selected from the group consisting of capillary force and at least one micropump in the fluid line; optically detecting the reaction zone of the device at a time point at which the liquid sample comprising at least one of the group consisting of an enzyme and acid has been transported from the fluid line to the reaction zone; and assessing that the particular analyte is present in the sample if the formation of gas bubbles occurs in the reaction zone.

15. The method as claimed in claim 14, wherein the optical capture is effected by means of an optical detection instrument selected from the group consisting of camera, microscope, photometer, refractometer and combinations thereof.

16. The method as claimed in claim 14, wherein the method encompasses a quantitative determination of the concentration of the analyte in the sample.

17. The method as claimed in claim 14, in further comprising the steps of applying at least one further liquid sample comprising a known concentration of the analyte to be determined to a receiving zone of the device; optically detecting the reaction zone of the device at a time point at which the liquid sample comprising at least one selected from the group consisting of the enzyme and acid has been transported from the fluid line to the reaction zone; and quantitatively determining the concentration of the analyte in the sample.

18. (canceled)

19. A method in which a device for detecting a particular analyte in a liquid sample by means of at least one conversion of the analyte selected from the group consisting of enzyme-catalyzed conversion of the analyte and acid-mediated conversion of the analyte, to yield at least one gas, is used for at least one of in vitro diagnosis of a disease, quality control of foodstuffs and testing of water quality, wherein the used device comprises at least one fluid line; at least one receiving zone for the receiving of a liquid sample; at least one zone selected from the group consisting a at least one enzyme zone comprising at least one particular enzyme suitable for catalyzing the conversion of the analyte to be determine to yield at least one gas, and at least one acidification zone comprising at least one acid; and at least one reaction zone for the formation of gas bubbles, wherein the reaction zone comprises or consists of a chamber which is fluidically connected to the at least one fluid line and which has liquid-tight walls; wherein the at least one fluid line is suitable for transporting the liquid sample from the receiving zone to the reaction via at least one zone selected from the group consisting of the at least one enzyme zone and the at least one acidification zone, by means of at least one selected from the group consisting of capillary forces and at least one micropump in the fluid line.

Description

DESCRIPTION OF THE DRAWINGS

[0061] It is intended that the subject matter of the invention be more particularly elucidated on the basis of the following figures and examples, without wishing to restrict said subject matter to the specific embodiments depicted here.

[0062] FIG. 1 shows the enzymes and/or acid for conversion of particular biomarkers which can be contained by the device according to the invention;

[0063] FIG. 2 shows the device;

[0064] FIG. 3 show the top plan view of the device; and

[0065] FIG. 4 shows a cross-section view of a detection instrument suitable for performing an optical capture of the reaction zone of the device.

DETAILED DESCRIPTION OF THE DRAWINGS

[0066] FIG. 1 shows, by way of example, possible enzymes and/or acids (optionally with oxidant) for the conversion of particular analytes (biomarkers) which can be contained by the device according to the invention and which can be used in the method according to the invention. In particular, the figure also describes the respective chemical reactions forming the basis of gas evolution.

[0067] FIG. 2 shows, by way of example, a possible device 1 according to the invention for the detection of a particular analyte in a liquid sample 2 by means of enzyme-catalyzed conversion of the analyte to yield at least one gas 7. The device 1 contains at least one fluid line 3 which is fluidically connected to at least one receiving zone 4 for the receiving of a liquid sample 2 (e.g., blood) and fluidically connected to at least one enzyme zone 5, the enzyme zone 5 containing at least one particular enzyme which catalyzes the conversion of the analyte to be determined to yield at least one gas 7. Furthermore, the device 1 contains at least one reaction zone 6 for the formation of gas bubbles 7, the reaction zone 6 having a fluidic connection to the fluid line 3 and being otherwise delimited by a gas-tight wall 10. The device 1 is characterized in that the at least one fluid line 3 is suitable for transporting the liquid sample 2 from the receiving zone 4 to the reaction zone 6 by means of capillary forces, the at least one particular enzyme being, in this case, cotransported from the enzyme zone 5 at least in part into the reaction zone 6. In this embodiment, the device 1 further contains a membrane 8 suited to the removal of biological cells (e.g., a membrane for the separation of blood cells from blood plasma) between the receiving zone 4 and the enzyme zone 5. Said membrane 8 ensures that no biological cells get from the fluid line 3 as far as the enzyme zone 5 by means of capillary forces. Moreover, said device 1 additionally contains an acidification zone 9 which contains at least one acid (e.g., HCl) and, in this case, coincides with the reaction zone 6. The acidification zone 9 ensures that the liquid sample, which contains enzyme in the reaction zone 6, is acidified. In the case of gases which dissolve in the liquid sample under acid formation (e.g., formation of H.sub.2CO.sub.3 in the case of the gas CO.sub.2), the acidification shifts the equilibrium in the direction of gas formation and a stronger (quantitative) escape of CO.sub.2 is thus effected. In brief, the presence of the acidification zone 9 can distinctly increase the detection sensitivity in the case of this type of gas.

[0068] FIG. 3 shows a top view of a reaction chamber 6 of a device according to the invention. What is depicted is a temporal course during the reaction, with the gas bubbles 7 growing in the course of time to form larger gas bubbles 7′. Besides the final volume expansion of the gas bubbles 7 to form the larger gas bubbles 7′, it is also possible to utilize the volume increase over time from the state of the original gas bubbles 7 up to the state of the larger gas bubbles 7′.

[0069] FIG. 4 shows a cross section of a detection instrument suitable for performing an optical capture of the reaction zone of the device according to the invention and for carrying out and outputting a quantitative determination of the concentration of the analyte in the sample. The gas bubbles 7 which are formed in the reaction zone 6 of the device according to the invention are completely illuminated by electromagnetic radiation 12 of a substantially coherent or noncoherent light source 11 (e.g., an LED, an OLED, a laser and/or a gas discharge lamp). An image sensor 13 (e.g., in the form of a diode array) which is situated under the reaction zone 6 captures the optical image 14 generated by the gas bubbles 7 (i.e., the characteristic interference pattern thereof). From said optical image 14, it is possible by means of a data processing program to calculate the product of number and volume of gas bubbles per unit of time, which is directly proportional to the reaction rate and to the concentration of the analyte in the sample. An optical device (not depicted here) can likewise additionally be situated between the image sensor and the reaction zone.

EXAMPLE 1—FUNDAMENTALS IN RELATION TO THE METHOD FOR DETECTING A PARTICULAR ANALYTE IN A LIQUID SAMPLE

[0070] The maximum reaction rate v.sub.max of an enzymatic reaction depends on the temperature and the pH of the solution in which the reaction is carried out. For a particular, constant pH, v.sub.max is dependent on the ambient temperature of the device according to the invention. If the ambient temperature is also constant (e.g., constant 25° C. room temperature), v.sub.max assumes a completely definite value. In this case, the measured reaction rate v of substrate (analyte) is dependent on the concentration of substrate (analyte) in the solution (Michaelis-Menten theory). What is applicable here is the equation

v=(v.sub.max.Math.[Analyte])/(k.sub.m+[Analyte])

where [Analyte] is the analyte concentration and k.sub.m represents the Michaelis-Menten constant of the enzyme.

[0071] In the case of a known v.sub.max under particular conditions and known Michaelis-Menten constant k.sub.m, it is thus possible by means of the measurement of the reaction rate of the substrate (analyte) to deduce the concentration of substrate (analyte) in the liquid sample.

[0072] The reaction rate is directly proportional to the gas volume produced per unit of time, i.e., proportional to the product of number and volume of detected gas bubbles. What is thus applicable is the relationship

v˜[Number(gas bubbles).Math.Volume(gas bubbles)]

[0073] Many of the gas bubbles which arise dissolve poorly in the liquid sample, meaning that the aforementioned relationship is fully applicable.

[0074] Since carbon dioxide is very highly soluble in aqueous solution and dissociates in part to yield carbonic acid, pH change is used for the conversion into the gaseous aggregate state. Since the sample chamber is delimited in all three dimensions by a gas-impermeable layer, gas bubbles are formed which cannot escape from the sample chamber. The amount and size of the gas bubbles is analyzed with the aid of an optical method. The amount and size of the gas bubbles correlates with quantity of the analyte in the sample.

EXAMPLE 2—METHOD FOR DETECTING UREA IN A LIQUID SAMPLE

[0075] For the detection of the analyte urea, the device according to the invention contains the enzyme urease in the enzyme zone. As can be seen in FIG. 1, the enzyme urease catalyzes the conversion of urea to yield the two gases ammonia and carbon dioxide.

[0076] An acidification zone on the device according to the invention ensures that, firstly, the carbon dioxide which arises does not go into solution as H.sub.3O.sup.+ and HCO.sub.3.sup.−, but escapes quantitatively. As a result, the sensitivity of detection of the device for urea is increased.

[0077] Moreover, the acidification causes the ammonia which arises to preferentially go into solution as NH.sub.4.sup.+ and OH.sup.−. However, since ammonia has anyway a high tendency to dissolve in aqueous media even at neutral pH, the acidification barely shifts the equilibrium in the direction of dissolved ammonia. In other words, the acidification barely reduces the production of ammonia gas, meaning that, with regard to the production of ammonia gas, the acidification barely causes an adverse effect on detection sensitivity.

[0078] The product of number and volume (amount) of carbon dioxide gas bubbles is thus dependent on the urea concentration in the liquid sample used. The amount of gas bubbles produced decreases with falling urea concentration.

[0079] The amount of gas bubbles can be recorded by means of a software-controlled microscope and be evaluated (e.g., with regard to their number and their geometric properties).

[0080] By way of example, the use of the device in so-called “point of care” urea diagnostics shall be described:

[0081] A drop of blood is applied to the at least one receiving zone of the device. The drop can, for example, be received directly from a (freshly) punctured finger of a person. In this case, the device advantageously contains a plasma-separating membrane, with the result that the blood cells are held back and only blood plasma can advance as far as the enzyme zone. The enzyme zone advantageously contains the enzyme urease in lyophilized form, since the long-term stability of the enzyme is very high in this form and, as a result, the device also ensures a long usability.

[0082] The blood plasma is drawn, along the fluid line by means of capillary forces, from the receiving zone to the enzyme zone, where it meets the lyophilized urease, which goes into solution in the blood plasma. The mixture of blood plasma and urease is promptly transported, via the acidification zone by means of capillary force, to the reaction zone, where carbon dioxide is released by the decomposition of urea. The amount of gas bubbles formed is recorded with the aid of an optical method (e.g., a software-controlled microscope), and this allows conclusions to be drawn regarding the concentration of urea in the blood drop used.

EXAMPLE 3—METHOD FOR DETECTING LACTATE IN A LIQUID SAMPLE

[0083] The enzyme lactate oxidase selectively converts lactate into pyruvate and hydrogen peroxide (see FIG. 1). In a second step, the hydrogen peroxide reacts, owing to its strongly reducing effect, with a potassium permanganate solution slightly acidified with sulfuric acid. The redox reaction leads, firstly, to the decolorization of the potassium permanganate solution, which has an intense purple color, and, secondly, to the formation of oxygen (see FIG. 1).

[0084] For reaction of the hydrogen peroxide with the potassium permanganate solution acidified with sulfuric acid, the device thus further requires an oxidation zone containing at least one oxidant (e.g., MnO.sub.4.sup.− due to dissolved KMnO.sub.4) and at least one acid (H.sub.3O.sup.+, for example due to dissolved H.sub.2SO.sub.4).

[0085] The product of number and volume (amount) of oxygen gas bubbles is thus dependent on the lactate concentration in the liquid sample used. The amount of gas bubbles produced decreases with falling lactate concentration.

[0086] The amount of gas bubbles can be recorded by means of a software-controlled microscope and be evaluated (e.g., with regard to their number and their geometric properties).