JP2004361189A - Online catecholamine sensing device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To perform continuous measurement by solving issues caused by foreign matter in measuring catecholamine of low concentration. <P>SOLUTION: This sensing system is equipped with a sampling probe 1 formed in a flow path for sampling bio-liquids, a pre-reactor 2 for decomposing easy-to-oxidize substances in the bio-liquids extracted by the probe 1, and an electrochemical detector 3 communicating with the pre-reactor 2 and given a film having molecule selectivity. The detector 3 uses a comb-shaped electrode 4 capable of amplifying a response by means of oxidation-reduction cycles of catecholamine, a reference electrode 5, and a counter electrode 6. The pre-reactor 2 is provided therein with uricase 9, catalase 10, and ascorbic-acid oxidizing enzyme 11 for decomposing uric acid and ascorbic acid, or an electrode 17 for oxidation/reduction having a thin-layer cell structure made by together bonding two substrates. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an on-line catecholamine sensing device, which is used for medical, physiological, or medical purposes to collect a small amount of a sample from a living body and continuously measure catecholamines contained in the sample with high sensitivity and selectivity. Catecholamine sensing devices.
[0002]
[Prior art]
In recent years, in the fields of medicine, medical treatment, and biology, a system combining liquid chromatography and an electrochemical detector has been mainly used for detecting catecholamines. This method can separate and detect catecholamines and their metabolites in the brain and blood, but cannot perform continuous measurement.
[0003]
For example, it has been reported that a system combining liquid chromatography and a comb-shaped array electrode (for example, see Patent Document 1) can detect catecholamines (dopamine) on the order of pM (for example, Non-Patent Documents). 1). In addition, a system combining capillary electrophoresis and an electrochemical detector has been reported.
[0004]
Hereinafter, a direct insertion type and a continuous liquid feeding type of this type of system will be described.
Research on a sensor device for continuously measuring catecholamine is also actively pursued (direct insertion type). First, in order to know the state of a minute tissue in a living body, a method of directly inserting a minute electrode such as a carbon fiber for measurement is used. In addition to catecholamines in the brain, an enzyme is immobilized on an electrode to selectively measure glucose, glutamic acid, and the like.
[0005]
For example, Whiteman et al. Detect catecholamines released from cells (for example, see Non-Patent Document 2). In such a measurement method, a Nafion membrane, a polypyrrole peroxide, or the like is modified on the electrode in order to eliminate the influence of an anionic electrochemically active substance (contaminant) such as ascorbic acid contained in a biological sample.
[0006]
Next, as a continuous liquid sending type, an online type sensing system that collects a biological sample, continuously sends the sample to a detector, and performs measurement has been reported (for example, see Non-Patent Document 3). It is easy to install a reactor for removing contaminants in the system, and measurement can be performed with high selectivity. Further, since the size of the detection electrode can be increased, high sensitivity can be achieved. However, since the sample is collected, the response speed and the immediate response are inferior to those of the direct insertion type microelectrode sensor.
[0007]
Therefore, in recent years, there has been reported an example in which the responsiveness is improved by using a very small amount of online sensors in which electrodes and flow paths are integrated on a silicon or glass substrate using a fine processing technique. Such an on-line sensor has a small dead volume, requires a small amount of a sample for measurement, improves spatial resolution, and provides a quick response, and monitors a neurotransmitter released from a cell in real time. (For example, see Non-Patent Document 4). Furthermore, a minute reactor for removing contaminants in advance is installed in a minute amount of online microsensor (for example, see Patent Document 2), and only selectivity is improved without impairing responsiveness ( For example, see Non-Patent Document 5).
[0008]
[Patent Document 1]
JP-A-9-292360
[Patent Document 2]
JP 2000-97899 A
[Non-patent document 1]
M. Takahashi, M .; Morita, O.M. Niwa and H.S. Tabei, Sensors and Actors B, 13-14 (1993) 336-339.
[0011]
[Non-patent document 2]
T. J. Schroeder, J .; A. Jankowski, K .; T. Kawagoe, R .; M. Wrightman, C.I. Lefrou and C.I. Amatore, Analytical Chemistry, 64, 3077-3083, (1992).
[0012]
[Non-Patent Document 3]
A. Koshy, E. et al. Zilkha, T .; P. Obrenovitch, H .; P. Bennetto, D .; A. Richards, L.A. Symon: Anal. Lett. , 26, 831 (1993).
[0013]
[Non-patent document 4]
O. Niwa, R, Kurita, T .; Horiuchi, K .; Torimitsu, Electroanalysis, 11, 356, (1999)
[0014]
[Non-Patent Document 5]
Hayashi, Kurita, Horiuchi, Niwa, Proceedings of the Society for Chemical Sensor Research, Vol. 17, pp97, 2001
[Problems to be solved by the invention]
Catecholamines have a problem in that the concentration in the living body is low (the number in the brain + nM, 1 nM or less in blood), and a sensor having a small electrode area such as carbon fiber has a very small response current and cannot be sufficiently detected.
[0016]
For this reason, there are an electrochemical amplification method of response using a comb-shaped array electrode and an enzyme amplification method using an enzyme. However, in the enzyme amplification method, glucose or the like having a concentration higher than the concentration in the living body is required, and these solutions have to be injected into the sample, which has a problem that the measurement is complicated.
[0017]
In addition, not only ascorbic acid but also metabolites of catecholamines having similar electrochemical properties such as DOPAC (3,4-dihydroxyphenylacetic acid) are present in the biological sample, and their concentration is higher than that of catecholamines. There is a problem that the response obtained from catecholamines is high and cannot be detected by other contaminants.
[0018]
Also, in order to suppress the effects of ascorbic acid, uric acid, and metabolites of catecholamines, a technique of forming a Nafion membrane or polypyrrole peroxide on an electrode is used.In blood, ascorbic acid and uric acid are converted to catecholamines. Since the concentration is relatively high, a method of measuring without being affected by contaminants by mainly combining separation techniques such as liquid chromatography and capillary electrophoresis is mainly used. However, since a sample cannot be introduced continuously, there is a problem that the method cannot be applied to monitoring of a disease state and a cell response that change every moment, a change in the concentration of a substance in the brain, and the like.
[0019]
The present invention has been made in view of such a problem, and it is an object of the present invention to provide an online catecholamine sensing device capable of continuous measurement in which measurement of low-concentration catecholamines can be performed in a manner that solves the problem of receiving contaminants. It is in.
[0020]
[Means for Solving the Problems]
In order to achieve the above object, the present invention provides a sampling probe for sampling a biological fluid, which is formed in a flow channel, and a biological fluid extracted by the sampling probe. A pre-reactor for decomposing the easily oxidizable substance, and an electrochemical detector which is connected to the pre-reactor and is provided with a membrane having molecular selectivity.
[0021]
According to a second aspect of the present invention, in the first aspect of the present invention, the prereactor and the electrochemical detector are integrated on a single chip in a microchannel. I do.
[0022]
According to a third aspect of the present invention, in the first aspect, the membrane having molecular selectivity is a membrane having a function of suppressing diffusion of anionic molecules onto an electrode. And
[0023]
According to a fourth aspect of the present invention, in the first aspect, the electrochemical detector is a comb-shaped electrode or a microelectrode array.
[0024]
According to a fifth aspect of the present invention, in the first aspect of the present invention, an enzyme for removing blood oxidizable substances such as uric acid and ascorbic acid is fixed or electrolyzed in the prereactor. Characterized by being provided with an electrode.
[0025]
The invention according to claim 6 is the invention according to claim 1, wherein a microdialysis probe or a glass capillary is connected as the sampling probe.
[0026]
According to a seventh aspect of the present invention, in the first aspect, a three-dimensional structure is provided inside the pre-reactor.
[0027]
According to an eighth aspect of the present invention, in the first aspect of the present invention, a flow path for changing pH is provided immediately before the electrochemical detector to change the sensitivity of the electrochemical detector. It is characterized by the following.
[0028]
According to a ninth aspect of the present invention, in the first or second aspect of the present invention, a merging channel is provided between the prereactor and the microchannel, and the merging channel and the microchannel are provided. And a minute projection is provided at the junction.
[0029]
Thus, the online catecholamine sensing device according to the present invention has a sampling probe with a dialysis membrane for removing macromolecules such as proteins and blood cells, and a molecular membrane for suppressing the diffusion of anionic molecules onto the electrode. Is characterized by a microarray-type electrode modified with a pre-reactor installed upstream of it to decompose highly concentrated and highly electrochemically active species, making it impossible with conventional technology. It is possible to measure catecholamines continuously, with high sensitivity and with high selectivity.
[0030]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following examples.
[0031]
[Example 1]
FIG. 1 is a configuration diagram for explaining Embodiment 1 of the online catecholamine sensing system of the present invention. In the drawing, reference numeral 1 denotes a sampling probe (a probe with a dialysis membrane for sampling; a microdialysis probe; an MD probe); Is a prereactor, 3 is an electrochemical detector (electrochemical flow cell for detection), 4 is a comb-shaped electrode, 5 is a reference electrode (Ag / AgCl), 6 is a counter electrode, 7 is a syringe for sending liquid, and 8 is a syringe. A pump, 9 indicates uricase, 10 indicates catalase, 11 indicates ascorbic acid oxidase, and 12 indicates a container.
[0032]
The on-line catecholamine sensing system of the present invention includes a sampling probe 1 formed in a flow channel for sampling a biological fluid, and a prereactor for decomposing an oxidizable substance in the biological fluid extracted by the sampling probe 1. And an electrochemical detector 3 which is connected to the pre-reactor 2 and is provided with a membrane having molecular selectivity. Further, a microdialysis probe or a glass capillary is connected as a sampling probe.
[0033]
The electrochemical detector 3 includes a comb-shaped electrode 4 capable of amplifying the response of catecholamines by a redox cycle (both the electrode width and the gap between the electrodes are 2 μm), a reference electrode 5 and a counter electrode 6. Used. The sampling probe 1 is connected with a syringe 7 for liquid feeding and a syringe pump 8. In the pre-reactor 2, a uricase 9, a catalase 10, and an ascorbate oxidase 11 are provided in a flow path of the pre-reactor 2 to decompose uric acid and ascorbic acid in a thin-layer cell structure in which two substrates are bonded together. ing.
[0034]
Catalase 10 is provided on the downstream side of uricase 9 in order to decompose hydrogen peroxide generated by an enzymatic reaction between uric acid and uricase 9. In addition, on the comb-shaped electrode 4, pyrrole is electrolytically polymerized and an excessively oxidized polypyrrole film is formed to suppress diffusion of anionic molecules to the comb-shaped electrode 4, thereby forming a molecule-selective film. . By imparting a negative charge by over-oxidation, the measurement can be performed without being affected by metabolites of catecholamines such as DOPAC due to electrostatic repulsion.
[0035]
Further, according to this method, since a film can be formed only on the comb-shaped electrode 4, the sensitivity of the comb-shaped electrode 4 can be maintained without inhibiting the diffusion of catecholamines between the electrodes.
[0036]
The online catecholamine sensing of the present invention was connected to a potentiostat (BAS) and a syringe pump (CMA) for measurement. The potential applied to the comb electrode 4 was set to 600 and −50 mV with respect to the reference electrode 5. A mixed solution of 1 nM dopamine, 50 μM ascorbic acid, 400 μM uric acid, and 10 nM DOPAC was prepared using bovine serum, placed in the container 12, and introduced into the system at a flow rate of 2 μl / min. For comparison, a similar experiment was performed with a system without an MD probe, an enzyme prereactor, and an electrode-modified membrane. In the system without the MD probe, the enzyme prereactor, and the electrode-modified membrane, a response current of about 750 nA was obtained at the oxidized electrode. This is because uric acid, ascorbic acid, and DOPAC are oxidized on the electrode.
[0037]
On the other hand, when the system according to the present invention was used, a response current of 0.02 nA was obtained. This was almost the same value as when only 10 nM dopamine was measured online using a similar comb-shaped electrode. That is, this indicates that the system according to the present invention eliminates the influence of the contaminants.
[0038]
As described above, catecholamines can be continuously measured with high sensitivity and selectivity by combining the MD probe, the pre-enzyme-reactor, and the electrochemical detector in combination with the liquid sending system.
[0039]
[Example 2]
FIG. 2 is a configuration diagram for explaining Embodiment 2 of the online catecholamine sensing system of the present invention. In the drawing, reference numeral 13 denotes a microelectrode, 14 denotes an insulating film, and 15 denotes a connection pad.
[0040]
In the second embodiment, an electrode in which a large number of microelectrodes 13 are arranged in an array is used instead of the interdigital electrode of the electrochemical detector of the first embodiment. The diameter of the electrodes was 2 μm, and the interval between the electrodes was 20 μm. A photoresist was used for the insulating film 14. The connection pad 15 is for connecting to a potentiostat. By using the microelectrode 13, the response current density increases due to the edge effect, and the background current decreases due to the decrease in the electrode area. Therefore, the S / N ratio is improved, which is suitable for high-sensitivity analysis.
[0041]
By forming this into an array, a larger current value can be obtained. The electrode was covered with a Nafion membrane to improve the selectivity of catecholamines for metabolites. In this case, measurement can be performed by applying an oxidation or reduction potential to the present electrode.
[0042]
As described above, the sensing system in which the MD probe, the pre-enzymatic modification reactor, and the microelectrode array are combined can continuously measure catecholamines with high sensitivity and selectivity.
[0043]
An experiment similar to that of Example 1 was performed, and it was confirmed that the present system can measure catecholamines with high sensitivity and selectively.
[0044]
[Example 3]
FIG. 3 is a configuration diagram for explaining a third embodiment of the online catecholamine sensing system of the present invention. In the figure, reference numeral 16 denotes a flow path, 17 denotes an electrode, 18 denotes a microprojection-shaped three-dimensional structure, and 19 denotes a potential. Reference numeral 20 denotes a connection pad for connecting to the stat, and reference numeral 20 denotes a resist film for forming a flow path.
[0045]
In the third embodiment, an electrochemical reactor having electrodes formed in a flow path in a thin-layer cell is provided instead of the pre-enzyme modification reactor which is the pre-reactor of the first embodiment. An electron transfer mediator film (osmium polymer) was applied on the electrode 17 to increase the electrolysis speed. Since both uric acid and ascorbic acid are easily oxidized on the electrode, they can be removed in the reactor. This electrochemical reactor has a three-dimensional structure 18 in the form of microprojections, and a thin film for an electrode is formed on the projections, so that the contact area with the sample solution increases and the reaction efficiency increases. Can be improved.
[0046]
An experiment similar to that of Example 1 was performed, and it was confirmed that the present system can measure catecholamines with high sensitivity and selectively.
[0047]
[Example 4]
FIG. 4 is a configuration diagram for explaining Embodiment 4 of the online catecholamine sensing system of the present invention, and shows a chip type online catecholamine sensing system to which an MD probe is connected. In the figure, reference numeral 21 is a prereactor, 22 is a detection electrode (comb-shaped electrode), 23 is a reference electrode, 24 is a counter electrode, 25 is an MD probe, 26 is a microchannel, 27 is a discharge tube, and 28 is a measurement. A container containing a sample for use, 29 is a syringe containing a phosphate buffer solution, and 30 is a syringe pump.
[0048]
For the detection electrode 22, the reference electrode 23, and the counter electrode 24, a carbon thin film manufactured by a dry method was used. This substrate is bonded to another substrate and has a very small cell volume. Uricase, catalase, and ascorbate oxidase are immobilized in the prereactor 21 as in Example 1 described above. As the detection electrode 22, a comb-shaped electrode similar to that used in Example 1 described above is used, and a film for eliminating anionic molecules is formed on this electrode.
[0049]
A silver paste was applied on the reference electrode 23. For the comb electrodes, 600 mV vs. 600 mV. Ag, -50 mV vs. Ag potential was applied. In the sample continuously collected by the MD probe 25, first, macromolecules such as proteins are removed by the dialysis membrane of the probe, and urea and ascorbic acid are removed in the prereactor 21. Only catecholamines and their metabolites reach the detection electrode 22, but due to electrostatic repulsion, catecholamines metabolites cannot reach the electrode surface and are discharged from the discharge tube 27.
[0050]
The on-line catecholamine sensing system of the present invention was connected to a potentiostat and a syringe pump to detect dopamine. Serum was prepared so that dopamine at a concentration of 1 nM, ascorbic acid at 50 μM, uric acid at 400 μM, and DOPAC at 30 nM were introduced into the system. On the other hand, the same experiment was performed on an online catecholamine sensing system without using an MD probe, an enzyme, and an anionic molecular membrane. When the above solution was introduced and measured, a current of about 700 nA was observed in the sensing system without any modification, whereas a current of about 20 pA was observed in the online catecholamine sensing system according to the present invention. This is because the effects of DOPAC, uric acid, and ascorbic acid were all eliminated, and only dopamine was detected.
[0051]
Further, by integrating the prereactor and the electrochemical cell for detection, the ineffective volume could be significantly reduced, and the measurement time could be shortened.
[0052]
As described above, the chip-type catecholamine sensing system combining the MD probe, the prereactor, and the detection electrode is capable of controlling the trace amount substance in a biological sample such as dopamine without being affected by metabolites and the like. It can be detected well.
[0053]
In addition, if this system is integrated on a single chip and the flow path between the prereactor and the electrochemical detector is made thin, the flow of liquid will be smooth and the sensitivity of the prereactor and the electrochemical detector will increase. It works.
[0054]
[Example 5]
FIG. 5 is a configuration diagram for explaining Embodiment 5 of the online catecholamine sensing system of the present invention, and is a diagram showing an online catecholamine sensing device provided with a merging channel. In the figure, reference numeral 31 denotes a tube for a merging channel, 32 denotes a syringe containing a low pH solution, and 33 denotes a syringe pump. Note that components having the same functions as the components shown in FIG. 1 are denoted by the same reference numerals.
[0055]
In the fifth embodiment, the on-line catecholamine sensing device of the first embodiment shown in FIG. 1 is provided with a confluent flow tube 31, a syringe 32 containing a low-pH solution, and a syringe pump 33. The junction flow path is provided between the prereactor 2 and the electrochemical detector 3. This is to prevent the enzyme immobilized in the prereactor 2 from being deactivated by introducing a low pH solution.
[0056]
Similarly to Example 1 described above, a mixed solution of adrenaline at a concentration of 10 nM, ascorbic acid at 50 μM, uric acid at 400 μM, and DOPAC at 30 nM was prepared using bovine serum, and introduced into the system at a flow rate of 2 μl / min. . Further, a 0.1 N sulfuric acid solution was introduced at a rate of 0.1 μl / min from the merging channel. A similar experiment was performed in a system in which a sulfuric acid solution was not introduced.
[0057]
First, the response current was 0.03 nA in the system into which the sulfuric acid solution was introduced. This was almost equivalent to the result obtained when a standard solution containing only adrenaline was measured. In this system, the detection limit was 5 nM. Next, when a sulfuric acid solution was introduced from the merging channel, the response current value increased to 0.05 nA.
[0058]
Thus, the response current increased despite the introduction of the sulfuric acid solution, despite the decrease in the adrenaline concentration in the sample, because the pH of the sample was reduced to 3 or less, and the redox efficiency of adrenaline was improved. This is because the response amplification factor has been improved. With the improvement of the oxidation-reduction efficiency, the detection limit was improved to 3 nM.
[0059]
As described above, by introducing a low-pH solution, such as a sulfuric acid solution, to a chip-type catecholamine sensing device that combines an MD probe, a prereactor, and a detection electrode, high sensitivity can be achieved in the range of pH in vivo such as adrenaline. Even for substances that are difficult to measure, the pH in the sample can be changed and measurement can be performed with high sensitivity.
[0060]
[Example 6]
FIG. 6 is a configuration diagram for explaining Embodiment 6 of the online catecholamine sensing system of the present invention, and is a diagram showing an online catecholamine sensing device in which the device shown in Embodiment 4 is chipped. In the figure, reference numeral 34 denotes a merging channel, 35 denotes a syringe containing a low-pH solution, 36 denotes a syringe pump, and 37 denotes a microprojection. Note that components having the same functions as the components shown in FIG. 4 are denoted by the same reference numerals.
[0061]
In the sixth embodiment, a merging channel 34 is added to the above-described fourth embodiment. The merging channel 34 is connected to a syringe 35 containing a low-pH solution and a syringe pump 36. The sample solution and the low pH solution were mixed at the junction. In order to efficiently mix the sample solution and the low pH solution, the microprojections 37 were formed using a photoresist. Using this device, an experiment was performed under the same conditions as in Example 4. As a result, a response current of 0.55 nA was obtained, which was larger than that of the device shown in Example 4. This is because a microprojection structure was formed at the junction and the sample solution and the low pH solution were efficiently mixed by the mixing effect.
[0062]
【The invention's effect】
As described above, according to the present invention, a sampling probe formed in a flow path for sampling a biological fluid, and a prereactor for decomposing oxidizable substances in the biological fluid extracted by the sampling probe And an electrochemical detector in communication with the prereactor and provided with a membrane having molecular selectivity, so that continuous measurement is possible and high sensitivity and high selective measurement are possible. Further, since the reactor and the detector can be easily replaced while the probe is left in the living body, continuous measurement can be performed for a long period of time. In addition, since the time until a response is reduced, real-time measurement can be performed. From this, it is possible to follow changes in the disease state and the like well.
[0063]
Furthermore, if this system is integrated on one chip and the flow path between the pre-reactor and the electrochemical detector is made thinner, the flow of liquid will be smoother, the reaction efficiency in the pre-reactor and the electrochemical detector This has the effect of increasing the sensitivity of the camera.
[Brief description of the drawings]
FIG. 1 is a configuration diagram for explaining Embodiment 1 of an online catecholamine sensing system of the present invention.
FIG. 2 is a configuration diagram for explaining Embodiment 2 of the online catecholamine sensing system of the present invention.
FIG. 3 is a configuration diagram for explaining Embodiment 3 of the online catecholamine sensing system of the present invention.
FIG. 4 is a configuration diagram for explaining Embodiment 4 of the online catecholamine sensing system of the present invention.
FIG. 5 is a configuration diagram for explaining Embodiment 5 of the online catecholamine sensing system of the present invention.
FIG. 6 is a configuration diagram for explaining Example 6 of the online catecholamine sensing system of the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Sampling probe 2 Prereactor 3 Electrochemical detector 4 Comb electrode 5 Reference electrode 6 Counter electrode 7 Syringe 8 for sending liquid 9 Syringe pump 9 Uricase 10 Catalase 11 Ascorbic acid oxidase 12 Container 13 Microelectrode 14 Insulating film 15 Connection Pad 16 Flow path 17 Electrode 18 Microprojection-like three-dimensional structure 19 Connection pad 20 Resist film 21 Prereactor 22 Detection electrode (comb electrode)
23 Reference electrode 24 Counter electrode 25 MD probe 26 Micro flow path 27 Discharge tube 28 Container 29 Syringe 30 Syringe pump 31 Merge flow path tube 32 Syringe 33 Syringe pump 34 Merge flow path 35 Syringe 36 Syringe pump 37 Micro projection

Claims (9)

Formed in the flow path, a sampling probe for sampling the biological fluid, a pre-reactor for decomposing oxidizable substances in the biological fluid extracted by the sampling probe, and communicated with the pre-reactor, molecules An online catecholamine sensing device, comprising: an electrochemical detector provided with a selective membrane. The online catecholamine sensing device according to claim 1, wherein the prereactor and the electrochemical detector are integrated on a single chip in a microchannel. The on-line catecholamine sensing device according to claim 1, wherein the membrane having molecular selectivity is a membrane having a function of suppressing diffusion of anionic molecules onto an electrode. The online catecholamine sensing device according to claim 1, wherein the electrochemical detector is a comb electrode or a microelectrode array. The on-line according to claim 1, wherein an electrode for fixing or electrolyzing an enzyme for removing blood oxidizable substances such as uric acid and ascorbic acid is provided in the pre-reactor. Catecholamine sensing device. The online catecholamine sensing device according to claim 1, wherein a microdialysis probe or a glass capillary is connected as the sampling probe. The online catecholamine sensing device according to claim 1, further comprising a three-dimensional structure inside the pre-reactor. The online catecholamine sensing device according to claim 1, wherein a flow path for changing pH is provided immediately before the electrochemical detector to change the sensitivity of the electrochemical detector. The merged flow path is provided between the prereactor and the minute flow path, and a minute projection is provided at a junction of the merged flow path and the minute flow path. Online catecholamine sensing device.

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