JP2017096727A - Cartridge for detecting protein or pathogens and automatic detector - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cartridge that detects protein or pathogens rapidly and sensitively, and an automatic detector having the cartridge.SOLUTION: The container 105 of a cartridge 100 includes an outer-air introduction port 111 and a vent hole 150 on an outer air introduction side, which are configured so that outer air introduced from the introduction port 111 passes through a support electrolyte solution held in the container and reaches the vent hole 150 on the outer air introduction side. A surface to which the element of a work electrode 101 is fixed lies downward so that the outer air can contact with one or more work electrodes 101 when the outer air passes through the support electrolyte solution 104.SELECTED DRAWING: Figure 6

Description

  The present invention relates to a protein or pathogen detection cartridge and an automatic detection apparatus. More specifically, the present invention relates to a protein or pathogen detection cartridge and an automatic detection device, each of which includes a conductive diamond electrode on which a protein or pathogen recognition element is immobilized.

  Conventionally, it has been desired to detect target pathogens, pathogenic bacteria, viruses and their proteins with high sensitivity. For example, influenza virus (IFV) may be a pandemic, and rapid and accurate detection of influenza virus is required. Currently used IFV detection methods include immunochromatography using antibodies as IFV recognition devices, detection by sugar chain arrays using sugar chains, RT-PCR using genes, and hemagglutination assays using red blood cells. and so on. However, these are time consuming and costly and require specialized knowledge and skills. In addition, since it is necessary to first collect a sample and prepare a test sample, a method for automatically performing from sample collection to detection has not been realized.

  Grabowska et al. Reported an IFV detection method using hybridization (Non-patent Document 1). This method uses a sensor in which two different oligonucleotide probes are immobilized on the surface of the Au electrode via gold thiol bonds, thereby simultaneously detecting both hemagglutinin (HA) and neuraminidase (NA) oligonucleotide targets. Is possible. Kamikawa et al. Reported IFV detection using magnetic nanoparticles covered with electroactive polyaniline and modified with H5N1-type HA monoclonal antibody on the surface (Non-patent Document 2). In this method, nanoparticles interacting with H5N1 virus are collected from serum with a strong magnetic force (MPC), and the amount of virus binding is quantified by electrochemical measurement.

  Sakurai et al. Reported a rapid and highly sensitive type classification of seasonal influenza using fluorescent immunochromatography (Non-patent Document 3). The immunochromatography method, which is a method for detecting IFV used in clinical practice, has a detection sensitivity of 1000 pfu and is difficult to detect at the early stage of infection. In the same document, Sakurai et al. Captured viruses in a sandwich type using both a virus supplement antibody and a detection antibody labeled with a sensitizer, and further performed detection using a densitometric analyzer and a fluorescence immunochromatography analyzer. It is reported that the improvement of the sensitivity 100 times that of the prior art has been achieved. However, complementary oligonucleotides for hybridization and antibodies to be immobilized on a substrate require enormous labor and cost for production, and have storage stability problems.

Hassen et al. Have reported quantification of influenza A virus using electrochemical impedance spectroscopy (Non-Patent Document 4). In this document, influenza A virus is detected using a device in which an antibody-sugar chain-removed avidin-thiol structure is immobilized on the gold electrode surface.

  On the other hand, boron-doped diamond electrodes have excellent characteristics as compared with other conventional electrode materials such as glassy carbon and platinum electrodes, and have attracted attention in recent years. In addition to the well-known properties of diamond, which have high thermal conductivity and extremely high hardness, boron-doped diamond electrodes have a wide potential window, small background current, high adsorption resistance, and are chemically inert. Has attractive properties. Boron-doped diamond electrodes are physically and chemically stable and have excellent durability.

  As a sensor provided with a diamond electrode, a diamond electrode capable of accurately quantifying catechol or a catechol derivative and a sensor provided with the diamond electrode have been reported (Patent Document 1). In this document, oxalic acid is electrochemically detected using a 4-pentenoic acid-modified diamond electrode.

  The conventional IFV detection apparatus needs to perform wavelength measurement after adding a hybridization reagent to a sample or adding gold nanoparticles. For viruses, various pathogenic bacteria, and pathogens, a device capable of early detection with high sensitivity without using antibodies or expensive devices is desired.

JP2007-292717 (Japanese Patent No. 4978858)

Grabowska et al., Anal. Chem., 85, 10167-10173 (2013) Kamikawa et al., Biosens Bioelectron., 26, 1346-1352 (2010) Sakurai et al., PLOS ONE DOI: 10.1371 / jounal.pone.0116715, 1-13 (2015) Hassen et al., Electrochim. Acta 56, 8325-8328 (2011)

  An object of the present invention is to provide a device for detecting a protein or a pathogen with high speed and high sensitivity, which solves the conventional problems.

  In order to solve the above-mentioned problems, the present invention provides a protein or pathogen detection cartridge and an automatic detection apparatus provided with a conductive diamond electrode on which a protein or pathogen recognition element is immobilized. By performing electrochemical measurement using an apparatus equipped with this cartridge, influenza virus can be detected quickly and with high sensitivity without adding a special reagent or gold nanoparticles to the specimen.

That is, the present invention includes the following.
[1] A cartridge for detecting a protein or pathogen comprising one or more working electrodes, a counter electrode, a reference electrode, and a container capable of holding a supporting electrolyte solution,
The one or more working electrodes each have a conductive diamond electrode on the surface of which the same or different types of elements that recognize proteins or pathogens are immobilized;
The working electrode, the counter electrode, and the reference electrode are configured to allow electrical continuity with the outside.
The working electrode, the counter electrode, and the liquid junction of the reference electrode are arranged in the container so as to be able to contact the supporting electrolyte solution when the supporting electrolyte solution is present in the container,
The container includes an outside air inlet, an outside air outlet side vent and an outlet port,
The introduction port and the ventilation port on the outside air outlet side are configured such that the outside air introduced from the introduction port passes through the supporting electrolyte solution held in the container and is led out to the ventilation port on the outside air outlet side. The outside air that has passed through the vent on the outside air outlet side is further led out to the outlet of the cartridge,
The container and the working electrode are configured so that the outside air can come into contact with the one or more working electrodes when passing through the supporting electrolyte solution, and when the outside air is in contact with the one or more working electrodes. The counter electrode and the reference electrode are configured so that the liquid junction of the counter electrode and the reference electrode is always in contact with the supporting electrolyte solution,
The supporting electrolyte solution and the internal electrolyte solution of the reference electrode are configured to be injected into the container,
When the outside air is brought into contact with the working electrode and when electrochemical measurement is performed with the working electrode, the surface on which the working electrode element is fixed is configured to face downward.
The outside air inlet, the outside air outlet, and the outside air outlet are arranged at a position higher than the highest liquid surface position of the supporting electrolyte solution.
A cartridge for detecting the protein or pathogen.
[2] The cartridge according to 1, wherein the vent on the inlet side of the outside air includes a bubble generation filter that makes the outside air a bubble.
[3] When the cartridge includes a plurality of working electrodes, the working electrode on the outside air inlet side is disposed at a lower position than the other working electrodes on the outlet port side, and the other working electrodes on the outlet port side are arranged. The cartridge according to 1 or 2, wherein is disposed at a higher position than the working electrode on the outside air inlet side.
[4] A protein comprising one or more working electrodes, a counter electrode, a reference electrode, and a container capable of holding a supporting electrolyte solution, and further comprising a liquid level up-and-down moving mechanism capable of raising and lowering the liquid level Or a cartridge for detecting pathogens,
The one or more working electrodes each have a conductive diamond electrode on the surface of which the same or different types of elements that recognize proteins or pathogens are immobilized;
The working electrode, the counter electrode, and the reference electrode are configured to allow electrical continuity with the outside.
The container has an outside air introduction port, an outside air outlet side ventilation port and an outlet port, and further has an air introduction port for a liquid level vertical movement mechanism,
The working electrode, the counter electrode, and the liquid junction of the reference electrode are arranged in the container so as to be able to contact the supporting electrolyte solution when the supporting electrolyte solution liquid level in the container rises,
The introduction port is configured to be able to blow the outside air introduced from the introduction port onto the surface of one or a plurality of working electrodes,
The outside air outlet side vent is arranged so that the outside air blown to one or a plurality of working electrodes can pass through, and the outside air that has passed through the outside air outlet side vent is further led out to the outlet of the cartridge. Configured,
When adsorbing protein or pathogen to the device, the level of the supporting electrolyte solution is lowered, and one or more working electrodes are not in contact with the supporting electrolyte solution.
When the level of the supporting electrolyte solution is lowered, the liquid junction of the counter electrode and the reference electrode may be in contact with the supporting electrolyte solution,
When detecting proteins or pathogens adsorbed on the element, the liquid level of the supporting electrolyte solution is raised, and the liquid junction of one or more working electrodes, counter electrodes and reference electrodes are in contact with the supporting electrolyte solution Has been
The supporting electrolyte solution and the internal electrolyte solution of the reference electrode are configured to be injected into the container,
When the outside air is brought into contact with the working electrode and when the electrochemical measurement is performed by the working electrode, the surface on which the working electrode element is fixed is configured to face downward.
The outside air inlet, the outside air outlet, and the outside air outlet are arranged at a position higher than the highest liquid surface position of the supporting electrolyte solution.
A cartridge for detecting the protein or pathogen.
[5] When the cartridge includes a plurality of working electrodes, the working electrode on the outside air inlet side is arranged at a higher position than the other working electrodes on the outlet port side, and the other working electrodes on the outlet port side are arranged. The cartridge according to 4, wherein is disposed at a position lower than the working electrode on the outside air inlet side.
[6] The cartridge according to 4 or 5, wherein the air inlet for the liquid surface vertical movement mechanism is located at a position higher than the highest liquid surface position of the supporting electrolyte solution.
[7] The cartridge according to any one of 1 to 6, wherein the cartridge is configured to have a sealed structure by injecting the supporting electrolyte solution from the counter electrode insertion port and attaching the counter electrode.
[8] Any one of 1 to 7 configured to inject the internal electrolyte solution for the reference electrode from the metal electrode insertion port of the reference electrode and attach the metal electrode so that the container can have a sealed structure. Cartridge.
[9] The cartridge according to any one of 1 to 8, further comprising an element capable of storing and reproducing.
[10] The cartridge according to 9, wherein the element capable of storing and reproducing can be recorded and reproduced from the outside of the cartridge via a substrate.
[11] The cartridge according to any one of 1 to 10, which has an introduction filter immediately after the outside air introduction port.

[12] An apparatus for detecting a protein or pathogen, comprising one or more cartridges according to any one of 1 to 11,
A working electrode, a counter electrode, and a reference electrode on the outer surface of the cartridge configured to be electrically conductive with the outside are electrically connected to the detection device;
When the cartridge includes an element capable of storing and reproducing, the element capable of storing and reproducing is electrically connected to the detection device,
An electrochemical measurement circuit capable of performing cyclic voltammetry measurement and / or electrochemical impedance measurement;
It has a display circuit that displays the measurement result, or an output terminal that outputs or transmits the measurement result.
It has a power circuit,
An outside air inlet for sucking outside air into the detection device, and the outside air inlet is connected to the outside air inlet of the cartridge;
An outside air outlet for discharging outside air from the detection device, the outside air outlet being connected to the outside air outlet of the cartridge;
It has an outside air suction pump connected to the outside air inlet,
Equipped with a control circuit,
An apparatus for detecting the protein or pathogen.
[13] The cartridge according to any one of 4 to 11, wherein the cartridge includes a liquid level vertical movement mechanism, the detection device includes a pump for the liquid level vertical movement mechanism, and the pump is connected to a control circuit of the detection device The apparatus according to 12, wherein:
[14] The device according to 12 or 13, wherein when the device includes a plurality of cartridges, the outside air inlet and the outside air outlet of the plurality of cartridges are connected to the device in series or in parallel.
[15] The device according to any one of 12 to 14, comprising a wireless or wired communication circuit.
[16] The apparatus according to any one of 12 to 15, further comprising a data recording / reproducing circuit for recording / reproducing data with respect to the element when the cartridge includes an element capable of storing / reproducing.
[17] The device according to any one of 12 to 16, further comprising an outside air filter between the outside air inlet of the device and the outside air inlet of the cartridge.
[18] Any of 12-17, comprising an outside air tank and a circulation pump, and comprising an outside air tank exhaust valve capable of closing an exhaust pipe connected to the outside air exhaust port between the outside air exhaust port and the outside air tank The device described in 1.

[19] A method for detecting a protein or a pathogen using the cartridge according to any one of 1 to 11 or the apparatus according to any one of 12 to 18.
[20] When the cartridge includes an element capable of storing and reproducing, from each element, information on a protein or pathogen to be measured by one or more working electrodes, initial characteristics of one or more working electrodes, and For cartridges that have already been used for measurement, the time used for measurement, the process of reading the detected numerical information,
A step of operating an outside air suction pump and feeding the sucked outside air into the cartridge;
An adsorption process in which inhaled outside air is blown onto a surface on which one or more working electrode elements are fixed, or in contact with one or more working electrodes as bubbles in a supporting electrolyte solution;
An exhaust stroke in which the outside air after the adsorption process passes through the outside air outlet side of the cartridge and is discharged from the outside air outlet and the outside air outlet of the device;
An electrochemical measurement process in which the external air suction pump is stopped and cyclic voltammetry measurement or electrochemical impedance measurement is performed while sequentially switching one or more working electrodes of one or more cartridges;
A step of recording a measurement result in each element of each cartridge when the cartridge has an element capable of storing and reproducing; and a recording information is communicated to the outside when the apparatus has a wireless or wired communication circuit Process,
20. The detection method according to 19, comprising.
[21] Using the detection device according to 18, which includes an outside air tank, a circulation pump, and an outside air tank exhaust valve,
When the cartridge has an element capable of storing and reproducing, from each element, information on the protein or pathogen to be measured by one or more working electrodes, initial characteristics of one or more working electrodes, and already measured For the cartridge used in the process, the time used for measurement, the process of reading the information of the detected numerical value,
Opening the outside air tank exhaust valve and operating the outside air suction pump to introduce unmeasured outside air into the outside air tank;
Closing the outside air tank exhaust valve and stopping the outside air suction pump;
A process of operating the circulation pump and sending the outside air in the outside air tank into the cartridge;
An adsorption process in which inhaled outside air is blown against a surface on which one or more working electrode elements are fixed, or in contact with one or more working electrodes as bubbles in the supporting electrolyte;
Passing the outside air after the adsorption step through the outside air outlet side of the cartridge and exhausting it from the outside air outlet port to the outside air tank;
An electrochemical measurement step in which the cyclic pump is stopped and cyclic voltammetry measurement or electrochemical impedance measurement is performed while sequentially switching one or more working electrodes of one or more cartridges;
A step of recording a measurement result in the element of each cartridge when the cartridge has an element capable of storing and reproducing; and
A step of communicating recorded information to the outside if the device has a wireless or wired communication circuit;
A method for detecting a protein or pathogen, comprising:

  Proteins or pathogens can be detected rapidly, sensitively and automatically using an apparatus equipped with the cartridge of the present invention. Compared to other electrochemical detection methods, the diamond electrode has an inactive surface, which can suppress nonspecific adsorption of proteins, etc., and has low noise and a wide potential window, enabling highly sensitive detection. . Further, the present invention can detect proteins or pathogens with high sensitivity without using expensive molecules such as antibodies, and it is necessary to add hybridization reagents and gold nanoparticles to the specimen. And rapid and highly sensitive detection is possible. In addition, it is not necessary to manually prepare the test sample, and secondary infection to the person in charge of the test can be prevented.

It is a perspective view of the cartridge of the present invention. It is a top view of the cartridge of the present invention. It is a front view of the cartridge of the present invention. It is a rear view of the cartridge of the present invention. It is the left view (a) and right view (b) of the cartridge of this invention. It is sectional drawing seen from the AA cross section in FIG. 2 of the cartridge 100 of this invention. The liquid level 130 can be set by the amount of the supporting electrolyte solution 104 injected into the container 105. FIG. 3 is a cross-sectional view of the cartridge of the present invention as seen from a BB cross section in FIG. It is sectional drawing seen from the AA cross section in FIG. 2 of the cartridge 200 of this invention. The vertical movement mechanism 201 is not expanded, and the liquid level 230 is low. It is sectional drawing seen from the AA cross section in FIG. 2 of the cartridge 200 of this invention. A state in which the liquid level 230 is raised by expanding the vertical movement mechanism 201 is shown. It is a perspective view of the detection part 300 provided with the element 110 of the working electrode of this invention. (a) is the figure which looked at the detection part 300 from diagonally upward, (b) is the figure which looked at the detection part 300 from diagonally downward. In (b), the surface 301 on which the elements of the working electrode are fixed is shown below the detection unit 300. FIG. 4A is a plan view of the detection unit 300. FIG. FIG. 11B is a cross-sectional view of the detection unit 300 as viewed from the GG cross section in FIG. FIG. 3 is a cross-sectional view of the cartridge 100 of the present invention as viewed from the CC cross section in FIG. 2. It is sectional drawing seen from the DD cross section in FIG. 2 of the cartridge 100 of this invention. It is sectional drawing seen from the EE cross section in FIG. 3 of the cartridge 100 of this invention. FIG. 5 is a cross-sectional view of the cartridge 100 of the present invention as viewed from the FF cross section in FIG. 3. An automatic detection device 500 including the cartridge 100 of the present invention is shown. The automatic detection apparatus 600 provided with the cartridge 100 of this invention, the external air tank 610, and the circulation pump 620 is shown. The automatic detection apparatus 700 provided with the cartridge 200 of this invention, the external air tank 610, the circulation pump 620, and the pump 701 for a vertical movement mechanism is shown. A scheme for solid phase synthesis of an azide group-introduced s2 (1-5) peptide dendrimer is shown. The detection of HA protein by CV measurement using peptide unmodified (left) and peptide modified (right) diamond electrodes is shown. Detection of influenza virus (IFV) by CV measurement using peptide unmodified (left) and peptide modified (right) diamond electrodes. The detection of HA protein by electrochemical impedance (EIS) measurement using a peptide-modified diamond electrode is shown. The result of the target HA protein and the control protein BSA in EIS measurement using a peptide-modified diamond electrode is shown. The square is HA (R ² = 0.989) and the circle is BSA (R ² = 0.784). The detection of IFV by EIS measurement using a peptide-modified diamond electrode is shown.

  Hereinafter, the present invention will be described in detail with reference to the drawings.

The protein or pathogen detection cartridge of the present invention The protein or pathogen detection cartridge of the present invention (hereinafter sometimes simply referred to as the cartridge of the present invention) is a conductive material in which an element that recognizes the protein or pathogen is immobilized on the surface. A conductive diamond electrode (hereinafter sometimes simply referred to as a conductive diamond electrode). Here, “recognizing” a protein or pathogen means that the element interacts, binds, or associates with the protein or pathogen. When the element recognizes a protein or pathogen, when an electric potential is applied, an electric current flows due to an electrochemical reaction. By measuring this current, a recognized protein or pathogen can be detected. As used herein, “detection” includes qualitative detection and quantitative detection.

  FIG. 6 illustrates a cartridge 100 as the cartridge of the present invention. The cartridge 100 of the present invention includes one or a plurality of working electrodes 101, a counter electrode 102, a reference electrode 103, and a container 105 that can hold a supporting electrolyte solution 104. This cartridge is for detecting proteins or pathogens. The number of the working electrode or electrodes that the cartridge of the present invention has is not particularly limited, and may be 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, etc. it can.

  Each of the one or more working electrodes 101 includes a conductive diamond electrode 110 having an element that recognizes the same or different type of protein or pathogen immobilized on the surface thereof.

  The working electrode 101, the counter electrode 102, and the reference electrode 103 are configured so as to be electrically connected to the outside.

  The working electrode 101, the counter electrode 102, and the liquid junction 123 of the reference electrode are arranged in the container 105 so as to be able to contact the supporting electrolyte solution 104 when the supporting electrolyte solution 104 is present in the container 105. Yes.

  The container 105 includes an outside air introduction port 111 and an outside air outlet side vent port 150, and the outside air inlet port 111 and the outside air outlet side vent port 150 hold the outside air introduced from the inlet port 111 in the container. It is configured to pass through the supporting electrolyte solution and be led out to the vent hole 150 on the outside air outlet side, and the outside air that has passed through the vent hole 150 on the outside air outlet side is further led out to the outlet port 155 of the cartridge. It is configured.

  In the cartridge, the container 105 and the working electrode 101 are configured so that the outside air can come into contact with the one or more working electrodes 101 when the outside air passes through the supporting electrolyte solution 104, and the outside air is operated here. The counter electrode 102 and the entire reference electrode are configured so that the counter electrode 102 and the liquid junction 123 of the reference electrode are always in contact with the supporting electrolyte solution 104 when in contact with the electrode. The entire reference electrode is a metal electrode (hereinafter simply referred to as a reference electrode) 103 of the reference electrode, a metal electrode insertion port 107 of the reference electrode, a liquid junction portion 123 of the reference electrode, a member 125 for holding the internal electrolyte solution, and When present, it refers to the entire one including the internal electrolyte solution 124.

  The cartridge is configured such that the supporting electrolyte solution 104 and the internal electrolyte solution 124 of the reference electrode 103 can be injected into the container 105. The internal electrolyte solution 124 is held by a member 125 for holding the internal electrolyte solution. When the outside air is brought into contact with the working electrode 101 and when electrochemical measurement is performed by the working electrode 101, the surface 301 on which the elements of the working electrode are fixed is configured to face downward. That is, the detector 300 is attached to the cartridge so that the surface 301 on which the working electrode elements are fixed faces downward.

  The outside air inlet 111, the outside air outlet 155, and the outside air outlet side vent 150 (specifically, the lower ends thereof) are arranged at positions higher than the highest liquid surface position of the supporting electrolyte solution 104. Conversely, regarding the cartridge of the present invention, the maximum liquid level position of the supporting electrolyte solution 104 is lower than the outside air inlet 111, the outside air outlet 155, and the outside air outlet 150 (specifically, the lower end thereof). Set to position.

  In an embodiment, the vent 113 on the outside air inlet side may include a bubble generation filter 120 that turns outside air into bubbles. The vent 113 on the outside air inlet side may have any shape such as a circle or a rectangle. In an embodiment, the vent 113 on the outside air inlet side may be a circle having a diameter of 1 to 10 mm, for example 3 to 4 mm. In one embodiment, the vent 113 on the outside air inlet side includes an inflow prevention mechanism so that the supporting electrolyte solution 104 does not flow into the outside air inlet 111 side. The bubble generation filter 120 may also serve as an inflow prevention mechanism. The inflow prevention mechanism may be a valve that prevents inflow of liquid.

  The dimensions of the cartridge of the present invention are not particularly limited. For example, when the vent 113 on the side of the outside air inlet is a circle having a diameter of 3 to 4 mm, the width through which bubbles in the container pass and the lateral width of the detection unit 300 are also included. To 3 to 5 mm, for example, about 3 to 4 mm. In addition, the cartridge of the present invention may have a longitudinal length of about 3 to 30 cm, a lateral length of about 4 to 40 cm, and a depth of about 1.5 to 15 cm in an embodiment.

  When the cartridge 100 includes a plurality of working electrodes 101 in an embodiment, the working electrode 140 on the outside air inlet side is disposed at a lower position than the other working electrodes 145 on the outside air outlet side, and the outlet port The other working electrode 145 on the side is arranged at a higher position than the working electrode 140 on the outside air inlet side. The low position and the high position here are as follows. When the outside air introduced from the outside air inlet 111 passes through the supporting electrolyte solution 104 and is led out to the vent 150 on the outside air outlet side, the lower side is viewed from the direction in which the outside air rises in the supporting electrolyte solution 104. The lower position is the upper position. Further, when the outside air introduced from the outside air inlet 111 passes through the supporting electrolyte solution 104 and is led out to the vent 150 on the outside air outlet side, the outside air is brought into contact with one or a plurality of working electrodes in order. The air inlet 113 on the inlet side is arranged at a position lower than the one or more working electrodes and at a position lower than the surface of the supporting electrolyte solution. It is arrange | positioned in the position higher than the working electrode of this, and is arrange | positioned in the position higher than the support electrolyte solution liquid surface.

  The cartridge of the present invention includes a cartridge provided with a liquid level vertical movement mechanism. In an embodiment, a cartridge 200 illustrated in FIG. 8 and FIG. 9 is given as a cartridge including the liquid level vertical movement mechanism 201.

  The liquid level up-and-down moving mechanism 201 can be, for example, a balloon made of a material that can expand and contract. By filling the inside with air, the balloon can be inflated and the liquid level 230 can be raised. Further, by sucking the air inside, the balloon can be deflated and the liquid level 230 can be lowered.

  In this embodiment, the container 105 includes an outside air inlet 111, an outside air outlet side vent 150 and an outlet 155, and further includes an air inlet 202 for a liquid level vertical movement mechanism. The outside air outlet side vent hole 150 is arranged so that the outside air blown to one or more working electrodes 101 can pass therethrough, and the outside air that has passed through the outside air outlet side vent hole 150 further passes to the outlet port 155 of the cartridge. It is configured to be derived.

  The working electrode 101, the counter electrode 102, and the liquid junction 123 of the reference electrode are arranged in the container so as to be able to contact the supporting electrolyte solution when the liquid level 230 of the supporting electrolyte solution 104 in the container 105 rises. Has been. An example is shown in FIG.

  When adsorbing proteins or pathogens contained in the outside air to the device, the liquid surface 230 of the supporting electrolyte solution 104 is lowered so that one or more working electrodes 101 do not come into contact with the supporting electrolyte solution. An example is shown in FIG. When the liquid level 230 of the supporting electrolyte solution 104 is lowered, the counter electrode 102 and the liquid junction 123 of the reference electrode may be in contact with the supporting electrolyte solution 104. When detecting proteins or pathogens adsorbed on the element, the liquid surface 230 of the supporting electrolyte solution 104 is raised, and the working electrode 101, the counter electrode 102, and the liquid junction 123 of the reference electrode are formed in the supporting electrolyte solution 104. It is configured to be in contact.

  When the cartridge includes a plurality of working electrodes 101 in the cartridge 200, in one embodiment, the working electrode 240 on the outside air inlet side is disposed at a higher position than the other working electrodes 245 on the outlet side, The other working electrode on the outlet side may be arranged at a lower position than the working electrode on the outside air inlet side. 8 and 9 illustrate such an embodiment. In one embodiment, the working electrode 240 on the outside air inlet side may be disposed at the same height as the other working electrode 245 on the outlet side. In one embodiment, the working electrode 240 on the outside air inlet side is disposed at a lower position than the other working electrode 245 on the outlet side, and the other working electrode on the outlet side is on the outside air inlet side. You may arrange | position in the high position compared with a working electrode. The low position here is a position on the side where the liquid level 230 is lowered by the liquid level vertical movement mechanism 201, and the high position is a position on the side where the liquid level 230 is elevated by the liquid level vertical movement mechanism 201. It is.

  In an embodiment, with respect to the cartridge 200, the air inlet 202 for the liquid level up-and-down moving mechanism can be disposed at a position higher than the highest liquid level position of the supporting electrolyte solution 104. In other words, the maximum liquid surface position of the supporting electrolyte solution 104 can be set to a position lower than the air inlet 202 for the liquid surface vertical movement mechanism. In one embodiment, the maximum liquid surface position of the supporting electrolyte solution 104 is lower than the outside air inlet 111, the outside air outlet 155, and the outside air outlet side vent 150 (specifically, the lower end thereof). In other words, the outside air inlet 111, the outside air outlet 155, and the outside air outlet side vent 150 (specifically, the lower end thereof) are arranged at a position higher than the highest liquid surface position of the supporting electrolyte solution 104. .

  In one embodiment, the cartridge of the present invention is configured to allow injection of the supporting electrolyte solution 104 and the reference electrode internal electrolyte solution 124. In an embodiment, the cartridge of the present invention may be configured such that the container 105 can be sealed by injecting the supporting electrolyte solution 104 from the counter electrode insertion port 106 and attaching the counter electrode 102. In one embodiment, the cartridge of the present invention is configured such that the container can be sealed by injecting the internal electrolyte solution 124 for the reference electrode from the metal electrode insertion port 107 of the reference electrode and attaching the metal electrode. It may be.

  In one embodiment, the cartridge of the present invention further includes an element 401 capable of storing and reproducing. The element 401 capable of storage / reproduction can be recorded / reproduced from outside the cartridge via the substrate 402. The substrate 402 can include wiring 403. The element 401 capable of storing and reproducing can be an integrated circuit having a memory chip, a semiconductor memory, a flash memory, a universal serial bus (hereinafter, USB) memory, an SD memory card, or the like. The element 401 capable of storing and reproducing includes information on proteins or pathogens to be measured by one or more working electrodes 101, initial characteristics of the one or more working electrodes 101, and cartridges already used for measurement. Information such as the time used for measurement and the detected numerical value can be stored. The substrate 402 can be a printed circuit board, such as a flexible printed circuit board. The wiring 403 can be appropriately adapted according to the type and standard of the element 401 that can be stored and reproduced. For example, a USB memory can have 4 wires (GND, D +, D-, VBUS).

  In an embodiment, the cartridge of the present invention may include an introduction filter 112 for dust prevention or the like immediately after the outside air introduction port 111. The introduction filter 112 is not particularly limited as long as it can pass virus particles.

  The cartridge of the present invention has a detection unit 300. This is illustrated in FIGS. The detection unit 300 includes a conductive diamond electrode 110 portion of the working electrode 101. The detection unit can include one or a plurality of conductive diamond electrodes 110. For convenience, the surface 301 on which the elements of the working electrode are fixed is the lower side of the detection unit 300. That is, there is a surface 301 on which the working electrode elements are immobilized on the lower side of the detection unit 300, and this is the side in contact with the outside air or electrolyte containing protein or pathogen (FIG. 11A).

  The working electrode 101, the counter electrode 102, and the reference electrode 103 of the cartridge of the present invention are configured so as to be electrically connected to the outside.

Automatic Detection Device of the Present Invention The present invention provides an automatic detection device (hereinafter sometimes simply referred to as the device of the present invention) for detecting proteins or pathogens. The apparatus of the present invention includes one or more cartridges of the present invention. Examples of the cartridge include the cartridge 100 or the cartridge 200. Examples of the automatic detection device of the present invention include an automatic detection device 500 including the cartridge 100 of the present invention, an automatic detection device 600 including the cartridge 100 of the present invention and an outside air tank 610, and a cartridge having a liquid level vertical movement mechanism 201. 200, an outside air tank 610, and an automatic detection device 700 including a pump 701 for a liquid level vertical movement mechanism.

  In the apparatus of the present invention, the working electrode 101, the counter electrode 102, and the reference electrode 103 on the outer surface of the cartridge that are configured to be electrically conductive to the outside are electrically connected to the detection apparatus.

  In the case where the cartridge of the present invention includes the element 401 capable of storing and reproducing, the element 401 capable of storing and reproducing may be electrically connected to the apparatus.

  The apparatus of the present invention includes an electrochemical measurement circuit 501 that can perform cyclic voltammetry measurement and / or electrochemical impedance measurement. The electrochemical measurement circuit 501 may include a potentiostat, an AC transmitter, a lock-in amplifier, and other attached circuits.

  The apparatus of the present invention may include a data display circuit 520 that displays the measurement result or an output terminal that outputs or transmits the measurement result. The data display circuit 520 may also serve as an operation circuit.

  In addition, the device of the present invention includes a power supply circuit 550. The input power to the power supply circuit 550 can be a battery or an AC power supply.

  The apparatus of the present invention includes an outside air inlet 505 for sucking outside air into the detection device, and the outside air inlet 505 is connected to the outside air inlet 111 of the cartridge. Further, the apparatus of the present invention includes an outside air exhaust port 507 for discharging outside air from the apparatus, and the outside air exhaust port 507 is connected to the outside air outlet 155 of the cartridge.

  The apparatus of the present invention further includes an outside air suction pump 506 connected to the outside air inlet 505.

  The apparatus of the present invention includes a control circuit 510. The control circuit 510 can control all operations such as measurement. The control circuit 510 can have a CPU. The control circuit 510 may have a function of recording the measurement result in a recordable / reproducible element 401 in each cartridge and communicating the measurement result to the outside by a wireless or wired communication circuit 530. The control circuit 510 may have a function of issuing an alarm when the number of detected viruses exceeds a certain number. The control circuit 510 can have a function of displaying an error on the display circuit 520 and issuing an alarm to the outside when an abnormality is found in the measurement result. The control circuit 510 stops the measurement when “the set number of measurements has been completed”, “has exceeded the detection capacity of the cartridge”, etc., and the measurement result can be recorded and reproduced in each cartridge. It is possible to have a function of recording in a simple element 401, informing the outside by a wireless or wired communication circuit 530, displaying it on the display circuit 520, and stopping the operation. The apparatus of the present invention may include a wireless or wired communication circuit 530.

  The apparatus of the present invention may include a changeover switch 560 that switches the connection between the plurality of working electrodes 101 and the electrochemical impedance measurement circuit 501. Accordingly, cyclic voltammetry measurement, electrochemical impedance measurement, and the like by the electrochemical impedance measurement circuit 501 can be performed while sequentially switching the plurality of working electrodes 101.

  In the apparatus of the present invention, when the cartridge is provided with a liquid level vertical movement mechanism, for example, in the case of 200, the apparatus of the present invention may include a pump 701 for the liquid level vertical movement mechanism. At this time, the pump 701 may be connected to the control circuit 510 of the detection device. The pump 701 for the liquid level vertical movement mechanism may have an intake port 702 and an exhaust port 703. An example is shown in FIG. The intake port 702 and the exhaust port 703 may be inside the apparatus of the present invention or may be connected to the outside.

  When the apparatus of the present invention includes a plurality of cartridges, the outside air inlet and the outside air outlet of the plurality of cartridges may be connected to the apparatus in series or in parallel. In series, among the n cartridges of the apparatus of the present invention, the outside air outlet of the first cartridge is connected to the outside air inlet of the second cartridge, and the outside air outlet of the second cartridge is the third. Is connected to the outside air inlet of the cartridge No. 1, and similarly connected to the outside air inlet of the nth cartridge. “Parallel” refers to connecting a plurality of cartridges included in the apparatus of the present invention to the outside air inlet and the outside air outlet of the apparatus without connecting the outside air introduction port and the outside air outlet port of each cartridge to each other.

  When the cartridge of the present invention includes the element 401 capable of storing and reproducing, the apparatus of the present invention may include a data recording / reproducing circuit 540 that records and reproduces data with respect to the element. The data recording / reproducing circuit 540 may be connected to the wiring 403 of the cartridge of the present invention.

  An outside air filter 508 may be attached between the outside air inlet 505 of the apparatus and the outside air inlet 111 of the cartridge. The outside air filter 508 is not particularly limited as long as it allows virus particles to pass therethrough, but for example, a filter that can remove cotton dust, sand particles, dust, and the like is preferable.

  In an embodiment, the apparatus of the present invention further includes an outside air tank 610 and a circulation pump 620, and closes an exhaust pipe 509 connected to the outside air outlet 507 between the outside air outlet 507 and the outside air tank 610. An outside air tank exhaust valve 630 that can be used is provided. Such an apparatus is illustrated in FIGS.

[Method for detecting protein or pathogen]
The present invention provides a method for detecting a protein or pathogen using the cartridge of the present invention or the apparatus of the present invention.

In an embodiment, the protein or pathogen detection method of the present invention includes the following steps.
When the cartridge includes the element 401 capable of storing and reproducing, from each element, information on the protein or pathogen to be measured by the one or more working electrodes 101, initial characteristics of the one or more working electrodes 101, and For cartridges that have already been used for measurement, the time used for measurement, the process of reading the detected numerical information,
A step of operating the outside air suction pump 506 to send the sucked outside air into the cartridge;
An adsorption step in which inhaled outside air is blown against the surface 301 on which one or more working electrode elements are immobilized, or in contact with one or more working electrodes as bubbles 121 in the supporting electrolyte solution 104;
An exhaust stroke in which the outside air after the adsorption process passes through the vent 150 on the outside air outlet side of the cartridge and is discharged from the outside air outlet 155 and the outside air outlet 507 of the apparatus;
Electrochemical measurement process for performing cyclic voltammetry measurement or electrochemical impedance measurement while sequentially switching one or more working electrodes of one or more cartridges by stopping the outside air suction pump 506, and the cartridge can be stored and reproduced A step of recording the measurement result in the element 401 of each cartridge when the device 401 is provided, and a step of communicating recording information to the outside when the apparatus is provided with a wireless or wired communication circuit 530;

  In the above method, an adsorption step in which inhaled outside air is blown against the surface 301 on which one or more working electrode elements are immobilized, or is brought into contact with one or more working electrodes as bubbles 121 in the supporting electrolyte solution 104. Can be set to an appropriate time such as 5 minutes, 10 minutes, 15 minutes, and 30 minutes.

In another embodiment, when the apparatus of the present invention includes an outside air tank 610, a circulation pump 620, and an outside air tank exhaust valve 630, the protein or pathogen detection method of the present invention includes the following steps.
When the cartridge includes the element 401 capable of storing and reproducing, from each element 401, information on the protein or pathogen to be measured by the one or more working electrodes 101, initial characteristics of the one or more working electrodes 101, And about the cartridge already used for the measurement, the process of reading the information of the time used for the measurement and the detected numerical value,
Opening the outside air tank exhaust valve 630 and operating the outside air suction pump 506 to introduce unmeasured outside air into the outside air tank 610;
Closing the outside air tank exhaust valve 630 and stopping the outside air suction pump 506;
A step of operating the circulation pump 620 and sending the outside air in the outside air tank into the cartridge;
An adsorption step in which inhaled outside air is blown against the surface 301 on which one or more working electrode elements are immobilized, or in contact with one or more working electrodes 101 as bubbles 121 in the supporting electrolyte solution 104;
A step of allowing the outside air after the adsorption step to pass through the vent 150 on the outside air outlet side of the cartridge and exhausting it from the outside air outlet 155 to the outside air tank 610;
An electrochemical measurement step in which the circulation pump 620 is stopped and cyclic voltammetry measurement or electrochemical impedance measurement is performed while sequentially switching one or more working electrodes of one or more cartridges;
A step of recording a measurement result in each element of each cartridge when the cartridge includes an element 401 capable of storing and reproducing; and
When the apparatus includes a wireless or wired communication circuit 530, communicating recorded information to the outside.

  In the above method, the step of operating the circulation pump 620 and feeding the outside air in the outside air tank into the cartridge can be performed for an appropriate time such as 5 minutes, 10 minutes, 15 minutes, 30 minutes, for example.

  The above is merely an example, and the present invention is not limited to these embodiments. For example, as a variation of the apparatus 700 shown in FIG. 18, an apparatus that does not include the outside air tank 610, the circulation pump 620, and the exhaust valve 630 is also assumed.

  The working electrode of the cartridge of the present invention has a substrate, a diamond layer on the substrate, and a modification layer on the diamond layer. The diamond layer on the substrate is a conductive diamond doped with a small amount of impurities. That is, a conductive diamond electrode is used as the working electrode. This conductive diamond electrode is preferably doped with a small amount of impurities. By doping with impurities, desirable properties as an electrode can be obtained. Examples of impurities include boron (B), sulfur (S), nitrogen (N), oxygen (O), and silicon (Si). For example, diborane, trimethoxyborane and boron oxide are used to obtain boron in a source gas containing a carbon source, sulfur oxide and hydrogen sulfide are obtained to obtain sulfur, oxygen or carbon dioxide is obtained to obtain oxygen, Ammonia or nitrogen can be added to obtain nitrogen, and silane or the like can be added to obtain silicon. In particular, a conductive diamond electrode doped with boron at a high concentration is preferable because it has advantageous properties such as a wide potential window and a small background current compared to other electrode materials. Therefore, the boron-doped diamond electrode will be described below as an example. A conductive diamond electrode doped with other impurities may be used. In this specification, unless otherwise specified, a potential and a voltage are used synonymously and are interchangeable. In this specification, the conductive diamond electrode may be simply referred to as a diamond electrode, and the boron-doped diamond electrode may be referred to as a BDD electrode.

The electrode portion of the working electrode has a diamond layer in which 0.01 to 8% w / w boron raw material mixed diamond is deposited on the substrate surface. The size of the substrate is not particularly limited, but a substrate having an area capable of measuring a sample solution in mL or μL is preferable. For example, the substrate may have a diameter of 1 to 10 cm and a thickness of 0.1 mm to 5 mm. The substrate Si substrate, a glass substrate or a quartz substrate such as SiO _2, ceramic substrate such as Al ₂ O _3, tungsten, may be a metal such as molybdenum. All or part of the surface of the substrate can be a diamond layer.

The size of the electrode part of the conductive diamond electrode can be appropriately designed depending on the measurement target. For example the electrode portions may be, for example, ^{0.1cm 2 ~10cm 2, 0.2cm 2 ~5cm} 2, or surface having an area of 0.5cm ² ~4cm ^2. All or part of the diamond layer can be used for electrochemical measurements. A person skilled in the art can appropriately determine the area and shape of the electrode portion according to the measurement target.

  The electrode portion of the working electrode has a diamond layer in which the surface of the Si substrate is vapor-deposited with high boron raw material mixed (0.01 to 8% w / w boron raw material as raw material preparation) diamond. A preferable boron raw material mixing rate is 0.05 to 5% w / w, particularly preferably about 0.3% w / w. After the diamond layer is formed on the substrate, a modification layer can be provided thereon.

The deposition process of diamond mixed with boron material on the substrate may be performed at 700 to 900 ° C. for 2 to 12 hours. The conductive diamond thin film is produced by a usual microwave plasma chemical vapor deposition (MPCVD) method. That is, a substrate such as a silicon single crystal (100) is set in a film forming apparatus, and a film forming gas using a high purity hydrogen gas as a carrier gas is allowed to flow. The deposition gas contains carbon and boron. When plasma is generated by applying microwaves to a film deposition system that flows high-purity hydrogen gas containing carbon and boron, carbon radicals are generated from the carbon source in the film-forming gas, and are formed on the Si single crystal. While maintaining the sp ³ structure and depositing while mixing boron, a diamond thin film is formed.

  The film thickness of the diamond thin film can be controlled by adjusting the film formation time. The thickness of the diamond thin film can be, for example, 100 nm to 1 mm, 1 μm to 0.1 mm, 1 μm to 100 μm, 2 μm to 20 μm, and the like.

  The conditions for the vapor deposition treatment of boron-doped diamond on the substrate surface may be determined according to the substrate material. As an example, the plasma output can be 500 to 7000 W, for example, 3 kW to 5 kW, preferably 5 kW. When the plasma output is within this range, the synthesis proceeds efficiently, and a high-quality conductive diamond thin film with few by-products is formed.

  The above electrodes are disclosed in Japanese Patent Application Laid-Open No. 2006-98281, Japanese Patent Application Laid-Open No. 2011-152324, Japanese Patent Application Laid-Open No. 2015-172401, and the like, and can be manufactured according to the descriptions in these documents.

  The conductive diamond electrode has high thermal conductivity, high hardness, chemically inert, a wide potential window, low electric capacity, low background current, and excellent electrochemical stability.

  The cartridge of the present invention includes a triode electrode. The resistance on the reference electrode side is set high, and no current flows between the working electrode and the reference electrode. Although a counter electrode is not specifically limited, For example, a silver wire and a platinum wire can be used. The reference electrode is not particularly limited, but a silver-silver chloride electrode (Ag / AgCl) is preferable from the viewpoint of stability and reproducibility. Unless otherwise specified herein, the measured voltage is measured with respect to a silver-silver chloride electrode (+0.199 V vs. standard hydrogen electrode (SHE)).

A silver-silver chloride electrode used as a reference electrode has a structure in which an AgCl-coated silver wire (Ag / AgCl) is immersed in an aqueous solution containing chloride ions (Cl ⁻ ). When an Ag / AgCl reference electrode is used as the reference electrode, the Ag / AgCl reference electrode is not particularly limited as long as it has a larger surface area than the working electrode (conductive diamond electrode). For example, referring to FIG. 6, the reference electrode 103 is immersed in the internal electrolyte solution 124, and a liquid junction portion 123 of the reference electrode is disposed between the internal electrolyte solution 124 and the supporting electrolyte solution 104 in the container 105. Has been. The liquid junction 123 of the reference electrode is electrically connected between the internal electrolyte solution 124 and the supporting electrolyte solution 104 by a porous Vycor glass, ceramic, or salt bridge. The salt bridge may be a glass tube or a filter paper, and may be gelled by agar. The salt bridge is usually filled with an inert electrolyte, such as sodium chloride.

  The working electrode, counter electrode, and reference electrode of the cartridge of the present invention are not particularly limited as long as the working electrode, the counter electrode, and the reference electrode are arranged so that they can be contacted simultaneously with the supporting electrolyte solution during electrochemical measurement.

  In the conductive diamond electrode of the present invention, an element for recognizing a protein or a pathogen is immobilized on the surface thereof. In some embodiments, immobilization means that the element is connected to the surface of the conductive diamond electrode by a covalent bond. The layer on which the element is fixed may be referred to as a modification layer. When the modified layer is brought into contact with the target protein or pathogen, the element recognizes the protein or pathogen. At this time, when a potential is applied, a current flows, and the protein or pathogen in the sample can be detected by measuring the current.

  Any pathogen can be detected by the cartridge or apparatus of the present invention as long as it is recognized by the element on the surface of the conductive diamond electrode and a current flows. Examples of pathogens include viruses and pathogenic bacteria. Pathogenic bacteria include, but are not limited to, Mycoplasma, Clostridium botulinum, Bordetella pertussis, Tetanus, Diphtheria, Vibrio cholerae, Shigella, Bacillus anthracis, pathogenic Escherichia coli, Staphylococcus, Salmonella, Clostridium perfringens, and Bacillus cereus. Absent.

  As the virus detected by the cartridge or apparatus of the present invention, any virus may be used as long as it is recognized by an element on the surface of the conductive diamond electrode and a current flows. Detected viruses include DNA viruses, RNA viruses, double-stranded DNA viruses, single-stranded DNA viruses, double-stranded RNA viruses, single-stranded RNA (+) strand viruses, and single-stranded RNA (-) strand viruses. Single-stranded RNA reverse transcription virus, double-stranded DNA reverse transcription virus, such as influenza virus, norovirus, rotavirus, rubella virus, measles virus, RS virus, herpes virus, hepatitis virus, adenovirus, foot-and-mouth disease virus, rabies virus, And human immunodeficiency virus. Influenza viruses include A, B, C, avian influenza viruses and various subtypes thereof.

  The protein detected by the cartridge or apparatus of the present invention may be any protein as long as it is recognized by an element on the surface of the conductive diamond electrode and a current flows. In certain embodiments, the protein to be detected is one having a tryptophan residue. In certain embodiments, the protein to be detected is a protein derived from the pathogen, pathogenic bacterium, or virus described above. Preferably, the virus-derived protein can be a virus surface protein. For example, if the detection target is an influenza virus, the element may recognize hemagglutinin (HA) or neuraminidase (NA) protein derived from the influenza virus. Examples of the protein to be detected further include M1 protein and M2 protein of influenza virus. When the detection target is Mycoplasma bacterium, the element can be recognized by Mycoplasma bacterium-derived P1 protein, membrane antigen protein, or ribosomal protein L7 / L12. In certain embodiments, the protein to be detected is a toxin protein. Toxin proteins include exotoxin, botulinum toxin, pertussis toxin, tetanus toxin (tetanospasmin), diphtheria toxin, alpha toxin of C. perfringens, cholera toxin, verotoxin, anthrax toxin (PA, EF, or LF), and Enterotoxins derived from Escherichia coli, Staphylococcus, Salmonella, Bacillus cereus, and the like, but are not limited thereto.

  The element for recognizing the protein or pathogen of the present invention may be any element as long as a current flows through the conductive diamond electrode when interacting with the target protein or pathogen.

  A device for recognizing a protein or pathogen used for the working electrode device of the present invention is schematically shown below. The element has one end immobilized on the surface of a conductive diamond electrode and the other end has a molecule that recognizes a protein or pathogen. Further, the element may optionally have a linker molecule.

Electrode- (L) -R
[In the formula, Electrode is a conductive diamond electrode, L is an optional linker, and R is a molecule that recognizes a protein or pathogen. ]

  In certain embodiments, the element that recognizes a protein or pathogen has a peptide that specifically interacts with the protein or pathogen of interest. Explaining in the above formula, the molecule R that recognizes a protein or pathogen may have a peptide. For example, a peptide having a length of 4 amino acids or more, 5 amino acids or more, 6 amino acids or more, 7 amino acids or more, 8 amino acids or more, 9 amino acids or more, 10 amino acids or more, or 15 amino acids or more can be used. For example, the peptide having a length of 100 amino acids or less or 50 amino acids or less can be used. The device can have one or more of the peptides. For example, the peptide s2 (1-5) that binds to the HA protein of influenza virus greatly increases HA binding by dendrimerization, and the 4-branched type is about 750 times that of s2 (1-5) alone. HA binding activity is obtained. Such a dendrimerized protein-binding peptide can be used in the device of the present invention.

  In one embodiment, the element that recognizes a protein or pathogen has a peptide that recognizes a protein derived from the above-mentioned pathogen, pathogenic bacterium, or virus. The amino acid sequence of such a peptide can be obtained from publicly known literatures or publicly known databases such as GenBank. The peptide may be optionally modified and may be dendrimerized as described above.

  In some embodiments, the element that recognizes a protein or pathogen has a peptide that recognizes an influenza virus. As the peptide for recognizing influenza virus, any known peptide can be used. For example, International Publication No. 2007/105565 pamphlet, International Publication No. 2010/024108 pamphlet, Japanese Patent Application Laid-Open No. 2010-209052 (these are referred to by reference). The contents of which are incorporated into the present specification), but are not limited thereto. Examples of peptides that recognize influenza virus include the following.

Peptide s2 (1-5) (ARLPR) (SEQ ID NO: 1)
Peptide s2 (ARLPRTMVHPKPAQP) (SEQ ID NO: 2)
GLAMAPSVGHVRQHG (SEQ ID NO: 3)
GLAMAPSVGHVRQHG (wherein the serine residue is modified with N-acetylgalactosamine through an O-glycoside bond) (SEQ ID NO: 4)
Peptides having these in part are also envisaged. The peptide may be appropriately modified. These are merely examples, and other peptides that recognize viruses, pathogens, and pathogens can also be used in the present invention.

  A peptide that recognizes a protein or pathogen is immobilized on the conductive diamond electrode of the present invention by an appropriate chemical method. In certain embodiments, the peptide may be immobilized on the diamond electrode via a linker molecule. In addition to known protein recognition peptides, equivalents having equivalent functions are envisaged and can be used in the present invention. The peptide can be chemically synthesized.

  The element that recognizes a protein or pathogen may be immobilized on the surface of the conductive diamond electrode using any appropriate method. In some embodiments, the molecule of interest can be linked to the electrode surface by electrolytic grafting. In some embodiments, a photoinduced radical reaction may be used for immobilization.

  If desired, a linker molecule can be used for immobilizing the device. The following are examples of molecules for forming the linker moiety.

A-L ₁ -B (-P)
[Wherein A is a functional group capable of reacting with a conductive diamond electrode, L ₁ is a linker moiety, B is a functional group capable of reacting with a molecule that recognizes a protein or pathogen, and P is optionally present. Represents a good protecting group. ]

  In this case, the molecule is first linked to the electrode surface by an appropriate reaction such as electrolytic grafting. That is, the functional group A is reacted to connect the electrode and L1. When the functional group B is protected by the protecting group P, the functional group B can be presented by performing a deprotection reaction thereafter. The molecule R that recognizes the protein or pathogen can then be linked to the linker moiety using any suitable ligation reaction.

  Examples of molecules R that recognize proteins or pathogens are given below.

C-R ¹
[Wherein R ¹ represents a moiety that recognizes a protein or a pathogen, and C represents a functional group capable of reacting with the functional group B. ]

  The linking reaction between the linker moiety and the molecule R that recognizes the protein or pathogen (linking reaction between the functional group B and the functional group C) is the Husgen cycloaddition reaction, the Gracer reaction, its coupling, Suzuki / Miyaura coupling reaction Etc., and those that form a covalent bond are preferred, but not limited thereto.

The functional group A that can react with the conductive diamond electrode can be a diazo group, an amino group, a carboxy group, a carbonyl group, an aldehyde group, a hydroxyl group, a nitro group, and the like. The linker portion L ₁ may have an aromatic ring, a carbocycle, a heterocyclic ring, such as a phenyl group, a naphthyl group, a polyether group, a polyethylene glycol group, or a hydrocarbon group. These may optionally be further substituted with an appropriate substituent such as an alkyl group, an aryl group, a halogen group, or a hydroxyl group. Further, the functional group B and the functional group C may be an alkynyl group, a boranyl group, a boryl group, a halogenated aryl group, an azide group, or the like. Further, when the Husgen cycloaddition reaction is used for the ligation reaction, the functional group B may have an alkynyl group and the functional group C may have an azide group. The functional group B may optionally be protected by a protecting group P. Protecting groups include trisubstituted silyl groups such as triisopropylsilyl group, trimethylsilyl group, butyldiphenylsilyl group, dimethylcumylsilyl group, benzyl group, lower alkoxycarbonyl group, halogeno lower alkoxycarbonyl group, benzyloxycarbonyl group, acetyl group Groups, acyl groups such as benzoyl groups, triphenylmethyl groups, tetrahydropyranyl groups, and the like, but are not limited thereto.

  In some embodiments, A can be a diazo group, L 1 can be a phenylene group, B can be an alkynyl group, and P can be a triisopropylsilyl group. The alkynyl group B can be presented by reacting A, immobilizing it on the electrode surface and deprotecting it. In one embodiment, the molecule R that recognizes a protein or pathogen may have a peptide group as R 1 and an azide group as functional group C. A molecule R that recognizes a protein or pathogen when azide group (functional group C) of R is reacted with an alkynyl group (functional group B) presented on the electrode surface to form a 1,2,3-triazole ring. Is immobilized on the electrode surface.

Electrochemical measurement method The target protein or pathogen can be detected using the cartridge or apparatus of the present invention. When the working electrode of the cartridge of the present invention is brought into contact with the sample air (outside air) or an electrolyte solution, the element on the surface of the working electrode recognizes a protein or pathogen. At this time, when a potential is applied to the working electrode, current can be observed. This can be measured by cyclic voltammetry or electrochemical impedance measurement.

Cyclic voltammetry Cyclic voltammetry is performed using a technique that varies (sweeps) the potential. Specifically, the current obtained when the electrode potential is swept from the initial potential (E _i ) to the reversal potential (E _λ ) at the sweep speed (v) and then reversed and then returned to Ei is observed. A current-potential graph (cyclic voltammogram) can be obtained by setting the initial potential E _i to a potential at which no electrode reaction occurs and the inversion potential E _λ to a potential at which the electrode reaction is diffusion-controlled. The initial potential, sweep speed, inversion potential, and the like can be set as appropriate.

  In this case, for a protein or pathogen sample with a known concentration or amount, a peak current value is determined, a calibration curve in which the relationship between the concentration or amount and the peak current density is plotted is prepared, From the peak current value, the concentration or amount of the protein or pathogen contained in the sample can be calculated.

Electrochemical impedance measurement For electrochemical impedance measurement, a potentiostat with an AC transmitter connected is used. A constant DC potential is applied to the electrode using a potentiostat, and an AC potential of ± 5 to 10 mV is superimposed and applied using an AC transmitter. Also, an AC wave having the same phase as the AC input from the transmitter to the potentiostat is input to the lock-in amplifier. As a result, the flowing current is a combination of the direct current and the alternating current, and the lock-in amplifier compares the alternating current component of the current with the alternating current from the transmitter, and outputs the impedance and the phase difference between the two. The AC frequency from the transmitter is changed little by little, and complex plane plots are performed based on the impedance and phase difference obtained at each frequency.

  In this case, for a protein or pathogen sample at a known concentration or amount, the electrode impedance (charge transfer resistance Rct) is determined by a Nyquist plot, and a calibration curve in which the relationship between the concentration or amount and Rct is plotted is prepared. From the Rct value of the measurement sample, the concentration or amount of the protein or pathogen contained in the sample can be calculated.

Chronoamperometry The electrochemical measurement using the apparatus of the present invention can also be performed by chronoamperometry. In chronoamperometry, the potential of the working electrode is stepped, and the change in current over time is measured. Chronoamperometry measurement can be performed by applying a constant step potential such as 0.1 to 3.0 V, 0.5 to 2.5 V, or the like.

  In this case, chronoamperometry measurements are performed at a constant applied voltage on a protein or pathogen sample of known concentration or amount. A calibration curve is created by recording the current value at a fixed time after voltage application and plotting the relationship between the concentration or amount of the protein or pathogen sample and the current value. Next, the current value at the predetermined time after the voltage application is measured for the measurement sample, and this is compared with the calibration curve, thereby calculating the concentration or amount of the protein or pathogen sample in the test sample solution.

  The test sample to be measured by the method of the present invention is not particularly limited, and examples thereof include air that may contain viruses or pathogens. For example, the atmosphere of a clinical facility such as a hospital where an influenza virus may exist, a school, a station, an airport, or any public facility may be mentioned. The outside air may be compressed or used for measurement as it is.

  In the present invention, an aqueous solvent is mainly used as a solvent for measurement. The solution to be measured usually contains a supporting electrolyte. The supporting electrolyte is an ionic substance and is not particularly limited, and examples thereof include phosphate buffered saline (PBS), potassium nitrate, and sodium sulfate. Preferably the supporting electrolyte is PBS.

  In the present specification, “detection” of a protein means that the target protein can be specifically measured in a sample containing a protein other than the target protein or a virus or bacteria. Further, “detection” of a pathogen means that the target pathogen can be specifically measured with respect to a sample containing a protein other than the target pathogen or a virus or bacteria.

  In the present specification, the viral load is expressed by the number of plaque forming units (pfu). In an embodiment, the pfu when describing the amount of influenza virus is that when using the influenza virus A / PR / 8/34 (H1N1) strain. The pfu measurement method can be estimated by measuring plaques formed when influenza virus is added to canine kidney-derived epithelial cells MDCK cells cultured in a monolayer. This is an index for convenience, and those skilled in the art can appropriately determine the corresponding pfu when using different influenza virus strains. Further, the pfu unit may be appropriately converted to the ng unit. In an embodiment, the device of the present invention can detect influenza virus with high sensitivity, for example, 20 pfu or more, or 40 pfu or more and 1000 pfu or less influenza virus.

  In one embodiment, the working electrode of the cartridge of the present invention comprises a conductive diamond electrode having an element that recognizes a protein or pathogen immobilized on the surface.

  In certain embodiments, the element that recognizes the protein or pathogen is an element that recognizes a virus or pathogen. The types of recognized viruses and pathogenic bacteria are as described above, and particularly include influenza viruses.

  In certain embodiments, the protein or pathogen recognizing element comprises a peptide that recognizes the pathogen protein. Recognized pathogen proteins are as described above, and particularly include HA proteins of influenza viruses. Examples of peptides that recognize pathogen proteins are also as described above.

  In one embodiment, the protein or pathogen recognizing element has a protein or pathogen recognizing molecule and a linker moiety, and the protein or pathogen recognizing molecule is immobilized on the surface of the diamond electrode via the linker moiety. Has been. Molecules that recognize proteins or pathogens, linker moieties, and immobilization methods are as described above.

Production Examples and Use Examples of Conductive Diamond Electrodes of the Present Invention The following procedures are intended to be examples only and are not intended to limit the technical scope of the present invention. Unless otherwise specified, the reagents are commercially available, or are obtained or prepared according to methods commonly used in the art and known literature procedures.

Abbreviations In this specification, the following abbreviations may be used.
IFV: Influenza virus
HA: Hemagglutinin
EIS: Electrochemical impedance spectroscopy
CV: Cyclic voltammetry
DMF: N, N-dimethylformamide
DCM: Dichloromethane
PyBOP: Benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate
DIEA: Diisopropylethylamine
PIP: Piperidine
TFA: trifluoroacetic acid
TIS: Triilopropylsilane
TIPS-Eth-Ar-N ₂ ⁺ BF ₄ ⁻ : 4-((Triisopropylsilyl) ethynyl) benzenediazonium tetrafluoroborate α-CHCA: α-cyano-4-hydroxycinnamic acid
THF: tetrahydrofuran
TBAF: Tetra-n-butylammonium fluoride
Fmoc: 9-fluorenylmethyloxycarbonyl group
AFM: Atomic force microscope

Materials and compounds Unless otherwise specified, commercially available compounds and chemicals were used without further purification. Those skilled in the art can modify the described procedures without departing from the spirit of the invention.

1. Synthesis of Peptide and Linker Two-branched peptide dendrimer (ARLPR) 2-K-KN3 into which azidolysine Lys (N ₃ ) was introduced at the end was synthesized using the Fmoc solid phase method. Peptide ARLPR is a hemagglutinin-binding peptide (Japanese Patent Laid-Open No. 2006-68020). In peptide synthesis by the solid phase method, a deprotection reagent is added to the peptide resin (solid phase support on which an amino acid is immobilized), and the N-α protecting group is removed. The activated amino acid is reacted therewith, and the chain length of the peptide is successively increased. The carboxy terminus of the peptide was amidated to eliminate unwanted charges. In addition, a branched structure was created by introducing Lys into the C-terminal side of the peptide. FIG. 19 shows a scheme for solid-phase synthesis of an azide group-introduced s2 (1-5) peptide dendrimer.

(A) Introduction of Lys (N ₃ ) into resin by manual synthesis Fmoc-NH-SAL resin (Watanabe Chemical, 0.59 mmol / g) 169 mg (0.1 mmol) was charged into a reaction column (PD-10, Amersham). To this was added 2 mL of DMF, and after gently shaking, DMF was removed from the bottom of the column with an aspirator. This operation was repeated three times to swell the resin. 2 mL of 20% (v / v) PIP / DMF was added to the reaction column, and after 1 minute, the solvent was removed with an aspirator. Similarly, 2 mL of 20% PIP / DMF was poured and shaken for 15 minutes to remove the solvent. The operation of pouring 2 mL of DMF and removing with an aspirator was repeated 4 times to wash the resin. Thereby, deprotection was performed.

Fmoc-Lys (N ₃ ) -OH (EUROGENTEC GROUP ANA SPEC, 53100-F025) 117 mg (0.3 mmol), PyBOP 156 mg (0.3 mmol), DMF 2 mL, DIEA 0.11 mL (0.6 mmol) were added to the reaction column for 1 hour. Shake. Thereby, a coupling reaction was performed.

  After 1 hour, the reaction solution was removed with an aspirator, and 2 mL of DMF was poured onto the reaction column, shaken lightly, and then removed with the aspirator four times (washing). Furthermore, the operation of adding 2 mL of DCM and removing it with an aspirator was repeated 4 times. The reaction column was placed in a desiccator and dried with a vacuum pump for 1 hour. When the sample was sufficiently dry it was stored at 4 ° C.

(B) Quantification of amino acid introduction rate
20 mg of Fmoc-Lys (N ₃ ) -NH-SAL resin introduced in (A) was accurately weighed into a sample tube, 2 mL of 20% (v / v) PIP / DMF was added, and the mixture was reacted at room temperature for 20 minutes. The supernatant was diluted 50 times with DMF, and the absorbance at 301 nm was measured. DMF was used for the measurement of the blank, and the amino acid introduction rate was calculated from the following formula from the molar extinction coefficient ε = 7800 of the Fmoc group at 301 nm.

Amino acid introduction rate (mol / g)
= (A ₃₀₁ , 1/50-A ₃₀₁ , DMF) x 50/7800 x (2 x 10 ^-3 ) x (1000/20)
= (0.644-0) × 0.641 × 10 ^-3
= 0.413 mmol / g

As a result of quantifying the amino acid introduction rate of the Fmoc-Lys (N ₃ ) -NH-SAL resin prepared in (A) above, the introduction rate was 0.413 mmol / g.

(C) Peptide elongation
PD-10 empty columns (17-0435-01, Amersham Biosciences) was charged with 242 mg (0.05 mmol) of Fmoc-Lys (N ₃ ) -NH-SAL resin, and the following (C-1) to (C-4) The elongation reaction of the peptide was carried out by the above procedure (the amount of resin used to make the peptide 0.1 mmol was used).

(C-1) Deprotection of Fmoc Group The resin with the first amino acid introduced was weighed onto a 121 mg (0.05 mmol) reaction column. After adding 2 mL of DMF to the reaction column and shaking lightly, DMF was removed from the lower part of the column with an aspirator. The operation of pouring 2 mL of DMF into the reaction column and removing it with an aspirator was repeated 4 times. 2 mL of 20% (v / v) PIP / DMF was poured into the reaction column, and after 1 minute, it was removed with an aspirator. Next, 2 mL of 20% (v / v) PIP / DMF was poured, shaken for 15 minutes, and removed. Thereafter, the operation of pouring 2 mL of DMF and removing it with an aspirator was repeated 5 times to wash the resin.

(C-2) Coupling Fmoc-AA-OH (each 0.3 mmol), PyBOP 157 mg (0.3 mmol), DMF 2 mL, DIEA 0.11 mL (0.6 mmol) were added to the reaction column, and shaken for 40 minutes. Amino acids were introduced in the order of lysine (Lys) → arginine (Arg) → proline (Pro) → leucine (Leu) → arginine (Arg) → alanine (Ala) from the C-terminal side. After 40 minutes, the reaction solution was removed with an aspirator, and 2 mL of DMF was poured into the reaction column and shaken lightly, and then removed with the aspirator four times. The amino acids used are shown in Table 1.

(C-3) Kaiser Test A reagent for the Kaiser test (code number 2590077, domestic chemistry) was used to determine whether the coupling reaction had been completed. Take about 1 mg of resin with a spatula, put in a microtube, add 10 μL each of reagents (1) ninhydrin / ethanol, (2) phenol / ethanol, and (3) KCN / pyridine, cover, and heat for about 1 minute with a dryer. did. Since the unreacted amino group remains when the color of the resin or solution becomes blue, the coupling reaction was performed once again with the same amino acid. When it became colorless or yellow, it was judged that the reaction was completed, and the above operations (C-1) to (C-3) were repeated until all amino acids were coupled.

(C-4) Deprotection After all extension reactions, pour 2 mL of 20% PIP / DMF onto the PD-10 column, stir gently with a spatula, remove 20% PIP / DMF with an aspirator, and 2 mL of 20% PIP / DMF. The Fmoc group was deprotected by removing it after pouring and shaking for 15 minutes. After adding 2 mL of DMF and removing with an aspirator 5 times, and adding t-butyl methyl ether and removing with an aspirator 2 times, cover the PD-10 column with aluminum foil and parafilm, and vacuum dry for 3 hours. It was.

(D) Cutting out (Cleavage and deprotection from resin)
In order to cut out the peptide from the synthesized resin, a cocktail solution was prepared with the composition shown in Table 2 and cut out. Since the azide group is easily decomposed by the reducing action of thiol, a cocktail solution having a thiol-free composition was used (see PE Schneggenburger et al., J. Pept. Sci., 16, 10-14 (2010)).

1 mL of the above cocktail solution was placed in a reaction vessel containing the peptide and left on ice for 2 hours under light-shielded conditions to prevent decomposition of the azide group by heat and light (usually left at room temperature for 8 hours without light) . After 2 hours, the reaction container was uncovered, pressurized from the upper mouth of the container, the contents of the reaction container were dropped into a 15 mL centrifuge tube, and washed twice with 200 μL of TFA. 2 mL of cold diethyl ether (peroxide free) was added, and after confirming that precipitation was possible, the volume was increased to 10 mL, and the mixture was vortexed. Subsequently, after centrifugation at 3500 rpm for 1 minute, the supernatant was removed, and the volume was again increased to 10 mL with cold diethyl ether. This operation was repeated a total of 5 times. By blowing N ₂ gas remaining peptide pellets centrifuge tube was volatilized cold diethyl ether (crude peptide).

(E) High performance liquid chromatography (HPLC)
To the crude peptide powder remaining in the 15 mL centrifuge tube, 300 μL of AN (acetonitrile) and 100 μL of MilliQ were added and completely dissolved (400 μL of 75% acetonitrile). A syringe and a 0.45 μm filter (Millex-LH, 4 mm, code SLLH H04 NL, Millipore) were connected to remove insoluble matters, and collected in a 1.5 mL tube. Again, 400 μL of 75% acetonitrile was added to the centrifuge tube and washed again, and collected in another 1.5 mL tube.

  An ODS-3 column ((i) φ4.6 × 250 mm, (ii) φ20 × 250 mm, GL Science) was used for HPLC, and the flow rate was 1 mL / min for (i) and 10 mL / min for (ii). 20 μL of peptide solution (co-wash solution) was injected, and elution conditions determined using the column (i) are shown below. At this time, fractions were collected every 30 seconds and analyzed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Solvent: A… 0.1% TFA / H ₂ OB… 0.1% TFA / AN
Gradient: B conc. 20% (10 minutes) + B conc. 20 → 60% (20 minutes) + B conc. 100% (10 minutes)
Wavelength: 220 nm

  Under the determined elution conditions, the peptide solution was subjected to HPLC using the column (ii), and the fraction containing the peak was collected every 15 seconds, and the fraction containing the target peptide was collected using MALDI-TOF MS, The product was isolated by lyophilization.

(F) MALDI-TOF MS
MALDI-TOF MS was measured using Ultraflex ™ (Bruker Daltonics). An N ₂ laser (337 nm) was used as the laser light source. As a matrix, α-cyano-4-hydroxycinnamic acid (α-CHCA, Sigma) was used. α-CHCA was suspended in 0.1% TFA / AN (3: 2, v / v) at a rate of 10 mg / mL, irradiated with ultrasonic waves, centrifuged, and this supernatant was used. For the calibration, a peptide calibration standard (code number 206195, Bruker) diluted according to the protocol was used.

  4 μL of α-CHCA solution and 2 μL of each fraction solution after HPLC were put into a 2 mL tube and pipetted, and 2 μL on a MALDI plate and air-dried. Calibration samples were also performed in the same manner. For Ultraflex ™, measurements were made in positive ion mode using reflectron mode.

The synthesized bifurcated peptide dendrimer (Lot. 140607) was confirmed by HPLC and MALDI-TOF MS. As a result of HPLC analysis, a sharp single peak was detected in 20 to 25 minutes, and it was confirmed that the target product had a high purity of 98% or more. In addition, the analysis results with MALDI-TOF MS showed an error of 0.1% or less, confirming that it was the target product (Exact Mass: 1486.94, calculation [M + H] ⁺ 1487.95, measurement [M + H] ⁺ 1487.95; calculation [M + Na] ⁺ 1509.84, measurement [M + Na] ⁺ 1509.98). The yield of this peptide was 7.7 mg, the yield was 10.8%, and the purity was> 98%. From the above results, it was judged that the target azide group-introduced peptide dendrimer was obtained.

Linker molecules _{TIPS-Eth-Ar-N 2} + BF ₄ ^- synthetic diamond linker molecule _{TIPS-Eth-Ar-N 2} + BF used in the presentation of the alkynyl group to the electrode ₄ of the ^- (4 - ((triisopropylsilyl) ethynyl ) Benzenediazonium tetrafluoroboric acid). First, the terminal alkyne and aryl halide are cross-coupled by the coupling, thereby obtaining an alkynylated aryl (aromatic acetylene) (see S. Anderson, Chem. Eur. J. 7, 4706-4714 (2001)). ). Palladium, copper and base were used as the catalyst. Furthermore, the amino group was diazoniumized for electrolytic grafting.

Experimental method (A) Synthesis of TIPS-Eth-Ar-NH ₂ by its coupling

The water in the three-necked flask was evaporated with a heat gun, vacuumed and then filled with argon (Ar). 4-Iodoaniline 1.0 g (4.57 mmol) and triethylamine 10 mL (71.7 mmol, d = 0.726 g / cm ³ ) were added. After evacuation and Ar filling were repeated 3 times each, degassing was performed, and while circulating Ar, copper iodide 8.9 mg (0.05 mmol), palladium acetate 20.3 mg (0.09 mmol), triphenylphosphine 50.6 mg (0.19 mmol) ) In order and added to a three-necked flask. Further, 1.2 mL (5.35 mmol, d = 0.813 g / cm ³ ) of triisopropylsilylacetylene was added, charged with Ar, and reacted at room temperature with stirring overnight. The reagents used are shown in Table 3.

Celite filtration A filter paper was placed on the Kiriyama funnel, and Celite was spread on the funnel. The reaction solution in the flask was filtered and washed with hexane. The reaction solid that could not be removed from the wall surface was subjected to ultrasonic treatment, dissolved or suspended in hexane, and filtered. The eggplant flask containing the filtrate was put on an evaporator, and the filtrate was concentrated (about 2 mL).

Silica gel column chromatography As a developing solvent, hexane: ethyl acetate = 10: 1 was used. Silica (Silica gel 60, Merck) 75 cc was dispersed in a developing solvent and packed in a column. The concentrated filtrate was gently and uniformly added on the sea sand, and the cock was opened and collected in a test tube.

Thin layer chromatography (TLC)
TLC of the fraction collected in the test tube was performed. Dichloromethane: hexane = 1: 1 was used as a developing solvent. TLC plate (TLC silica gel 60 F ₂₅₄ , (105714, Merck Millipore)) was spotted with a solution of iodoaniline (R _f = 0.29) as a raw material in dichloromethane and a solution before silica gel column chromatography. Confirmed by post-UV irradiation. Next, the solution in the test tube was spotted on the TLC plate and developed in order, and the test tube sample in a range where the reaction product (R _f = 0.39) could be confirmed was collected in an eggplant flask and concentrated by an evaporator (about 2 mL). The eggplant flask containing the concentrated reaction solution (TIPS-Eth-Ar-NH ₂ ) was depressurized with a pump and vacuum dried for 1 hour. After measuring the yield, 1H-NMR measurement was performed.

(B) TIPS-Eth-Ar -N 2 + BF 4 by diazotization ^- Synthesis of

TIPS-Eth-Ar-NH by diazotization of _{2 TIPS-Eth-Ar-NH} 2 + BF 4 - Scheme of Synthesis advance NaNO ₂ 0.4 g of (6.0 mmol) was dissolved in H ₂ O in 2 mL, refrigerated at 4 ° C. I left it. The eggplant flask containing TIPS-Eth-Ar-NH ₂ (1.1 g, 4.0 mmol) obtained in (A) was placed in a bath containing methanol (MeOH) and liquid nitrogen, and the temperature in the flask was −5 ° C. It cooled until it fell down. Add 4 mL of H ₂ O here and mix well with a stirrer until dispersed, then 3.36 mL of HBF 4 (16 mmol, d = 1.4 g / cm ³ )
Was added. ₂ mL of NaNO ₂ aqueous solution that has been cooled in advance is set to −5 to −10.
Several drops were added while maintaining the temperature. After stirring at −5 ° C. for 20 minutes, the mixture was returned to room temperature and stirred for 20 minutes.
The reagents used are shown in Table 4.

Suction filtration
NaBF ₄ 4 mg (36.4 nmol) was dissolved in 80 mL of H ₂ O to prepare a 5% NaBF ₄ aqueous solution. The 5% NaBF ₄ aqueous solution, MeOH, diethyl ether, and H ₂ O used as the washing solution were cooled in advance at 4 ° C. The filter paper was placed on the Kiriyama funnel, and the filter paper was applied with H ₂ O while sucking. All the reaction liquid was filtered and washed with H ₂ O. A 5% NaBF ₄ solution was washed in this order, followed by MeOH and diethyl ether, and the remaining reaction product was recovered by sonication. After the powder on the filter paper was dried in a desiccator under reduced pressure for 1 hour, the yield was measured.

The yield and yield of the product, and the results of analysis by ¹ H-NMR are shown below.
(A) TIPS-Eth-Ar-NH ₂
Yield: 1.01 g (4.0 mmol, Lot. 140801)
Yield: 87%
¹ H-NMR (400 MHz, CDCl ₃ , TMS): δ (ppm) = 7.28 (2H, d), 6.58 (2H, d), 3.78 (2H, s), 1.11 (2H, s)

(B) TIPS-Eth-Ar-N ₂ ⁺ BF ₄ ⁻
Yield: 0.11 g (0.32 mmol, Lot. 140826)
Yield: 8%
¹ H-NMR (400 MHz, CDCl ₃ , TMS): δ (ppm) = 8.58 (2H, d), 7.79 (2H, d), 1.11 (2H, s)

From the above ¹ H-NMR analysis, regarding TIPS-Eth-Ar—NH ₂ , the proton ratios of the respective peaks matched, and thus the target compound could be synthesized. The _{TIPS-Eth-Ar-N 2} + BF 4 - relates, synthetic and peaks of 3.77ppm derived amino group disappears, since the proton ratio of each of the other peaks were consistent likewise desired compound Was confirmed.

2. Preparation of boron-doped diamond (BDD) electrode Briefly, a diamond film was synthesized on a Si substrate by chemical vapor deposition using microwave plasma. Methane was used as the carbon source and trimethylborane was used as the boron source. The concentration of trimethylborane to be doped in the raw material was 0.3% w / w. The surface morphology was characterized using a scanning electron microscope. The quality of the thin film was confirmed by Raman spectroscopy. The BDD electrode produced in this way was used. This will be specifically described below.

Preparation of conductive diamond thin film by vapor phase synthesis method (A) Pretreatment of Si substrate Si substrate (diameter 5cm, thickness 1mm) is placed in a petri dish containing diamond powder so that the mirror surface is facing down, and Si substrate is used for 20 minutes. The substrate surface was scratched by rotating it manually. Thereafter, the Si substrate was dipped in a beaker containing 2-propanol and cleaned by ultrasonic irradiation for 20 minutes. Finally, the solvent was volatilized with N ₂ gas and dried.

(B) Synthesis of diamond film on Si substrate Diamond film synthesis on Si substrate by chemical vapor deposition (CVD method, chemical vapor deposition method) using microwave plasma is performed by Plasma Deposition System (AX6500, Sekitechno). TRON Co., Ltd.). Four types of gases were used: methane, trimethylborane, hydrogen, and oxygen. The boron concentration was set to 0.3% w / w and reacted for 5 hours. Table 7 shows the synthesis conditions.

Raman spectroscopy
Using a confocal Raman optical microscope for 532 nm (ST-BX51, Seki Technotron Co., Ltd.), the chemical bonding state of the substrate surface after film formation was analyzed. Naphthalene was used for calibration, and laser light was irradiated 5 times for 5 seconds. In order to confirm that the film was formed uniformly, analysis was performed at any nine locations on the diamond electrode.

As a result, a boron-boron bond peak was observed in the vicinity of 520 cm ⁻¹ . In the vicinity of 1333 cm- ¹ , a Raman peak of carbon with sp ³ structure was observed. On the other hand, no Raman peak of carbon with sp ² structure was observed in the vicinity of 1560 cm ⁻¹ . From this, it was judged that a high-purity diamond film was uniformly synthesized.

SEM Observation The surface and cross-sectional shape of the diamond film were measured using a scanning electron microscope FE-SEM (JSM-7600F, JEOL Ltd.). A diamond electrode (Lot. 140530) ultrasonically cleaned with ultrapure water, ethanol, and acetone for 5 minutes each was cut into 0.5 cm squares and fixed to a sample stage with silicon grease (Shin-Etsu Chemical Co., Ltd.). The acceleration voltage was set to 5.0 kV for surface observation and 2.0 kV for cross-sectional observation. SEM observation confirmed the synthesis of polycrystalline BDD.

3. Modification of boron-doped diamond (BDD) A scheme for presenting alkynyl groups by electrolytic grafting is shown below.

(A) Preparation of electrode and solution The cell, O-ring, Pt wire, and diamond electrode were sonicated in the order of H ₂ O, then ethanol (EtOH), and then acetone for 5 minutes each. First, 3.87 g (0.01 mol) of TBA · PF ₆ was weighed into a volumetric flask, and acetonitrile (AN) was added to prepare a 100 mM tetrabutylammonium hexafluorophosphate (TBAPF ₆ ) solution. First, HA-peptide was allowed to interact, and then CV measurement was performed. The three-electrode method (working electrode: diamond electrode, counter electrode: Pt, reference electrode: Ag / AgCl) was used for CV measurement.

(B) Reaction between linker molecule and diamond electrode (electrolytic grafting)
Volumetric flask _{TIPS-Eth-Ar-N 2} + BF 4 - to 18.6 mg (0.05 mol) weighed and, 10mM TIPS-Eth-Ar- N 2 + BF 4 dissolved in an electrolytic solution TBAPF ₆ / AN 5mL (100mM) ^- solution was prepared. This whole quantity was put into the cell. The control PC was set as shown in Table 8, and cyclic voltammetry (electrolytic grafting) was performed 5 times at -0.7 to + 0.6V.

(C) Deprotection of triisopropylsilyl (TIPS) group After completion of measurement, first put 9.5 mL of THF in the cell, and then add 0.5 mL of THF solution of TBAF (1 mol / L), and let stand for 20 minutes. Was deprotected (see YR Leroux et al., J. Am. Chem. Soc., 132, 14039-14041 (2010)).

Immobilization of azide group-introduced peptide A reaction solution having a peptide concentration of 0.1 nM was prepared. The alkynyl group-presenting diamond electrode was sonicated with MilliQ, EtOH, and acetone for 5 minutes each. A reaction solution was prepared so that the peptide concentration was 0.1 mM with the composition shown in Table 9 (peptide charge amount 100 times), and a reaction solution was prepared by diluting it to 0.1 nM with MeOH: H ₂ O = 1: 1. (Peptide charge 0.01 times).

A diamond electrode was immersed in each reaction solution and allowed to react while shaking at room temperature for 24 hours. After 24 hours, the reaction solution was removed and sonicated with MilliQ, EtOH, and acetone for several seconds each. The electrode was dried by blowing N ₂ gas, and stored in a sealed container containing silica gel at 4 ° C.

As the electrode after the reaction, a 1 mM K ₃ [Fe (CN) ₆ ] / Na ₂ SO ₄ aqueous solution was prepared, and cyclic voltammetry (CV) measurement was performed on each electrode for 3 cycles to confirm the surface state. In addition, CV measurement was performed for 3 cycles of PBS, which is a solvent for HA and IFV, and changes in the background were confirmed. In addition, the contact angle was observed with these electrodes, and the wetting characteristics of the surface were examined.

4). Detection of hemagglutinin protein (HA) and influenza virus (IFV) using boron-doped diamond (BDD) electrode Cyclic voltammetry (CV) measurement method Measurement of HA protein using unmodified and peptide-modified BDD electrodes First, the HA-peptide The interaction was followed by CV measurements. A three-electrode method (working electrode: diamond electrode, counter electrode: Pt, reference electrode: Ag / AgCl) was used. The interaction with H1HA was performed for 15 minutes, and the measurement was performed in a PBS solution.

  First, a cell was assembled with a peptide-immobilized electrode (peptide charge × 0.01), and the background was measured with PBS for 3 cycles. Thereafter, about 60 μL of 500 nM HA / PBS was added to the cell so that the electrode portion was immersed, and the cells were allowed to interact for 30 minutes. After 30 minutes, the HA solution was removed and washed with PBS three times. The cells were filled with PBS, and cyclic voltammetry measurement was performed for 3 cycles. These operations were performed again at different locations on the electrode, and the interaction was observed at a total of two locations. The measurement conditions shown in the table below were used.

  In the same manner as described above, 50 to 500 nM HA solution diluted with PBS was allowed to interact for 30 minutes each. CV measurement was performed in 3 cycles each while washing the electrode with PBS before changing the concentration. Measurement conditions were as shown in Table 10.

  The results are shown in FIG. The left side of FIG. 20 shows the measurement of HA in the solution when using a diamond electrode without peptide modification. An increase in current density was observed in the 500 nM HA solution. The right side of FIG. 20 shows a case where a peptide-modified diamond electrode is used. In the first cycle, a significant increase in current density was observed compared to the unmodified diamond electrode, and HA could be detected. From the second cycle onward, it can be seen that the current density is not much different from that of PBS alone, and most of the HA protein can be detected in the first cycle.

Measurement of IFV using unmodified and peptide-modified BDD electrodes Cells were assembled with peptide-immobilized electrodes (peptide charge x 0.01), and the background with PBS was measured for 3 cycles at 3 locations. Thereafter, 1 mL (200 pfu) of a 200 pfu / mL virus solution was added to adjust the electrode part so that it was immersed and allowed to interact for 15 minutes. After 15 minutes, the virus solution was removed and the cells were washed with PBS three times. The cells were filled with PBS, and cyclic voltammetry measurement was performed at three positions for 3 cycles each.

  The results are shown in FIG. FIG. 21 left is a measurement of IFV in solution when using a diamond electrode not modified with peptide. An increase in current density was observed in the 200 pfu IFV solution. The right side of FIG. 21 shows the result of using a peptide-modified diamond electrode. A marked increase in current density was observed compared to an unmodified diamond electrode, and IFV could be detected. In this way, IFV could be detected with high sensitivity.

Electrochemical impedance (EIS) measurement method HA and IFV detection by EIS measurement using peptide-modified BDD electrodes The conditions for impedance measurement were based on SK Arya et al., Sens. Actuators, B, 194, 127-133 (2014). .

First, a redox substance solution was prepared. K ₃ [Fe (CN) ₆ ] 0.164 g (0.5 mmol) and K ₄ [Fe (CN) ₆ ] 0.211 g (0.5 mmol) were weighed separately in a volumetric flask, dissolved in 50 mL of PBS, and 10 mM K ₃ [ Fe (CN) ₆ ] / PBS and 10 mM K ₄ [Fe (CN) ₆ ] / PBS were prepared. These were mixed at 1: 1 (v / v) to give 5 mM [Fe (CN) ₆ ] ^{3- / 4-} / PBS.

  Next, the control PC was set as shown in Table 11. As the initial potential (E Start), the potential (natural potential) already generated when the working electrode, the counter electrode, and the reference electrode are respectively attached is used, so that the value displayed on the control PC was input as needed. The number of sampling (Frequency), frequency domain (Frequency Scan), amplitude (Amplitude), etc. were determined from S. K. Arya et al., Sens. Actuators, B, 194, 127-133 (2014).

Similarly to the CV measurement, a three-electrode method (working electrode: conductive diamond electrode, counter electrode: Pt, reference electrode: Ag / AgCl) was used, and HA solutions of various concentrations were allowed to interact. First, a cell was assembled with a peptide-immobilized electrode (peptide charge × 100), 5 mM [Fe (CN) ₆ ] ³⁻ / ^4- / PBS was added, and the background was measured for 3 cycles. Thereafter, about 60 μL of an HA solution diluted with PBS (−) was added to the cell with PBS to adjust so that the electrode portion was immersed, and allowed to interact for 15 minutes. After 15 minutes, the solution was removed and washed with PBS three times, and 5 mM [Fe (CN) ₆ ] ^{3− / 4−} / PBS was filled in the cell and measured for 3 cycles. This operation was performed with HA solution (5-500 nM, 1-100 μg / mL, respectively), IFV solution (1-140 pfu), and bovine serum albumin (BSA) solution (5-500 nM).

  The HA EIS measurement results using a conductive diamond electrode are shown in FIGS. HA could be detected specifically. In FIG. 23, there was no correlation between concentration and response for the irrelevant protein BSA (control), whereas the response for HA was well proportional to the concentration. From these results, HA could be detected with high sensitivity and specificity.

FIG. 24 shows the EIS measurement result of IFV using a conductive diamond electrode. R _ct increases linearly with 40pfu or less, could be detected IFV concentration-dependent manner. In addition, it was possible to detect with high sensitivity even in the low viral load region of 0-40pfu.

  From the above, it was possible to detect 20 pfu of influenza virus using a conductive diamond electrode having an element for recognizing protein or pathogen immobilized on the surface. Non-specific binding of proteins existing in the living body such as albumin was not observed, and high-sensitivity detection was possible without using a sensitizer-labeled antibody that was essential in the conventional method.

  The cartridge of the present invention includes a conductive diamond electrode having an element for recognizing such a protein or pathogen immobilized on the surface. The apparatus of the present invention includes the cartridge, and can detect a protein or a pathogen by electrochemical measurement.

100 Cartridge 101 Working Electrode 102 Counter Electrode 103 Reference Electrode 104 Support Electrolyte Solution 105 Container 106 Counter Electrode Insertion Port 107 Reference Electrode Metal Electrode Insertion Port 110 Conductive Diamond Electrode 111 Outside Air Inlet 112 Inlet Filter 113 Ventilation Port 120 on the Outside Air Inlet Side Bubble generating filter 121 Bubble 123 Liquid junction part 124 of reference electrode Internal electrolyte solution 125 Member 130 for holding the internal electrolyte solution Liquid level 140 Working electrode 145 on the outside air inlet side Working electrode 150 on the outside air outlet side On the outside air outlet side Vent 155 Outside air outlet 200 Cartridge 201 with liquid level up / down mechanism 202 Liquid level up / down mechanism 202 Air inlet 230 for liquid level up / down mechanism Liquid level 240 Working electrode 245 on outside air inlet side On the outside air outlet side Working electrode 300 Detecting unit 301 The working electrode element is fixed. Surface 401 that can be stored and reproduced 402 Substrate 403 Wiring 500 Device 501 equipped with cartridge 100 of the present invention 501 Electrochemical measurement circuit 505 Outside air inlet 506 Outside air inlet pump 507 Outside air outlet 508 Outside air filter 509 Exhaust pipe 510 Control circuit 520 Data display circuit 530 Communication circuit 540 Data recording / reproducing circuit 550 Power supply circuit 560 Changeover switch 600 Device 610 provided with cartridge 100 and outside air tank Outside air tank 620 Circulating pump 630 Outside air tank exhaust valve 700 Cartridge 200, outside air tank, and liquid level up and down Device 701 with moving mechanism Pump 702 for liquid level vertical movement mechanism Intake port 703 for pump of pump 701 Exhaust port for pump of 701

Claims (21)

A cartridge for detecting proteins or pathogens, comprising one or more working electrodes, a counter electrode, a reference electrode, and a container capable of holding a supporting electrolyte solution,
The one or more working electrodes each have a conductive diamond electrode on the surface of which the same or different types of elements that recognize proteins or pathogens are immobilized;
The working electrode, the counter electrode, and the reference electrode are configured to allow electrical continuity with the outside.
The working electrode, the counter electrode, and the liquid junction of the reference electrode are arranged in the container so as to be able to contact the supporting electrolyte solution when the supporting electrolyte solution is present in the container,
The container includes an outside air inlet, an outside air outlet side vent and an outlet port,
The introduction port and the ventilation port on the outside air outlet side are configured such that the outside air introduced from the introduction port passes through the supporting electrolyte solution held in the container and is led out to the ventilation port on the outside air outlet side. The outside air that has passed through the vent on the outside air outlet side is further led out to the outlet of the cartridge,
The container and the working electrode are configured so that the outside air can come into contact with the one or more working electrodes when passing through the supporting electrolyte solution, and when the outside air is in contact with the one or more working electrodes. The counter electrode and the reference electrode are configured so that the liquid junction of the counter electrode and the reference electrode is always in contact with the supporting electrolyte solution,
The supporting electrolyte solution and the internal electrolyte solution of the reference electrode are configured to be injected into the container,
When the outside air is brought into contact with the working electrode and when electrochemical measurement is performed with the working electrode, the surface on which the working electrode element is fixed is configured to face downward.
The outside air inlet, the outside air outlet, and the outside air outlet are arranged at a position higher than the highest liquid surface position of the supporting electrolyte solution.
A cartridge for detecting the protein or pathogen.   2. The cartridge according to claim 1, wherein the vent on the inlet side of the outside air includes a bubble generation filter that makes the outside air a bubble.   When the cartridge is provided with a plurality of working electrodes, the working electrode on the outside air inlet side is disposed at a lower position than the other working electrodes on the outlet side, and the other working electrode on the outlet side is disposed on the outside air. The cartridge according to claim 1, wherein the cartridge is disposed at a higher position than the working electrode on the inlet side. A protein or pathogen comprising a container capable of holding one or a plurality of working electrodes, a counter electrode, a reference electrode, and a supporting electrolyte solution, and further having a liquid level up and down mechanism capable of raising and lowering the liquid level A cartridge for detecting,
The one or more working electrodes each have a conductive diamond electrode on the surface of which the same or different types of elements that recognize proteins or pathogens are immobilized;
The working electrode, the counter electrode, and the reference electrode are configured to allow electrical continuity with the outside.
The container has an outside air introduction port, an outside air outlet side ventilation port and an outlet port, and further has an air introduction port for a liquid level vertical movement mechanism,
The working electrode, the counter electrode, and the liquid junction of the reference electrode are arranged in the container so as to be able to contact the supporting electrolyte solution when the supporting electrolyte solution liquid level in the container rises,
The introduction port is configured to be able to blow the outside air introduced from the introduction port onto the surface of one or a plurality of working electrodes,
The outside air outlet side vent is arranged so that the outside air blown to one or a plurality of working electrodes can pass through, and the outside air that has passed through the outside air outlet side vent is further led out to the outlet of the cartridge. Configured,
When adsorbing protein or pathogen to the device, the level of the supporting electrolyte solution is lowered, and one or more working electrodes are not in contact with the supporting electrolyte solution.
When the level of the supporting electrolyte solution is lowered, the liquid junction of the counter electrode and the reference electrode may be in contact with the supporting electrolyte solution,
When detecting proteins or pathogens adsorbed on the element, the liquid level of the supporting electrolyte solution is raised, and the liquid junction of one or more working electrodes, counter electrodes and reference electrodes are in contact with the supporting electrolyte solution Has been
The supporting electrolyte solution and the internal electrolyte solution of the reference electrode are configured to be injected into the container,
When the outside air is brought into contact with the working electrode and when the electrochemical measurement is performed by the working electrode, the surface on which the working electrode element is fixed is configured to face downward.
The outside air inlet, the outside air outlet, and the outside air outlet are arranged at a position higher than the highest liquid surface position of the supporting electrolyte solution.
A cartridge for detecting the protein or pathogen.   When the cartridge includes a plurality of working electrodes, the working electrode on the outside air inlet side is disposed at a higher position than the other working electrodes on the outlet port side, and the other working electrodes on the outlet port side are disposed on the outside air. The cartridge according to claim 4, wherein the cartridge is disposed at a position lower than the working electrode on the inlet side.   The cartridge according to claim 4 or 5, wherein the air inlet for the liquid level up-and-down moving mechanism is located at a position higher than the highest liquid level position of the supporting electrolyte solution.   The cartridge according to any one of claims 1 to 6, wherein the cartridge is configured to have a sealed structure by injecting a supporting electrolyte solution from a counter electrode insertion port and attaching the counter electrode.   The internal electrolyte solution for the reference electrode is injected from the metal electrode insertion port of the reference electrode, and the metal electrode is attached so that the container can have a sealed structure. The cartridge according to item.   The cartridge according to claim 1, further comprising an element capable of storing and reproducing.   The cartridge according to claim 9, wherein the element capable of storing and reproducing can be recorded and reproduced from the outside of the cartridge via a substrate.   The cartridge according to any one of claims 1 to 10, further comprising an introduction filter immediately after the outside air introduction port. A device for detecting proteins or pathogens comprising one or more cartridges according to any one of claims 1 to 11, comprising
A working electrode, a counter electrode, and a reference electrode on the outer surface of the cartridge configured to be electrically conductive with the outside are electrically connected to the detection device;
When the cartridge includes an element capable of storing and reproducing, the element capable of storing and reproducing is electrically connected to the detection device,
An electrochemical measurement circuit capable of performing cyclic voltammetry measurement and / or electrochemical impedance measurement;
It has a display circuit that displays the measurement result, or an output terminal that outputs or transmits the measurement result.
It has a power circuit,
An outside air inlet for sucking outside air into the detection device, and the outside air inlet is connected to the outside air inlet of the cartridge;
An outside air outlet for discharging outside air from the detection device, the outside air outlet being connected to the outside air outlet of the cartridge;
It has an outside air suction pump connected to the outside air inlet,
Equipped with a control circuit,
An apparatus for detecting the protein or pathogen.   The cartridge according to any one of claims 4 to 11, wherein the cartridge includes a liquid level vertical movement mechanism, the detection device includes a pump for the liquid level vertical movement mechanism, and the pump is provided in a control circuit of the detection device. 13. The device according to claim 12, which is connected.   The apparatus according to claim 12 or 13, wherein when the apparatus includes a plurality of cartridges, the outside air inlet and the outside air outlet of the plurality of cartridges are connected to the apparatus in series or in parallel.   15. A device according to any one of claims 12 to 14, comprising a wireless or wired communication circuit.   The apparatus according to any one of claims 12 to 15, further comprising a data recording / reproducing circuit that records and reproduces data with respect to the element when the cartridge includes an element capable of storing and reproducing.   The device according to any one of claims 12 to 16, further comprising an outside air filter between the outside air inlet of the device and the outside air inlet of the cartridge.   The external air tank exhaust valve provided with an external air tank and a circulation pump, and having an external air tank exhaust valve capable of closing an exhaust pipe connected to the external air exhaust port between the external air exhaust port and the external air tank. The device according to item.   A method for detecting a protein or a pathogen using the cartridge according to any one of claims 1 to 11 or the apparatus according to any one of claims 12 to 18. When the cartridge has an element capable of storing and reproducing, from each element, information on the protein or pathogen to be measured by one or more working electrodes, initial characteristics of one or more working electrodes, and already measured For the cartridge used in the process, the time used for measurement, the process of reading the information of the detected numerical value,
A step of operating an outside air suction pump and feeding the sucked outside air into the cartridge;
An adsorption process in which inhaled outside air is blown onto a surface on which one or more working electrode elements are fixed, or in contact with one or more working electrodes as bubbles in a supporting electrolyte solution;
An exhaust stroke in which the outside air after the adsorption process passes through the outside air outlet side of the cartridge and is discharged from the outside air outlet and the outside air outlet of the device;
An electrochemical measurement process in which the external air suction pump is stopped and cyclic voltammetry measurement or electrochemical impedance measurement is performed while sequentially switching one or more working electrodes of one or more cartridges;
A step of recording a measurement result in each element of each cartridge when the cartridge has an element capable of storing and reproducing; and a recording information is communicated to the outside when the apparatus has a wireless or wired communication circuit Process,
The detection method according to claim 19, comprising: Using the detection device according to claim 18, comprising an outside air tank, a circulation pump, and an outside air tank exhaust valve, the following steps:
When the cartridge has an element capable of storing and reproducing, from each element, information on the protein or pathogen to be measured by one or more working electrodes, initial characteristics of one or more working electrodes, and already measured For the cartridge used in the process, the time used for measurement, the process of reading the information of the detected numerical value,
Opening the outside air tank exhaust valve and operating the outside air suction pump to introduce unmeasured outside air into the outside air tank;
Closing the outside air tank exhaust valve and stopping the outside air suction pump;
A process of operating the circulation pump and sending the outside air in the outside air tank into the cartridge;
An adsorption process in which inhaled outside air is blown against a surface on which one or more working electrode elements are fixed, or in contact with one or more working electrodes as bubbles in the supporting electrolyte;
Passing the outside air after the adsorption step through the outside air outlet side of the cartridge and exhausting it from the outside air outlet port to the outside air tank;
An electrochemical measurement step in which the cyclic pump is stopped and cyclic voltammetry measurement or electrochemical impedance measurement is performed while sequentially switching one or more working electrodes of one or more cartridges;
A step of recording a measurement result in the element of each cartridge when the cartridge has an element capable of storing and reproducing; and
A step of communicating recorded information to the outside if the device has a wireless or wired communication circuit;
A method for detecting a protein or pathogen, comprising:

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